<figure><img src="image_83.png" /></figure>

Review

# Fascinating Fasciclin: A Surprisingly Widespread Family of Proteins that Mediate Interactions between the Cell Exterior and the Cell Surface

Georg J. Seifert

Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Science, Muthgasse 18, 1190 Vienna, Austria; georg.seifert@boku.ac.at; Tel.: +431-47654-94144

Received: 9 April 2018; Accepted: 17 May 2018; Published: 31 May 2018

**Abstract:** The Fasciclin 1 (FAS1) domain is an ancient structural motif in extracellular proteins present in all kingdoms of life and particularly abundant in plants. The FAS1 domain accommodates multiple interaction surfaces, enabling it to bind different ligands. The frequently observed tandem FAS1 arrangement might both positively and negatively regulate ligand binding. Additional protein domains and post-translational modifications are partially conserved between different evolutionary clades. Human FAS1 family members are associated with multiple aspects of health and disease. At the cellular level, mammalian FAS1 proteins are implicated in extracellular matrix structure, cell to extracellular matrix and cell to cell adhesion, paracrine signaling, intracellular trafficking and endocytosis. Mammalian FAS1 proteins bind to the integrin family of receptors and to protein and carbohydrate components of the extracellular matrix. FAS1 protein encoding plant genes exert effects on cellulosic and non-cellulosic cell wall structure and cellular signaling but to establish the modes of action for any plant FAS1 protein still requires biochemical experimentation. In fungi, eubacteria and archaea, the differential presence of FAS1 proteins in closely related organisms and isolated biochemical data suggest functions in pathogenicity and symbiosis. The inter-kingdom comparison of FAS1 proteins suggests that molecular mechanisms mediating interactions between cells and their environment may have evolved at the earliest known stages of evolution.

**Keywords:** arabinogalactan proteins; cellulose; pectin; matricellular proteins; SOS5; periostin; Mpb83

## 1. A Brief History of FAS1 Domain Proteins

The first FAS1 protein was identified in an insect model for central nervous system development, the grasshopper *Schistocerca americana*. In order to identify cell surface molecules potentially involved in the formation of axon bundles (fascicles), monoclonal antibodies (mAbs) recognizing cell surface antigens on specific fascicles were characterized. One of these antibodies recognized a 70 kDa glycoprotein named Fasciclin 1 (SaFas1 (Appendix A)) [1]. The genes coding for grasshopper SaFas1 and *Drosophila melanogaster* DmFas1 were cloned soon afterwards [2] and a homologous fruit fly gene called Midline fasciclin (*DmMfas*) was identified later [3]. In the fruit fly, a *DmFas1* knockout affected neuronal branching as well as synaptic function [4] and laser ablation of the grasshopper ortholog *SaFas1* led to disrupted cell adhesion of pioneer axons [5]. The crystal structure of DmFas1 provided the prototype for the structurally novel FAS1 domain [6]. In the meantime, molecular techniques and sequence comparison tools revealed the widespread occurrence of homologous proteins defined by the FAS1 domain (IPR000782; PF02469). The *Homo sapiens* genome encodes four FAS1 domain proteins named transforming growth factor-β induced protein (HsTgfbi), Periostin (HsPn), Stabilin-1 (HsStab1) and Stabilin-2 (HsStab2). The *HsTgfbi* gene (Appendix B) was identified in human adenocarcinoma cells as a transcript that was induced 20-fold by transforming growth factor-β [7]. Likewise, *HsPn*, was cloned based on its expression in an osteoblast cell line [8] and subsequently
found to be enriched in the periosteum [9]. Finally, HsStab1 and HsStab2 were identified using two different antibodies binding to a subpopulation of endothelial cells and to the hyaluronic acid (HA) clearance receptor, respectively [10]. Due to their association with a multitude of clinical conditions, human and mammalian FAS1 proteins have been the focus of numerous detailed studies that are instructive for a general understanding of FAS1 domain proteins. Unexpectedly for proteins associated with cell adhesion, FAS1 domain proteins exist not only in animals but also in plants, fungi and prokaryotes. The first plant FAS1 protein was discovered using antibody interference in the alga *Volvox carteri* [11], a simple model for multi-cellularity consisting of just two cell types. When specific mAbs raised against a crude membrane preparation were added to volvox cultures they inhibited embryo development. The cognate protein was named algal cell adhesion molecule (CAM) based on its apparent role in the formation of intercellular contacts during early embryogenesis. The existence and physiological role of algal-CAM, which contains two FAS1 domains, raised the exciting possibility of a cell adhesion mechanism conserved between animals and plants. In higher plants FAS1 domain proteins were also identified by the biochemical and bioinformatic analysis of a group of highly O-glycosylated hydroxyproline-rich glycoproteins called arabinogalactan-proteins (AGPs) [12,13]. The bioinformatic investigation of the *Arabidopsis thaliana* genome revealed the existence of many fasciclin-like AGPs (FLAs) in plants [12–14]. At the same time a different investigation mapped one of several *Arabidopsis thaliana* salt overly sensitive (sos) mutations to the Salt overly sensitive 5 (*AtSos5*) gene encoding the AtFla4 protein [15]. In a mutant screen in a crop plant, the rice locus Microspore and tapetum regulator 1 (*OsMtr1*) was found to be required for male reproductive development and to encode a tandem FAS1 glycoprotein [16]. In fungi, FAS1 proteins have been identified in transcriptomics or proteomics studies in the Shiitake mushroom *Lentinula edodes* [17] and the rice pathogen *Magnaporthe oryzae* [18], while in the fission yeast *Schizosaccharomyces pombe* the FAS1 domain protein SpFsc1 was identified in a screen for autophagy related loci [19]. Apparently, FAS1 proteins already existed before the evolution of eukaryotes. The best-known eubacterial FAS1 proteins are Mpb70 and Mpb83, which were identified in *Mycobacterium bovis* culture filtrates [8,20–24]. Database queries reveal FAS1 proteins in both eubacteria and archaea, suggesting the inception of the domain preceded the existence of last universal common ancestor (LUCA) [25]. FAS1 proteins are often implicated in the interaction between the cell and the extracellular matrix (ECM). Considering the diversity of ECM architectures and compositions FAS1 domain proteins are surprisingly widespread between different kingdoms of uni- and multicellular life. However, despite their seemingly boundless presence throughout the tree of life, FAS1 proteins are not ubiquitous, especially in microbes whose genomes rapidly adapt to differing life styles. This suggests that FAS1 domain proteins are not essential for life per se but are suited for specialized cellular interactions that for some organisms are not required. I will next describe what is known about the structure of the FAS1 domain itself and discuss diverse additional structural features of FAS1 proteins in various kingdoms. This will be followed by a review of the biological roles of mammalian and plant FAS1 domain proteins, including the relationship of structure to function, which should help elucidate the mechanisms of FAS1 proteins in plant development.

## 2. The Structure of the Fasciclin 1 Domain

### 2.1. The Fasciclin 1 Domain

The FAS1 domain extends to approximately 140 amino acids. Although sequence conservation between different FAS1 proteins can be quite low, there exist two more highly conserved sequence stretches of around 15 residues called H1 and H2 and a conserved central YH motif (Figure 1A). Therefore, to identify FAS1 domain proteins in sequence databases, domain enhanced lookup time accelerated BLAST (DELTA-BLAST) should be used [26]. Using X-ray crystallography and NMR spectroscopy, several studies have elucidated the structures of isolated FAS1 domains or of entire FAS1
proteins [27–33]. The FAS1 domain is globular and contains a central structural fold of two β-sheets oriented at an almost perpendicular angle, varyingly described as β-wedge or β-sandwich (Figure 1B).

<figure><img src="image_84.png" /></figure>

**Figure 1.** The FAS1 domain across kingdoms of life. (A) Delta-BLAST alignment of some FAS1 domains mentioned in this article. Note the conservation of the N and C-proximal H1 and the H2 region as well as the central YH motif. The sequences used by Delta-BLAST were HsTgfbi (NP_000349.1), HsPn (gi 93138709), DsFas1 FAS1-3 (1O70_A), Algal-CAM (gi 75282282), AtFLA11 (gi 116247778), AtFLA4 (gi 75206907), MbMpb83 (gi 614094354), and SmNex18 (gi 81635876). Red/blue indicates highly/not conserved residues; color bits threshold set to 2.5; (B) The general topology of the FAS1 domain features is reminiscent of the “thumbs-up” gesture. The secondary structure of HsTgfbi FAS1-1 is annotated omitting the three N-terminal helices for clarity; (C) The crystal structure of HsTgfbi FAS1-1 [33] in ribbon display.

In the human HsTgfbi structure, the first β-sheet encompasses strands β1-β2-β8-β6/7 [33] (Figure 1C). The two inner strands are oriented parallel and the two outer ones antiparallel. The second β-sheet consists of β3-β4-β5. There are three α-helices at the N-terminus (not shown in Figure 1B), three more (α4 to α6) between β1 and β2 and a less highly conserved α-helix (α') between β2 and β3. The FAS1 domain (Figure 1B, redrawn from [25]) is a member of the “β-grasp fold” superfamily [25] and may be imagined as a “thumbs-up” gesture of the right hand with the palm representing the first β-sheet (light green in Figure 1B), the bent index, middle and ring fingers symbolizing the second β-sheet (dark green in Figure 1B) and the thumb and pinkie resembling α4 to α6 (light brown in Figure 1B) and α' (dark brown in Figure 1B), respectively. Among all FAS1 proteins, structure to function relations have been most intensely studied for HsTgfbi (reviewed in [34]). Therefore, the elucidation of the entire HsTgfbi crystal structure [33] could be seen as the “Rosetta stone” for a better mechanistic understanding of the many biological roles of FAS1 proteins in different organisms including plants. Several studies identified individual regions and amino acid residues on the four
Tgfbi FAS1 domains that are critical for function. The main approach was to use in vitro cell adhesion as a functional assay (Figure 2).

<figure><img src="image_85.png" /><figcaption>**Figure 2.** Cell adhesion assay. (A) On an uncoated or control-coated (red) plastic substrate, cell adhesion is inefficient; (B) cells adhere rapidly when plastic is coated with an adhesion protein such as Tgfbi or Pn (green); (C) to identify the receptor for the adhesion protein, integrin isotype specific antibodies (red) are co-incubated; (D) mammalian FAS1 proteins are thought to bind to different types of dimeric integrins (blue and yellow) that mediate mechanical contact between the cytoskeleton (grey) and the ECM as well as transduce intracellular signals using numerous associated proteins (pink and nude).</figcaption></figure>

Briefly, cells more efficiently adhere to surfaces coated with adhesion proteins such as Tgfbi than to control-coated surfaces (Figure 2A,B). That this adhesion is dependent on the family of ECM receptors called integrins can be tested by adding integrin antibodies that block the Tgfbi-stimulated cell adhesion (Figure 2C). To identify sites on Tgfbi that might mediate cell adhesion, peptides corresponding to conserved Tgfbi sequence motifs were added. For instance, the NKDIL and the EPDIM peptides that are part of the FAS1-2 and the FAS1-4 (see following section) domains, respectively, both interfered with Tgfbi stimulated cell adhesion. By contrast, the KADHH peptide in the corresponding region of FAS1-1 had no effect [35]. In a study of a different cell type expressing a different integrin, the NKDIL and EPDIM peptides did not interfere with adhesion but an 18-amino acid peptide that covered the YH motif did [36]. A HsPn specific mAb identified a corresponding integrin interaction region on FAS1-2 [37]. The cell adhesion assay is too crude to demonstrate *direct* binding between FAS1 proteins and integrin; however, in combination with the crystal structure of HsTgfbi it showed that different integrins interact with different surface regions of the FAS1 domains [33]. Although integrins are not known in plants, given the complex biological roles of some FAS1 proteins of plants, this insight should be valuable for the prediction of their molecular function.

## 2.2. *Single and Tandem Fasciclin 1 Domains*

While prokaryotic representatives typically contain a single FAS1 domain, many eukaryotic family members contain two or more arranged in tandem (numbered FAS1-1, FAS1-2 etc. from N-to C-terminus). The two insect family members Fas1 and Mfas, and mammalian Tgfbi and Pn each contain four FAS1 domains, while there are seven in the two mammalian stabilins (Figure 3).
<figure><img src="image_86.png" /><figcaption>**Figure 3.** Domain organization and post translational modifications of six representative FAS1 proteins discussed in this article. Proteins are shown with their N-termini on the left. As opposed to the C-terminally attached GPI-anchors Mpb83 is acylated near its N-terminus. Note that the annotation of AG II glycosylation is tentative and that the annotation of N-glycans is based on bioinformatic predictions. Abbreviations used in this Figure, GPI: glycosylphosphoinositidyl; TM: transmembrane; AG II: arabinogalactan type II; CROPT: Cysteine-Rich domain of Pn and TGFBIp; EGF: epidermal growth factor; PAST: domain rich in Pro, Ala, Ser and Thr.</figcaption></figure>

In plants, there exist both single FAS1 FLAs as well as family members with two domains in tandem. FAS1 tandem proteins also exist in fungi, where dual and five-fold tandems have been reported [18,19]. The tandem FAS1 domains structurally interact with each other, as was found in a structure of DmFas1 domains FAS1-3 an FAS1-4 [27] and of whole HsTgfbi [33]. It was suggested that the C-proximal domain in a tandem pair might bind to interaction partners such as integrins while the N-proximal domains might compete with this binding acting either as negative regulators or as safeguards against unwanted ligand binding [28]. A recent structure to function study of *AtFla4* in *Arabidopsis thaliana* showed that the C-proximal FAS1 domain was necessary and sufficient for genetic function [38]. By contrast, some studies showed cooperativity between all FAS1 domains [39]. To which degree the intramolecular interaction between neighboring FAS1 domains might affect the intermolecular interaction with other binding partners in vivo remains an open question.

## 2.3. Membrane Association

Most FAS1 domain proteins contain additional structural features and post-translational modifications some of which are shared between phylogenetically distinct family members. Practically all FAS1 proteins have an N-terminal secretion peptide; however, the final localization is determined by other signals. Mycobacterial Mpb83 is lipid-anchored to the plasma membrane by Cys acylation of the LAGC motif close to the N-terminus [40,41]. In plants, most FAS1 proteins are anchored to the outer sheet of the plasma membrane by a glycosylphosphoinositidyl (GPI) lipid anchor [14]. This modification is attached to proteins in the endoplasmic reticulum in exchange of a C-terminal signal peptide and exists in FAS1 proteins of plants, insects and fungi [17,18,42]. However, only one of the two *D. melanogaster* FAS1 proteins, DmFas1, is GPI-anchored [43]. The two mammalian stabilins and SpFsc1 from fission yeast are type 1 transmembrane proteins with cytosolic tails at their C-termini [10,19]. By contrast, Mpb70 from mycobacteria, mammalian Tgfbi and Pn, DmMfas and a group of FLAs are soluble proteins without any membrane anchoring motif [14,34]. Hence most FAS1 proteins are either
bound to the plasma membrane or are soluble. In case of Mpb83 both surface anchored and soluble forms exist. Likewise, GPI-anchored proteins can be released from the plasma membrane by the action of phospholipases. The GPI anchor is probably needed for efficient transport of GPI-anchored proteins to the plasma membrane and potentially for their controlled release. In plants the GPI-anchor acts as a sorting signal for plasmodesmata [44]. Whether in non-animal eukaryotes such as plants and fungi that lack a cholesterol-rich plasma membrane, the GPI modification functions similar to animals to sequester proteins into membrane microdomains is under debate [45].

## 2.4. Glycosylation

Secreted proteins are often glycosylated. This is also the case for many FAS1 proteins, but not for all of them. For instance, although Mpb70 and Mpb83 share 75% sequence identity, but the latter is O-glycosylated close to its N-terminus with three threonine-linked mannose residues [46]. Another example is the N-linked glycosylation of four animal FAS1 proteins, mammalian Tgfbi and Pn and insect Fas1 and Mfas that are similar in size and all have four FAS1 domains, however, they carry zero, one, five and eight N-glycans, respectively. Likewise, in plant FAS1 proteins, N-glycosylation of FAS1 domains ranges from two to eight N-glycans [14,16]. A distinguishing feature of FAS1 proteins in plants is the presence of regions rich in proline, alanine, serine and threonine (PAST). Especially sequences containing the repetitive pattern (P-A/S/T)ₙ, whose Pro residues are post-translationally converted to Hyp and galactosylated are typical for AGPs [47]. Indeed, the first FLAs were identified in precipitates obtained with β-glucosyl Yariv reagent [12,13], which binds to β(1→3) galactose chains present on many AGPs [48,49]. However, the peptide patterns for O-glycosylation are not as precisely defined as the N-X-T/S N-glycosylation motif [50], and biochemical identification of O-glycosylation on defined proteins is not as straightforward as the molecular weight shift caused by enzymatic removal of N-glycans. Hence one should be aware of potential discrepancies as to which FAS1 proteins are indeed FLAs, e.g., Algal-CAM contains an extensive PAST-rich N-terminal region and so does OsMtr1, and after removing N-glycans the apparent molecular weights of both proteins were larger than expected for the apoprotein [11,16]. Therefore, both proteins might well be members of the FLA family. By contrast, other FAS1 proteins such as the wheat locus named TaFLA9 lack any PAST-rich regions [51] and might be wrongly annotated as FLAs. However, the AtFla4 protein is decorated with AGP-like glycan epitopes despite the absence of canonical (P-A/S/T)ₙ repeats [38]. Clearly, the simple presence or absence of PAST-rich regions is an inaccurate predictor for AGP-like O-glycosylation. The O-glycans of FLAs and other AGPs are known as type II arabinogalactans (AG II). They consist of Hyp-linked galactose that is the basis of a (1-3) β-linked galactan backbone with (1-6) β-linked galactose branches and kinks. AG II contains terminal modifications, mainly by L-arabinofuranose, L-rhamnose, L-fucose, D-glucuronic acid, 4-methyl-D-glucuronic acid and D-xylose [52–54]. Although pharmacological and genetic interference with AG II function has revealed many biological roles for these carbohydrates [55,56], models of how AGPs function remain sparse. An AGP of low abundance was shown to form a covalent link with both hemicellulose and pectin [57]. In fractionated cell walls, AGP-associated carbohydrate epitopes are abundant in pectic and hemicellulosic fractions and some are even present in the cellulose fraction [58]. Moreover, AGPs are self-adhesive in a calcium-dependent fashion [59], and can bind to pectin via carboxyl-calcium ion bridges [60]. Such results indirectly suggest that AG II-modified proteins FLAs might at least partially be linked to cell wall polysaccharides via their O-glycans.

## 2.5. Additional Domains

For human FAS1 proteins, O-glycosylation has not been reported [43]; however, they contain additional domains that expand their spectrum of molecular interactions. The N-terminal region of mature HsTgfbi, HsPn and DsMfas folds into a structure called Cysteine-Rich domain of Pn and TGFBIp (CROPT) [33], that mediates the interaction with various forms of collagen, fibronectin and the heterophilic binding between HsPn and HsTgfbi (reviewed in [43,61]). The human stabilins contain
three dual FAS1 domain tandems and one isolated FAS1 domain, that each are separated by a varying number of Epidermal Growth Factor (EGF) repeats and an X-link domain. These domains are thought to be involved in phosphatidylserine and hyaluronic acid binding, respectively, but are not present in FAS1 proteins other than mammalian stabilins [62,63]. The C-terminal domain (CTD) of HsTgfbi contains the RGD motif that, although crucial for integrin binding of many adhesion proteins, is not required for the adhesion stimulating properties of HsTgfbi [64,65]. However, the RGD motif might modulate binding of HsTgfbi to specific integrins [39]. The CTD of HsPn, displays a high degree of intrinsic disorder and is both differentially spliced [66] and proteolytically regulated [67]. Interestingly, compared to full-length forms, HsPn forms lacking the CTD show relatively enhanced binding to ECM proteins [68]. The extreme C-terminus is highly basic and is thought to bind to heparin. Differential splicing and proteolysis might switch between forms of HsPn that more readily binds to HS and forms that more strongly interact with matrix proteins [61,67]. Compared to vertebrates, differential splicing is less frequent in plants; however, a great degree of regulatory fine-tuning might be provided by differential gene expression of the large number of plant genes encoding FAS1 proteins.

## 2.6. Groups of Fasciclin 1 Proteins in Plants

Compared to other organisms, plants contain the greatest number of FAS1 protein encoding genes. The majority of them are annotated as FLAs. The numbers of FLA encoding loci vary considerably between different plant genomes such as cotton (17 and 19 loci in *Gossypium barbadense* and *G. hirsutum*, respectively [69,70]), *Eucalyptus grandis* (18 [71]), *Arabidopsis thaliana* (21 [14]), *Cannabis sativa* (23 [72]), *Brassica rapa* (33 [73]), wheat *Triticum aestivum* (34 [51]), rice *Oryza sativa* (27 [74]) and cottonwood *Populus trichocarpa* (35 [75]). Apparently, diversity in this gene family varies even between closely related species (e.g., between the two *Gossypium* species or between *Arabidopsis thaliana* and *Brassica rapa*). An important step to understand the diversity of this complex gene family was sub-classification according to the presence of one or two FAS1 domains, their sequence similarity, the presence of GPI-anchors and the organization of PAST-regions [14]. Inclusion of FAS1 domain proteins that are not yet annotated as AGPs, such as OsMtr1 and Algal-CAM, might slightly enlarge the FLA family. The present FLA sub-classification has four groups: group A members are GPI-anchored and contain a single FAS1 domain flanked by two PAST-regions, group B are not GPI-anchored and sport a tandem of two FAS1 domains separated by a PAST-region. Group C contains GPI-anchored, single and tandem FAS1 proteins, with the single FAS1 domains phylogenetically closest to the N-proximal FAS1 domains of that group. Moreover, group C has PAST regions inserted close to their C-termini and between FAS1 domains. Lastly, group D contains FLAs that are not closely related to any other group and might represent separate branches of diversification.

# 3. Biological Aspects of FAS1 Domain Proteins across the Tree of Life

## Human and Mammalian Fasciclin 1 Proteins

FAS1 proteins have been the subject of experimentation in a variety of species; however, by far the most work has been published about the four human representatives [reviewed in [34,43,76-80]]. Particularly *HsPn*, *HsTgfbi* and *HsStab2* have been implicated in a plethora of medically relevant conditions ranging from normal development [76,81] to wound healing [77], bone regeneration [82,83] and cancer [67]. A Medline search shows 624, 1229, 105 and 718 entries for Tgfbi, Pn, Stab1 and Stab2, respectively (2 March 2018, Title/Abstract, synonyms see Appendix B). A detailed medical discussion is beyond the scope of this article; however, selected highlights are presented to provide a critical viewpoint on the molecular function of FAS1 domain proteins in plants. Missense mutations in the *HsTgfbi* locus are known to cause different types of corneal dystrophy [84]. In many cases of this disease, mutated HsTgfbi forms aggregates in the corneal stroma. This is due to the thermal and pH instability of the protein and to its high abundance in the cornea [85]; however, the condition might not help us to understand HsTgfbi function. A complete *Tgfbi* gene knockout in mice caused neither corneal
abnormalities nor any other major defects [86], suggesting compensation by other ECM molecules. Although *Pn* knock-out mice showed that this locus is essential for normal osteogenesis and dental development [87,88], the two closely-related *Pn* and *Tgfbi* loci function identically in many ways [43]. The most intriguing common property of mammalian FAS1 proteins is their interaction with the integrin family of ECM receptors (Table 1). Hetero-dimeric integrins are expressed in a cell-type-specific fashion and bind to many different ECM proteins such as fibronectin, laminin and fibrinogen. The cell adhesion assay described above (Figure 2) suggests that FAS1 domains might bind to integrins; however, to establish the mode of binding, alternative assays such as co-immunoprecipitation of recombinant proteins and Foerster resonance energy transfer are required (Table 1).

<table><caption>**Table 1.** Mammalian Fas1 proteins interact with integrin receptors.</caption><thead><tr><th>Integrin Type</th><th>TGFBI</th><th>PN</th><th>STAB-2</th></tr></thead><tbody><tr><td>α1β1</td><td>[64] I,A</td><td></td><td></td></tr><tr><td>α2β1</td><td>[89] C,F</td><td></td><td></td></tr><tr><td>α3β1</td><td>[35] A,I,P<br>[90] A,I<br>[91] A,I</td><td></td><td></td></tr><tr><td>α4</td><td>[92] I</td><td></td><td></td></tr><tr><td>α5</td><td>[92] I</td><td></td><td></td></tr><tr><td>α5β1</td><td></td><td>[93] A,I,G</td><td></td></tr><tr><td>α6β4</td><td>[94] A,I</td><td>[95] I</td><td></td></tr><tr><td>α7β1</td><td>[96] A,I</td><td></td><td></td></tr><tr><td>αvβ3</td><td>[97] A,I,P<br>[98] A,I<br>[99] C,F</td><td>[100] I,A<br>[101] I<br>[88] I<br>[102] A,D<br>[103] G<br>[104] I<br>[105] I</td><td></td></tr><tr><td>αvβ5</td><td>[106] A,I,P<br>[36] A,I,P</td><td>[100] I,A<br>[101] I<br>[102] A,D<br>[104] I</td><td>[107] C,T</td></tr><tr><td>β3</td><td></td><td>[108] G,A</td><td></td></tr><tr><td>αMβ2</td><td>[65] A,C,I</td><td>[109] A,I</td><td>[110] I,G</td></tr></tbody></table>

Assay type: G: genetic interference (siRNA, transfection, mutants & epistasis analysis etc.); A: cell adhesion (coating of plastic surfaces); I: immuno-interference (e.g., anti-integrin antibodies inhibit PN mediated responses); P: peptide interference (peptide of Fas1 domain interferes with integrin dependent binding); C: Co-IP (endogenous or added recombinant Fas1 protein); F: immunofluorescence co-localization; D: DNA-aptamer inhibition; T: FRET.

The integrin complexes are hubs of mechano-chemical signaling and interact with the cytoskeleton and with numerous signaling pathways including focal adhesion kinase (FAK), mitogen activated protein kinase (MAPK), Target of rapamycin (mTOR), and β-catenin that influence cell division and migration, differentiation and programmed cell death [76]. FAS1 proteins not only bind and influence integrins as a stationary component of the ECM but also act as mobile paracrine factors. An example for the latter is the role of HsPn in glioblastoma. In this invasive brain cancer, glioblastoma stem cells (GSCs) secrete HsPn that acts as chemoattractant for circulatory monocytes (MCs)—precursors of macrophages (Figure 4A).
<figure><img src="image_87.png" /><figcaption>**Figure 4.** Multiple roles of HsPn: (A) Circulatory monocytes (MC) are attracted by glioblastoma stem cells (GSC) that secrete HsPn (dark green) to invade the site of the primary tumor and differentiate into tumor associated macrophages (TAM); (B) GSC-secreted Pn via integrin αvβ3 signaling triggers the release of growth factors (GF) that establish a metastatic niche; (C) molecular interactions of Pn in the ECM. Blue double arrows indicate binding of HsPn. Grey double arrows indicate binding of other components. Green arrows indicate activating reactions: the metalloprotease Bone morphogenetic protein Bmp-1 proteolytically activates Lysine oxidase 1 (Lox-1) in turn enhancing collagen crosslinking.</figcaption></figure>

MCs are induced to invade the site of the primary tumor and to differentiate into tumor associated macrophages (TAMs) by HsPn binding to MC-expressed integrin. In turn, TAMs secrete growth factors that further stimulate cell division in GSCs generating a metastatic niche [105] (Figure 4B). A similar model for Pn function has previously been proposed for other malignancies such as cholonangiocarcinoma and renal carcinoma [93,104]. Both HsPn and HsTgfbi directly bind to several ECM components and modulate the biosynthesis of the ECM proteins (Figure 4C). Owing to its versatile binding properties, mammalian Tgfbi was previously known as collagen-associated protein containing the RGD sequence (RGD-CAP) [111], while HsPn is known to bind to collagen and fibronectin [61]. The latter interaction affects the correct secretion and deposition of these fibrous proteins [112] and additional interactions facilitate the intramolecular crosslinking of collagen in the ECM. Via its FAS1-1 and FAS1-3 domain Pn binds to Bone morphogenetic protein-1 (Bmp-1), a metalloprotease that proteolytically activates lysine oxidase-1 (Lox-1), which, in turn, enhances collagen crosslinking [113,114]. The peptide growth factor Bone morphogenetic protein-2 (Bmp-2) that is not related to Bmp-1 also binds to Pn albeit at a different site [113]. The physiological role of this interaction remains unknown. Another matrix protein interacting with Pn is tenascin-C that forms a remarkable six-armed structure named hexabrachion and it was suggested that the link between tenascin-C and collagen formed by Pn might adapt the ECM to mechanical stress [68]. While some of the ligands of HsPn and HsTgfbi bind to the CROPT domain or the C-terminal domain, the interaction with integrin occurs via FAS1 domains. In overview, the two secreted FAS1 proteins HsTgfbi and HsPn exemplify how FAS1 domains accommodate a variety of protein-protein interactions enabling a single protein to act both in ECM integration and in intercellular signaling. By contrast, the membrane anchored
stabilins mostly act as receptors for self ligands and as cell-cell interaction signals. One of their biological functions is in the removal of unwanted material from extracellular spaces and circulation by professional scavenger cells. Despite their similarity at the domain and sequence level, HsStab1 and HsStab2 are associated with distinct ligands and processes. HsStab1 acts as an intracellular sorting receptor as well as an endocytotic cell surface receptor for ligands such as acetylated low-density lipoprotein (acLDL) [115], secreted protein acidic and rich in cysteine (SPARC) [116,117], placental lactogen (PL) [118] and a chitinase-like protein called SI-CLP [119,120]. The FAS1-7 domain of HsStab1 binds to PL and SI-CLP while acLDL and SPARC interact with domains other than FAS1. HsStab1 can guide its various ligands on the secretory route from the Golgi into the lysosomes after endocytosis and through transcytosis (reviewed in [79]). HsStab2 binds ligands such as acLDL and advanced glycation products as well as various glycosaminoglycans such as HA, chondroitin sulfate and dermatan sulfate as well as the anticoagulant heparin and is required for the physiological clearance of these materials from circulation [121]. The polysaccharides bind to the X-link domain on Stab2 [122]. Both HsStab1 and HsStab2 have also been implicated in the phagocytosis of unwanted cells [123]. With their EGF domains, the stabilins recognize the "eat-me!" signal phosphatidylserine (PS) present at the surface of apoptotic cells and aged erythrocytes. HsStab2 utilizes its FAS1 domain in this process to bind to and cooperate with αvβ5 integrin [107]. The interaction between HsStab2 and PS is important for the fusion of uninucleate myoblasts during myofiber formation with PS functioning as a "fuse-me!" signal [124,125]. Finally, the interaction of HsStab2 FAS1 domains with integrins mediated both homotypic and heterotypic cell to cell adhesion [110,126]. In summary, each of the four human FAS1 proteins fulfils a variety of different biological functions. While secreted HsTgfbi and HsPn act in cell-ECM adhesion, ECM structuring and intercellular signaling, the membrane-anchored HsStab1 and HsStab2 proteins act in endocytosis, intracellular trafficking and cell to cell recognition.

## 4. Biological Functions of Plant Fasciclin 1 Proteins

The varying gene numbers for FLAs in different plant species shows that gene duplication and gene loss are common. On the one hand, this suggests that the large repertoire of FAS1 proteins might be fine-tuned at the level of differential gene expression analogous to differential mRNA splicing in vertebrates. On the other hand, gene redundancy makes the investigation of FLA function more cumbersome. However, the non-redundant *AtFla1*, *AtFla3*, *AtFla4*, *OsMtr1* and the pair of *AtFla11* and *-12* provide a glimpse of the role of FAS1 proteins in plants.

### 4.1. *The AtFla4 AtFei Pathway*

A mutant screen in *Arabidopsis thaliana* that discovered several key regulators of salt stress response [127] also identified a mutant that carried a missense mutation in the *AtFla4* locus [15] often called *atsos5* and here referred to as *atfla4* for consistency. In *atfla4* root elongation was almost completely stalled after transfer to growth media containing moderate levels of NaCl and the mutant root developed a characteristic radially swollen tip. Likewise, elevated levels of sucrose induced this phenotype in *atfla4* [128], which is reminiscent of many conditional mutants defective in cell wall polysaccharide biosynthesis [129]. Although conditional root swelling is the most dramatic aspect of the *atfla4* phenotype, the mutant's abnormally thickened hypocotyls and inflorescence stems, its fat roots under salt-free conditions, larger leaves, shorter siliques and its abnormal seed coat mucilage indicate that *AtFla4* acts non-conditionally throughout the plant [15,128,130–134]. Cell walls in *atfla4* roots were shown to lack the pectin-rich middle lamella that is essential for intercellular adhesion, suggesting a potential function of *AtFla4* in pectin biosynthesis or structure [15]. However, double knockouts in the two similar leucine-rich receptor-like kinases (LRR-RLKs) *AtFei1* and *AtFei2* were not only phenotypically identical to *atfla4* but also non-additively interacted with it [128]. This suggested that *AtFla4* and the two *AtFei* loci act in a linear genetic pathway. The *atfei1* *atfei2* double mutants were hypersensitive to cellulose biosynthesis inhibition and showed a decrease in crystalline cellulose production under restrictive conditions. As these phenotypic features were reminiscent of cellulose
biosynthetic mutants it was proposed that the Fei-RLKs and by association AtFla4, might function in the biosynthesis of this important polymer [128]. The phenotype of both *atfla4* and *atfei1 atfei2* was modulated by variety of growth regulators. Firstly, the *atfei1 atfei2* root phenotype was suppressed by inhibition of the biosynthesis and oxidation of ACC but not of the perception and signaling of its product ethylene [128]. This suggested that, in line with *AtFla4*, the *AtFei* loci might negatively regulate a novel ethylene-independent ACC signaling pathway that is activated by cell wall defects [135]. Both AtFei proteins interacted with ACC synthase in yeast two hybrid assays but did not phosphorylate them in vitro [128]. Secondly, abscisic acid (ABA) signaling acted synergistically with *AtFla4* to regulate root elongation and abiotic stress response as the *atfla4* phenotype was suppressed by exogenous ABA and by ABA-oversensitive mutants and a *atfla4*-like phenotype was induced by ABA inhibition [130]. Interestingly, *AtFla4* did not interact with two NADPH respiratory burst oxidases that are required for stress responses triggered by cellulose synthase inhibition [136,137]. Finally, both the *atfei1 atfei2* double and the *atfla4* single mutants non-additively interacted with the IAA-alanine resistant 4 (*Atlar4*) locus [138]. These studies implicate the *AtFla4 AtFei* pathway with a variety of intracellular signaling pathways; however, they do not provide a model for the relation between the cell wall and AtFla4. Insights into this problem were stimulated by the characterization of the seed coat mucilage phenotype of *atfla4* and *atfei2*. During seed maturation, pectic mucilage polymers are secreted into a pocket between the plasma membrane and the primary cell wall. Upon hydration of the mature seed the mucilage polymers rapidly swell and rupture the primary walls. However, the mucilage normally adheres to the ruptured primary cell wall [139]. There exist numerous mutants affected in aspects of seed coat mucilage biosynthesis and maturation. They are instructive models for cell wall biosynthesis throughout the plant [140]. Because in *atfla4* and *atfei2* mutants and in the cellulose synthase mutant *atcesA5* seed coat mucilage adhesion was found to be defective it was initially suggested that *AtFla4* and *AtFei2* might act in cellulose biosynthesis [131,132]. However, the subsequent comparative analysis of *atfla4*, *atfei2*, *atcesA5* and other mucilage adherence mutants showed distinct roles of *AtFla4* and *AtFei2* on the one hand and *AtCesA5* on the other hand [133,134]. Rather than influencing cellulose or hemicellulose structure, *AtFla4* acted on the pectin network, which was clearly distinct from the genetic actions of *AtCesA5* and of the xylan biosynthetic locus *Mucilage-modified5* (*AtMum5*) [133,141,142]. In contrast to mutants in the latter genes, *atfla4* non-additively interacted with two pectin-related loci. Firstly, *Mucilage-modified2* (*AtMum2*) encodes a β-galactosidase acting on galactan side chains of pectic rhamnogalacturonan type I (RG I) and its loss of function prevents the extrusion of mucilage upon hydration [143]. The extrusion defect of *atmum2* was suppressed in the *atfla4 atmum2* double mutant [133]. Secondly, Flying saucer 1 (*AtFly1*) encodes a transmembrane E3 ubiquitin ligase thought to regulate the degree of pectin methyl esterification. Loss of *AtFly1* function led to ectopic adhesion between primary cell walls and mucilage [144] and *atfla4* suppressed this phenotype [133,134]. A possible explanation for these observations is that AtFla4 might physically interact with galactan side chains on rhamnogalacturonan type I (RGI) pectin structures that are normally remodeled by the β-galactosidase encoded by *AtMum2*, to control timely mucilage maturation and adherence. This hypothesis is consistent with the localization of *AtFla4* in the mucilage pocket during seed coat development [133,134]. How does AtFei2 fit into this scenario, and can the characterization of the seed coat mucilage phenotype be extrapolated to the whole plant? In the case of seed coat mucilage, AtFei2 might attach AtFla4 to the plasma membrane and thereby contribute to the architecture of the network—an entirely structural explanation. However, it cannot be ruled out that the interaction between the hypothetical AtFla4-pectin matrix and AtFei2 might also convey signals to the cell interior [133,134]. While AtFla4 localized to the developing mucilage pocket and the adjacent plasma membrane of seed coat epidermal cells, AtFla10 and AtFla17 but not AtFla4 were identified in a proteome of mature mucilage [145]. Whatever the biological function of these FLAs in mature mucilage, due to the *atfla4* single mutant phenotype they apparently are not functionally equivalent to AtFla4. It is unclear whether the mode of action of *AtFla4* in seed coat mucilage is identical to its mechanism throughout the plant; however, the overlapping genetic role with the *AtFei* genes in both tissues
suggests a common mechanism. The AtFla4 protein might interact with various cellular components. It was hypothesized that AtFla4 might bind to the AtFei RLKs [128,146]. The localization of the majority of yellow fluorescent protein-tagged AtFla4 at the plasma membrane is compatible with this possibility [38]. However, AtFla4 was also released into the apoplast, where it might interact with cell wall carbohydrates [38,133]. The interaction of AtFla4 with cell wall polysaccharides remains to be investigated; however, both covalent and non-covalent interactions between pectic polymers and AG II glycan have previously been demonstrated (see above). In fact, it was shown that protein O-galactosylation is required for the function of AtFla4. Several galactosyl transferases (GALTs) mediate the initial glycosylation of hydroxyl proline residues of AGPs. Two of them called AtGalt2 and AtGalt5 together are responsible for the biosynthesis of the bulk of AGPs and their mutation caused a root and seed phenotype identical to *atfla4* [147]. Intriguingly, the phenotypes of *atgalt2 atgalt5 atfla4 atfei1 atfei2* quintuple and the *atfla4 atfei1 atfei2* triple mutants were identical [146]. It was hypothesized that AtFla4 might physically interact with the Fei RLKs via its O-glycans. This hypothesis was seemingly contradicted by a structure to function study where the deletion of potential O-glycosylation sites on AtFla4 did not interfere with genetic complementation of *atfla4* [38]. However, the AtFla4 level in the transgenic plants might have been higher than normal or cryptic O-glycosylation sites might still have been present. How AtFla4 and AtFei interact and if O-linked glycosylation plays a direct or indirect role for AtFla4 and AtFei function remains to be tested. Taken together, *AtFla4* is a unique genetic paradigm for FAS1 domain proteins in plants. A speculative model (Figure 5) combined from previous studies proposes that after its release from the plasma membrane and via its O-glycan AtFla4 possibly interacts with pectic polymers and contributes to biophysical properties such as swelling and interpolymer connectivity.

<figure><img src="image_88.png" /><figcaption>**Figure 5.** Hypothetical roles of AtFla4 in cell wall structure and signaling. AtFla4 might interact with the pectic network with covalent or non-covalent interactions of its glycans (yellow discs and green stars: O-glycans which are yet to be precisely defined, N-glycans are omitted). It might mechanically link pectin with AtFei1 and AtFei2 receptor kinases and the plasma membrane. The signals transduced by the receptor kinases might involve ACC synthase (ACS) and Abcisic Acid Insensitive 1 (ABI1). How signaling connects the kinases with cellulose synthase (CesA) and pectin biosynthesis is unclear.</figcaption></figure>

In parallel AtFla4 might also interact with AtFei1 and AtFei2 via its FAS1 domain to link pectin with AtFei1 and -2 and indirectly to ACC synthase. The access of the C-terminal Fas1 domain to the AtFei receptor domain might be modulated by the interaction to the cell wall. Under standard conditions relatively firm binding of AtFla4 to AtFei might suppress ACS. Under cell wall stress conditions binding of AtFla4 to AtFei might be reduced and the ACS suppression by AtFei might be relaxed leading to ACC production and decreased cellulose production and other responses. However, any postulated physical interactions of *AtFla4* remain to be demonstrated.
## 4.2. A Conserved Function of *AtFla11* and *AtFla12* in Secondary Cell Walls

One of the functionally and economically most interesting aspects of some FLAs is their role for secondary cell wall formation and structure. Secondary cell walls are by far the most abundant re-growing fiber and energy feedstock on this planet and its major constituents—cellulose, xylans and lignin—are among the most abundant biopolymers. Their sustainable and socially compatible production is thought crucial in the global transition to a carbon neutral economy. *AtFla11* and *AtFla12* (group A; [14]) and their orthologs (Appendix C) from diverse fiber crops and model plants were noted in *Arabidopsis thaliana* for their expression during the secondary thickening of stems [148], their co-expression with secondary cell–wall–specific cellulose synthase loci [149] and their expression in sclerenchyma cells [150]. A correlative and causative association of *AtFla11* and *AtFla12* orthologs with secondary cell wall biosynthesis has been observed in many plant species. The *Zinnia elegans* *ZeFla11* gene was found to be expressed in trans-differentiating cell cultures and in a specific subset of secondary cell wall forming vessel cells and adjacent parenchyma cells [151]. *AtFla11* and -12 orthologs from poplar, eucalyptus and willow were highly expressed in tension wood (TW), a reaction wood forming at the upper side of branches and stems growing sideways. TW consists of almost pure cellulose deposited in the gelatinous secondary cell wall layer (G-layer) in elongated G-fiber cells and shows a characteristically low-cellulose microfibril angle (MFA, with respect to the growth axis), which entails superior tensile strength [152–154]. The three *AtFla11/12* eucalyptus orthologs *EgFla1*, -2 and -3 were highly expressed in stems correlating with secondary cell wall formation [71]. Also in flax and hemp *AtFla11/12* orthologs were highly expressed in fiber forming tissues [72,155]. A cotton *AtFla12* ortholog was expressed at a higher level and for a longer time period in *Gossypium barbadense* compared to *G. hirsutum* correlating with greater fiber strength and length of the former [69]. Hence, *AtFla11* orthologs might be natural genetic determinants for fiber crops. So, throughout the higher plants, *AtFla11/12* and their orthologs correlate with secondary cell wall formation, specifically the deposition of cellulose microfibrils aligned along the axis of mechanical stress. What is the role of FLAs in this process?

In *Arabidopsis thaliana* there exists a genetic redundancy between *AtFla11* and -12; however, the *atfla11 atfla12* double mutant was affected in the mechanical properties of its secondary cell–wall–rich stems [156]. In *atfla11 atfla12* tensile strength and stiffness were reduced and MFA was increased. Moreover, the chemical composition and cell wall structure showed a reduction of galactose, arabinose and cellulose and a concomitant increase in lignin content. The authors speculated that the FLAs via their FAS1 domains might form a heteromeric higher-order network strengthening the interaction between cellulose microfibrils [156]. The most highly expressed hybrid aspen locus called *PtFLA6* was silenced with antisense RNA. This also reduced the transcript level of many other *PtFla* genes [157,158]. Although no apparent morphological phenotype was observed, the *PtFla6* knockdown resulted in reduced flexural strength and stiffness in stems of transgenic plants. Moreover, the content of AGPs, cellulose and lignin was reduced alongside a reduction of transcript levels for numerous genes involved in cellulose and lignin formation [157]. When TW formation was stimulated in the *PtFla6* knockdown plant, the relative frequency of abnormal G-fibers increased, but no other changes were observed [158]. Taken together, the complex response to *PtFla6* interference is more compatible with a regulatory role than it is with a direct structural role in the cellulose layer. In *Eucalyptus grandis*, *EgFla1*, -2 and -3 were experimentally overexpressed in cambial sectors or in whole tobacco plants [71]. Despite their sequence similarity, each transgene exerted a specific effect on phenotypic parameters such as MFA, cell wall thickness, cell wall composition and xylem cell type specification. This meant that structurally even very similar *AtFla11/12* orthologs exerted different effects in the same cellular context [71]. The function of *AtFla11/12* orthologs has also been investigated during cotton fiber formation. In this system, trichome cells are initiated at the outer integument of the ovule during anthesis (2 d before to 5 d after anthesis; dpa). Then the trichomes undergo a period of rapid cell elongation (3–20 dpa), which is followed by secondary wall formation (16–40 dpa) and maturation (40–60 dpa). Hence, each cotton fiber is a single trichome cell made up of almost pure cellulose [159]. Its formation is the result of
three distinct developmental processes: (1) specification and initiation, (2) expansion and (3) cell wall thickening and maturation. Two *AtFla11/12* orthologs in the cotton species *Gossypium hirsutum* named *GhAgp4* (Appendix D) and *GhFla1* were suppressed using RNAi [159,160]. *GhFla1* was specifically expressed in elongating fibers with peak expression at 10 dpa [70] and indeed, fibers of RNAi lines were much shorter than controls [159]. However, *GhFla1* RNAi suppression also resulted in delayed and reduced trichome initiation and, moreover, resulted in decreased RNA levels of other fiber-expressed FLAs and of numerous genes implicated with cell wall biosynthesis. The complex alterations at the transcript level correlated with alterations in cell wall composition including decreased cellulose formation. By contrast, the overexpression of *GhFla1* resulted in increased initiation and elongation of cotton fibers as well as upregulation of other FLAs and cell wall biosynthetic loci and in an increased cellulose content [159]. These data confirmed and extended a previous study where the *GhAGP4* locus was silenced by RNAi. Like the *GhFla1* silencing, this reduced fiber initiation, elongation and quality, and affected the level of transcripts related to cell wall biosynthesis [160]. The observations made in cotton are difficult to reconcile with a biomechanical role of *GhFla1* and *GhAgp4* as an adhesive between secondary cell wall cellulose microfibrils because not only secondary cell walls were affected by the interference but also the fundamentally different processes of initiation and elongation that happen before. The apparent involvement of these genes in all three phases of fiber formation and their control of mRNA levels for an entire battery of other genes rather suggests that this class of FAS1 proteins fulfils a role in developmental control comparable to a peptide hormone such as the proteoglycan xylogen [161].

What do we know about possible molecular interactors and subcellular localization of *AtFla11/AtFla12* orthologs? Intriguingly, cotton orthologs of *AtFla7* and *AtFla11* were co-precipitated with active secondary cellulose synthase *GhCesA8* that was effectively extracted by inclusion of cellulase in the extraction buffer [162]. This supports the hypothesis that group A FLAs physically interact with the cellulose synthase complex. This hypothesis offers an attractive explanation for co-expression between group A FLAs and secondary cell wall formation related genes and for changes in MFA and mechanical properties in group A *FLA* loss of function genotypes. If one assumes that nascent cellulose fibers in combination with cellulose synthase are both bound by FLAs, this model could also explain the localization of PtFLA6 in secondary cell walls [157,158]. However, this model offers no simple explanation for the observation that the transcriptional program of secondary cell wall formation as well as cellular differentiation was heavily influenced by the experimental manipulation of FLAs [157], or for the many effects of FLA overexpression. This problem might be solved with a role of FLAs in signaling. So far, there has been one report that suggested that a RLK might physically interact with FLAs. A pull-down experiment using the *Arabidopsis thaliana* sucrose-induced receptor kinase (AtSirk1) as bait identified AtFla8 and AtFla9 among the 16 interaction partners. Interestingly, among several potential substrates of AtSirk1, there was the primary cell wall specific cellulose synthase catalytic subunit 3 (AtCesA3) [163]. At present, there is no genetic evidence linking AtFla8 and AtFla9 with primary cell wall cellulose synthesis; however, the evidence for their physical interaction with an RLK that acts on cellulose synthase offers an interesting alternative mechanism for how FLAs might influence cell wall biosynthesis. Using their potential to interact with multiple binding partners they might combine receptor kinases with substrates such as cellulose synthase. Taken together, several modes of action can be envisaged for *AtFla11/12* and its orthologs (Figure 6).

Firstly, AtFla11/12 might be directly secreted to the cell wall and bind to polymers and cross-link them. Secondly these FAS1 proteins might act as extracellular signals or be part of a receptor complex to influence transcriptional events downstream. Thirdly, they might augment cellulose deposition by acting as a physical part of the cellulose synthase machinery. Fourth, by interacting with both cellulose synthase and receptor kinase they might facilitate or modulate post-translational regulation of cellulose biosynthesis (Figure 6).
<figure><img src="image_89.png" /><figcaption>**Figure 6.** Putative role of *AtFla11*, *AtFla12* and their orthologs in secondary cell wall formation. Firstly they might act as secreted structural proteins in secondary cell walls binding to cellulose or other glycans. Secondly, they might be part of outside-in signaling, in connection with an unknown receptor that leads to complex changes of gene expression. Thirdly, they might bind to cellulose synthase (CesA) and directly influence or stabilize its action. Finally, they might act as adaptors between CesA and receptor kinases to modulate CesA phosphorylation.</figcaption></figure>

### 4.3. A Cascade of FLAs Acts in Male Gametophyte Development

The FAS1 proteins encoded by the *AtFla3* and the *OsMtr1* genes, respectively, are required for male gametophyte development; however, the mRNA pattern in *Arabidopsis thaliana* suggests that several FLAs might sequentially act in this process (Figure 7).

<figure><img src="image_90.png" /><figcaption>**Figure 7.** FAS1 proteins act sequentially in male gametophyte development: (A) *OsMtr1* acts in early stage of tapetum development. It is specifically expressed and secreted in microspore cells (MS) and influences the development of tapetum cells; (B) *AtFla3* acts at the late stage of the formation of the cellulose- and pectin-rich intine layer of pollen cell walls; (C) RNA-Seq data of *Arabidopsis thaliana* at different floral stages [164,165] show that six FAS1 genes are specifically expressed in anthers at different stages of floral development. *f1* indicates the first open flower. The expression patterns of *AtFla20* that might be the ortholog of *OsMtr1* and of *AtFla3* are consistent with roles in early and late pollen development, respectively.</figcaption></figure>
No functional pollen was found in the rice microspore and tapetum regulator 1 (*osmtr1*) mutant [16]. *OsMtr1* expression was specifically detected in early microspore development during meiosis and tetrad formation and during the release of young microspores (Figure 7A). However, despite *OsMtr1* never being expressed in the tapetum, a nutritive maternal tissue that surrounds microspores, the *osmtr1* mutation dramatically affected tapetum development. This suggested that *OsMtr1* might act non-cell autonomously. The *osmtr1* mutant tapetum failed to undergo programmed cell death, which normally supports pollen wall formation in developing microspores. Tetrads formed normally in *osmtr1* yet, mutant microspores showed defective development as soon as they were released possibly due to the lack of cell wall material contributed by the adjacent tapetum. The localization of OsMtr1 in the microspores and the extracellular space between them and the tapetum suggested that OsMtr1 might act as a secreted signal to coordinate microspore development with tapetum degeneration (Figure 7A). Molecular interactors such as a hypothetical *OsMtr1* receptor on tapetum cells remain to be identified [16]. The expression of *AtFla3* in pollen during anthesis and pollen germination combined with the high level of expression in anthers is unique among all FLAs and *AtFla3* function was investigated using RNAi and overexpression [166]. The *AtFla3* RNAi lines were partially male-sterile due to a reduced ability to form functional microspores. Histological analysis showed that *AtFla3* RNAi affected the formation of intine, the innermost layer of the pollen wall, which consists mainly of cellulose and pectin. The earliest time point at which intine abnormalities were observed was the late uninucleate stage, suggesting that *AtFla3* might act in the formation of intine in microspore development. Abnormal *AtFla3* RNAi pollen showed defective cellulose staining, suggesting that *AtFlaA3* was required for cellulose deposition in the intine layer. More detailed analyses are required to test whether any other cell wall polymers are affected and if full loss of function of the *AtFla3* locus causes a more complete defect of pollen function. During *Arabidopsis thaliana* floral development at least six FAS1 domain loci are specifically expressed in anthers during separate developmental stages [164,165]. Like its closest rice homologue *OsMtr1*, *AtFla20* is expressed at an early stage, where it overlaps with *AtFLA21* and *At5g16920*—an FAS1 protein not annotated as FLA (Figure 7C). The mRNA levels of *AtFla14* and *AtFla5* sequentially peak at intermediate stages and *AtFla3* is expressed immediately before and during flower opening (anthesis) in mature and germinating pollen, as previously reported [166]. How most of these FAS1 domain proteins are involved in normal pollen wall formation remains to be investigated. Nonetheless, their expression pattern during pollen formation appears to reflect the complex development and composition of the pollen wall.

### 4.4. Plant FAS1 Proteins Have Many Potential Functions

Several other FLAs are known to perform at least partially non-redundant genetic roles. Firstly, the loss of function mutation of *AtFla1* caused a phenotype in the growth regulator induced formation of shoots and roots from the callus [167]. Two alleles in two different wild-type backgrounds were isolated and the wild-type background on its own not only had a dramatic influence on callus derived organ formation but it essentially inverted the genetic effect of *AtFla1* on the process. By contrast, the negative effect of the *AtFla1* locus on root length and lateral root number was constant between the two tested wild-type backgrounds. It is presently unclear how *AtFla1* might influence organ formation but initial promoter studies showed expression of *AtFla1* early during the formation of organized tissue from callus. The protein might therefore be required for pattern formation [167]. Secondly, in a gene-editing study in the non-model plant *Brassica carinata*, *BcFla1*, a group A FLA that is more similar to *AtFla13* than to *AtFla11*, was implicated in root hair growth, where tip-focused deposition of primary cell wall materials occurs [168]. In a third example, in maize the expression level of group A *ZmFla* genes was inversely correlated with seed abortion. Consistently, two different mutations in the group A *AtFla9* locus that showed abnormally low and high *AtFla9* RNA levels displayed increased and decreased seed abortion, respectively [169]. Lastly, a comparative study of 30 different wheat (Triticum aestivum) varieties found an association between the expression of a FAS1 protein and grain milling properties [170]. According to this report, the locus *TaFla8* [51] coding for a FAS1 domain
that does not contain any PAST-rich domains, is specifically expressed in grain. Interestingly, its level was relatively low in varieties displaying beneficial milling properties compared to varieties scoring poorly with respect to milling efficiency. Hence, human selection of wheat varieties with improved milling properties might have inadvertently reduced the basal expression level of *TaFla8*, which might normally strengthen cell walls.

## 5. Fungal FAS1 Proteins

In a screen for genes specifically expressed in fruiting bodies of the Shiitake mushroom *Lentinula edodes* the *LeFlp1* locus coding for a GPI-anchored single FAS1 domain protein was identified and its specific expression during fruiting body formation suggested a developmental role [17]. In another study the *MoFlp1* locus of the rice blast fungus *Magnaporthe oryzae* was shown to encode a GPI-anchored vacuolar tandem FAS1 protein [18]. Its disruption caused defects in the formation of conidia, which, according to the authors, hinted at a potential role of *MoFlp1* in autophagy. Interestingly, the fission yeast *Schizosaccharomyces pombe* FAS1 domain protein SpFsc1 was identified in a genetic screen for autophagy-related loci [19]. SpFsc1 consists of five FAS1 domains in tandem and a transmembrane domain. The protein was detected in the vacuolar membrane and was shown to specifically act in a late step in autophagy, the fusion between the phagosomes with the vacuolar membrane. The mechanistic details including the role of the FAS1 domains in this process remain to be elucidated. In summary, studies on FAS1 domain proteins in fungi are sparse; however, they provide a stimulating variation of the hypothetical modes of action of this protein family.

## 6. Bacterial FAS1 Proteins

The most thoroughly studied eubacterial FAS1 proteins are the homologues Mpb70 and Mpb83 from *Mycobacterium* (reviewed in [171]). In various genomes of obligate pathogenic, opportunistic and non-pathogenic mycobacteria [172], both non-pathogenic and pathogenic species contained *Mpb70*/*Mpb83* homologues, while the genomes of opportunistic mycobacteria did not. This suggests a relation of *Mpb70*/*Mpb83* to mycobacterial lifestyle and pathogenesis. While Mpb83/Mpb70 was hypothesized to undergo “homophilic” interactions with mammalian Pn thereby interfering with cell adhesion in bones [171] this idea is not compatible with the secreted nature of Pn and is derived from cell aggregation data [8,173], which could have alternative explanations. Moreover, no intermolecular interactions between Fas1 domains have been reported. However, both host cell to ECM adhesion and host cell to mycobacterium adhesion might be modulated by binding of Mpb70/Mpb83 to molecules presented at the host cell surface. Indeed, Mpb83 was demonstrated to bind to Toll-like receptors 1 and -2 (HsTlr1 and -2), two LRR-RLKs present on human monocytes [41]. The binding of recombinant Mpb83 to HsTlr2 triggered mitogen activated protein kinase (MAPK) signaling and as a consequence induced the production of matrix metalloprotease-9 (MMP-9) and cytokines [174]. The direct interaction between Tlr2 and Mpb83, as shown by surface plasmon resonance [41], is an intriguing finding, given the presence of more than 200 LRR-RLKs in plant genomes [175]. A role of bacterial FAS1 proteins in the interaction between bacteria and their eukaryotic hosts is also observed in symbiotic communities. In the first example, the FAS1 protein SmNex18 (locus SMa1077) of the nitrogen-fixing legume symbiont *Sinorhizobium meliloti* was shown to be expressed specifically during nodulation, the initial stage of symbiosis between bacterium and plant [176,177]. Secondly, a FAS1 protein was found in *Nostoc cyanobacteria* that form symbiotic bacterial–fungal communities called lichen. *Nostoc punctiforme* isolates living in lichen highly expressed an unnamed FAS1 protein (locus ACC81089) that was only a minor compound in a closely related free living strain [178]. Moreover, FAS1 proteins featured prominently in the metaproteome of phyllospheric (i.e., plant leaf dwelling) bacteria [179] but were absent from rhizospheric (root-dwelling) bacteria [180]. Likewise, while present in many different genera of archaea, the FAS1 domain is not necessarily ubiquitous within the same genus. To give an example, among the 13 completely sequenced *Methanobacterium* isolates, only *M. paludis* isolated from peatland contained FAS1 domain protein sequences [181]. On the one hand, such
circumstantial observations support the hypothesis that FAS1 proteins are important for the interaction between prokaryotic colonizers and eukaryotic hosts; on the other hand, they also show that FAS1 proteins are not essential.

## 7. Concluding Remarks

Can we define a common denominator for the function of FAS1 proteins? The characterization of HsTgfbi structure and function demonstrates the versatility of the FAS1 domain in binding multiple ligands, which might be a conserved feature. The frequent assumption that FAS1 proteins are involved in "cell adhesion," however, is more context-dependent. It is derived from insightful experiments performed with animal cell cultures; however, it might be misleading for "hard-shelled" cells such as bacteria, fungi and plants that usually adhere to their ECM as a result of turgor pressure and build up adhesive materials such as pectin. By contrast, the designation of Tgfbi and Pn as matricellular proteins, defined as non-structural ECM-components that interact with cell surface receptors as mediators between the cell and its microenvironment [77,182], seems a more productive concept for approaching the common function of FAS1 proteins in all kingdoms of life including plants. The crucial challenge for future work on plant FAS1 proteins will be the identification of molecular interactors both of the FAS1 domain and of their abundant but enigmatic glycan modifications.

**Funding:** This research was funded by the Austrian Science Fund FWF (Grant number I1182-B22).

**Conflicts of Interest:** The author declares no conflict of interest.

## Appendix A

Since many genes and proteins from different organisms are discussed in this review, I adopted universal nomenclature rules according to a highly popular textbook [183]. Gene symbols are written in italics, with the first letter in uppercase indicating a wild-type allele and in lowercase indicating a mutant allele. Proteins are written in non-italics. If the text relates to genes or proteins from a certain species, a two-letter prefix (Hs for *Homo sapiens*, At for *Arabidopsis thaliana* etc.) is used.

## Appendix B

Because many human proteins were independently discovered several times, and maybe also for asserting the status of discovery, there is an abundance of synonyms. Tgfbi is also known as β-ig H3, βIG, BIGH3, kerato-epithelin, RGD-CAP and MP78/70. Pn is synonymous with Osteoblast specific factor-2 (Osf-2). Stab1 has also been named Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) and FEEL-1 is an acronym for fasciclin, EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1. Frequent Stab2 synonyms are lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and hyaluronic acid receptor (HARE).

## Appendix C

The term "*AtFla11/12* ortholog" is here used to designate FLAs from other plant species that are more similar in protein sequence to *AtFla11* or *AtFla12* than to any other *Arabidopsis thaliana* protein.

## Appendix D

The nomenclature in the cotton literature is somewhat confusing. Some FLAs are named AGPs and GhFla1 refers to two entirely different protein sequences. The GhFla1 that was mentioned by Li et al. (2010) [160] being most similar to *AtFla9/6/13* and the *GhFla1* silenced by Huang et al. (2013) [159] is an ortholog of *AtFla11/12*. The *GhAgp4* locus (gb EF470295) silenced by Li (2010) [160] is identical to the sequence annotated as *GhFla12*.
References

1. Bastiani, M.J.; Harrelson, A.L.; Snow, P.M.; Goodman, C.S. Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. *Cell* **1987**, *48*, 745–755. [CrossRef]

2. Snow, P.M.; Zinn, K.; Harrelson, A.L.; McAllister, L.; Schilling, J.; Bastiani, M.J.; Makk, G.; Goodman, C.S. Characterization and cloning of fasciclin I and fasciclin II glycoproteins in the grasshopper. *Proc. Natl. Acad. Sci. USA* **1988**, *85*, 5291–5295. [CrossRef] [PubMed]

3. Hu, S.; Sonnenfeld, M.; Stahl, S.; Crews, S.T. Midline fasciclin: A drosophila fasciclin-I-related membrane protein localized to the CNS midline cells and trachea. *J. Neurobiol.* **1998**, *35*, 77–93. [CrossRef]

4. Zhong, Y.; Shanley, J. Altered nerve terminal arborization and synaptic transmission in drosophila mutants of cell adhesion molecule fasciclin I. *J. Neurosci.* **1995**, *15*, 6679–6687. [CrossRef] [PubMed]

5. Jay, D.G.; Keshishian, H. Laser inactivation of fasciclin I disrupts axon adhesion of grasshopper pioneer neurons. *Nature* **1990**, *348*, 548–550. [CrossRef] [PubMed]

6. Clout, N.J.; Tisi, D.; Hohenester, E. Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I. *Structure* **2003**, *11*, 197–203. [CrossRef]

7. Skonier, J.; Neubauer, M.; Madisen, L.; Bennett, K.; Plowman, G.D.; Purchio, A.F. cDNA cloning and sequence analysis of β-g-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-β. *DNA Cell Biol.* **1992**, *11*, 511–522. [CrossRef] [PubMed]

8. Takeshita, S.; Kikuno, R.; Tezuka, K.; Amann, E. Osteoblast-specific factor 2: Cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. *Biochem. J.* **1993**, *294*, 271–278. [CrossRef] [PubMed]

9. Horiuchi, K.; Amizuka, N.; Takeshita, S.; Takamatsu, H.; Katsuura, M.; Ozawa, H.; Toyama, Y.; Bonewald, L.F.; Kudo, A. Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor β. *J. Bone Miner. Res.* **1999**, *14*, 1239–1249. [CrossRef] [PubMed]

10. Politz, O.; Gratchev, A.; McCourt, P.A.; Schledzewski, K.; Guillot, P.; Johansson, S.; Svineng, G.; Franke, P.; Kannicht, C.; Kzhyshkowska, J.; et al. Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues. *Biochem. J.* **2002**, *362*, 155–164. [PubMed]

11. Huber, O.; Sumper, M. Algal-CAMS: Isoforms of a cell adhesion molecule in embryos of the alga *Volvox* with homology to *Drosophila* fasciclin I. *EMBO J.* **1994**, *13*, 4212–4222. [PubMed]

12. Schultz, C.J.; Johnson, K.L.; Currie, G.; Bacic, A. The classical arabinogalactan protein gene family of arabidopsis. *Plant Cell* **2000**, *12*, 1751–1768. [CrossRef] [PubMed]

13. Schultz, C.J.; Rumsewicz, M.P.; Johnson, K.L.; Jones, B.J.; Gaspar, Y.M.; Bacic, A. Using genomic resources to guide research directions. The arabinogalactan protein gene family as a test case. *Plant Physiol.* **2002**, *129*, 1448–1463. [CrossRef] [PubMed]

14. Johnson, K.L.; Jones, B.J.; Bacic, A.; Schultz, C.J. The fasciclin-like arabinogalactan proteins of Arabidopsis. A multigene family of putative cell adhesion molecules. *Plant Physiol.* **2003**, *133*, 1911–1925. [CrossRef] [PubMed]

15. Shi, H.; Kim, Y.; Guo, Y.; Stevenson, B.; Zhu, J.K. The Arabidopsis *Sos5* locus encodes a putative cell surface adhesion protein and is required for normal cell expansion. *Plant Cell* **2003**, *15*, 19–32. [CrossRef] [PubMed]

16. Tan, H.; Liang, W.; Hu, J.; Zhang, D. MTR1 encodes a secretory fasciclin glycoprotein required for male reproductive development in rice. *Dev. Cell* **2012**, *22*, 1127–1137. [CrossRef] [PubMed]

17. Miyazaki, Y.; Kaneko, S.; Sunagawa, M.; Shishido, K.; Yamazaki, T.; Nakamura, M.; Babasaki, K. The fruiting-specific *LeFlp1* gene, encoding a novel fungal fasciclin-like protein, of the basidiomycetous mushroom *Lentinula edodes*. *Curr. Genet.* **2007**, *51*, 367–375. [CrossRef] [PubMed]

18. Liu, T.B.; Chen, G.Q.; Min, H.; Lin, F.C. *MoFlp1*, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in *Magnaporthe oryzae*. *J. Zhejiang Univ. Sci. B* **2009**, *10*, 434–444. [CrossRef] [PubMed]

19. Sun, L.L.; Li, M.; Suo, F.; Liu, X.M.; Shen, E.Z.; Yang, B.; Dong, M.Q.; He, W.Z.; Du, L.L. Global analysis of fission yeast mating genes reveals new autophagy factors. *PLoS Genet.* **2013**, *9*, e1003715. [CrossRef] [PubMed]
20. Nagai, S.; Matsumoto, J.; Nagasuga, T. Specific skin-reactive protein from culture filtrate of *Mycobacterium bovis* BCG. *Infect. Immun.* **1981**, *31*, 1152-1160. [PubMed]

21. Nagai, S.; Miura, K.; Tokunaga, T.; Harboe, M. Mpb70, a unique antigenic protein isolated from the culture filtrate of BCG substrain Tokyo. *Dev. Biol. Stand.* **1986**, *58*, 511–516. [PubMed]

22. Terasaka, K.; Yamaguchi, R.; Matsuo, K.; Yamazaki, A.; Nagai, S.; Yamada, T. Complete nucleotide sequence of immunogenic protein Mpb70 from *Mycobacterium bovis* BCG. *FEMS Microbiol. Lett.* **1989**, *49*, 273-276. [CrossRef] [PubMed]

23. Radford, A.J.; Wood, P.R.; Billman-Jacobe, H.; Geysen, H.M.; Mason, T.J.; Tribbick, G. Epitope mapping of the *Mycobacterium bovis* secretory protein Mpb70 using overlapping peptide analysis. *J. Gen. Microbiol.* **1990**, *36*, 265-272. [CrossRef] [PubMed]

24. Ulstrup, J.C.; Jeansson, S.; Wiker, H.G.; Harboe, M. Relationship of secretion pattern and Mpb70 homology with osteoblast-specific factor 2 to osteitis following *Mycobacterium bovis* BCG vaccination. *Infect. Immun.* **1995**, *63*, 672–675. [PubMed]

25. Burroughs, A.M.; Balaji, S.; Iyer, L.M.; Aravind, L. Small but versatile: The extraordinary functional and structural diversity of the β-grasp fold. *Biol. Dir.* **2007**, *2*, 18. [CrossRef] [PubMed]

26. Boratyn, G.M.; Schaffer, A.A.; Agarwala, R.; Altschul, S.F.; Lipman, D.J.; Madden, T.L. Domain enhanced lookup time accelerated blast. *Biol. Dir.* **2012**, *7*, 12. [CrossRef] [PubMed]

27. Clout, N.J.; Hohenester, E. A model of FAS1 domain 4 of the corneal protein βig-h3 gives a clearer view on corneal dystrophies. *Mol. Vis.* **2003**, *9*, 440–448. [PubMed]

28. Moody, R.G.; Williamson, M.P. Structure and function of a bacterial fasciclin I domain protein elucidates function of related cell adhesion proteins such as TGFBIp and periostin. *FEBS Open Biol.* **2013**, *3*, 71–77. [CrossRef] [PubMed]

29. Carr, M.D.; Bloemink, M.J.; Dentten, E.; Whelan, A.O.; Gordon, S.V.; Kelly, G.; Frenkiel, T.A.; Hewinson, R.G.; Williamson, R.A. Solution structure of the *Mycobacterium tuberculosis* complex protein Mpb70: From tuberculosis pathogenesis to inherited human corneal disease. *J. Biol. Chem.* **2003**, *278*, 43736–43743. [CrossRef] [PubMed]

30. Basaiawmoit, R.V.; Oliveira, C.L.; Runager, K.; Sorensen, C.S.; Behrens, M.A.; Jonsson, B.H.; Kristensen, T.; Klintworth, G.K.; Enghild, J.J.; Pedersen, J.S.; et al. SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the Arg124His mutation. *J. Mol. Biol.* **2011**, *408*, 503–513. [CrossRef] [PubMed]

31. Yun, H.; Kim, E.H.; Lee, C.W. ¹H, ¹³C, and ¹⁵N resonance assignments of FAS1-IV domain of human periostin, a component of extracellular matrix proteins. *Biomol. NMR Assign.* **2017**, *12*, 95–98. [CrossRef] [PubMed]

32. Underhaug, J.; Koldso, H.; Runager, K.; Nielsen, J.T.; Sorensen, C.S.; Kristensen, T.; Otzen, D.E.; Karring, H.; Malmendal, A.; Schiott, B.; et al. Mutation in transforming growth factor β induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization. *Biochim. Biophys. Acta* **2013**, *1834*, 2812–2822. [CrossRef] [PubMed]

33. Garcia-Castellanos, R.; Nielsen, N.S.; Runager, K.; Thogersen, I.B.; Lukassen, M.V.; Poulsen, E.T.; Goulas, T.; Enghild, J.J.; Gomis-Ruth, F.X. Structural and functional implications of human transforming growth factor β-induced protein, TGFBIp, in corneal dystrophies. *Structure* **2017**, *25*, 1740–1750. [CrossRef] [PubMed]

34. Thapa, N.; Lee, B.H.; Kim, I.S. TGFBIp/βig-h3 protein: A versatile matrix molecule induced by TGF-β. *Intern. J. Biochem. Cell. Biol.* **2007**, *39*, 2183–2194. [CrossRef] [PubMed]

35. Kim, J.E.; Kim, S.J.; Lee, B.H.; Park, R.W.; Kim, K.S.; Kim, I.S. Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-β-induced gene, βig-h3. *J. Biol. Chem.* **2000**, *275*, 30907–30915. [CrossRef] [PubMed]

36. Kim, J.E.; Jeong, H.W.; Nam, J.O.; Lee, B.H.; Choi, J.Y.; Park, R.W.; Park, J.Y.; Kim, I.S. Identification of motifs in the fasciclin domains of the transforming growth factor-β-induced matrix protein βig-h3 that interact with the αvβ5 integrin. *J. Biol. Chem.* **2002**, *277*, 46159–46165. [CrossRef] [PubMed]

37. Orecchia, P.; Conte, R.; Balza, E.; Castellani, P.; Borsi, L.; Zardi, L.; Mingari, M.C.; Carnemolla, B. Identification of a novel cell binding site of periostin involved in tumour growth. *Eur. J. Cancer* **2011**, *47*, 2221–2229. [CrossRef] [PubMed]

38. Xue, H.; Veit, C.; Abas, L.; Tryfona, T.; Maresch, D.; Ricardi, M.M.; Estevez, J.M.; Strasser, R.; Seifert, G.J. *Arabidopsis thaliana* FLA4 functions as a glycan-stabilized soluble factor via its carboxy-proximal fasciclin 1 domain. *Plant J.* **2017**, *91*, 613–630. [CrossRef] [PubMed]
39. Son, H.N.; Nam, J.O.; Kim, S.; Kim, I.S. Multiple FAS1 domains and the RGD motif of TGFBI act cooperatively to bind αvβ3 integrin, leading to anti-angiogenic and anti-tumor effects. *Biochim. Biophys. Acta* **2013**, *1833*, 2378-2388. [CrossRef] [PubMed]

40. Vosloo, W.; Tippoo, P.; Hughes, J.E.; Harriman, N.; Emmas, M.; Beatty, D.W.; Zappe, H.; Steyn, L.M. Characterisation of a lipoprotein in *Mycobacterium bovis* (BCG) with sequence similarity to the secreted protein MPB70. *Gene* **1997**, *188*, 123-128. [CrossRef]

41. Chambers, M.A.; Whelan, A.O.; Spallek, R.; Singh, M.; Coddeville, B.; Guerardel, Y.; Elass, E. Non-acylated *Mycobacterium bovis* glycoprotein MPB83 binds to TLR1/2 and stimulates production of matrix metalloproteinase 9. *Biochem. Biophys. Res. Commun.* **2010**, *400*, 403-408. [CrossRef] [PubMed]

42. Rittenour, W.R.; Harris, S.D. Glycosylphosphatidylinositol-anchored proteins in *Fusarium graminearum*: Inventory, variability, and virulence. *PLoS ONE* **2013**, *8*, e81603. [CrossRef] [PubMed]

43. Mosher, D.F.; Johansson, M.W.; Gillis, M.E.; Annis, D.S. Periostin and TGF-β-induced protein: Two peas in a pod? *Crit. Rev. Biochem Mol. Biol.* **2015**, *50*, 1-13.

44. Zavaliev, R.; Dong, X.; Epel, B.L. Glycosylphosphatidylinositol (GPI) modification serves as a primary plasmodesmal sorting signal. *Plant Physiol.* **2016**, *172*, 1061-1073. [PubMed]

45. Tapken, W.; Murphy, A.S. Membrane nanodomains in plants: Capturing form, function, and movement. *J. Exp. Bot.* **2015**, *66*, 1573-1586. [CrossRef] [PubMed]

46. Michell, S.L.; Whelan, A.O.; Wheeler, P.R.; Panico, M.; Easton, R.L.; Etienne, A.T.; Haslam, S.M.; Dell, A.; Morris, H.R.; Reason, A.J.; et al. The mpb83 antigen from *Mycobacterium bovis* contains O-linked mannose and (1→3)-mannobiose moieties. *J. Biol. Chem.* **2003**, *278*, 16423-16432. [CrossRef] [PubMed]

47. Kieliszewski, M.J. The latest hype on Hyp-O-glycosylation codes. *Phytochemistry* **2001**, *57*, 319-323. [CrossRef]

48. Kitazawa, K.; Tryfona, T.; Yoshimi, Y.; Hayashi, Y.; Kawauchi, S.; Antonov, L.; Tanaka, H.; Takahashi, T.; Kaneko, S.; Dupree, P.; et al. β-galactosyl Yariv reagent binds to the β-1,3-galactan of arabinogalactan proteins. *Plant Physiol.* **2013**, *161*, 1117-1126. [CrossRef] [PubMed]

49. Paulsen, B.S.; Craik, D.J.; Dunstan, D.E.; Stone, B.A.; Bacic, A. The Yariv reagent: Behaviour in different solvents and interaction with a gum arabic arabinogalactan-protein. *Carbohydr. Pol.* **2014**, *106*, 460-468. [CrossRef] [PubMed]

50. Shimizu, M.; Igasaki, T.; Yamada, M.; Yuasa, K.; Hasegawa, J.; Kato, T.; Tsukagoshi, H.; Nakamura, K.; Fukuda, H.; Matsuoka, K. Experimental determination of proline hydroxylation and hydroxyproline arabinogalactosylation motifs in secretory proteins. *Plant J.* **2005**, *42*, 877-889. [CrossRef] [PubMed]

51. Faik, A.; Abouzouhair, J.; Sarhan, F. Putative fasciclin-like arabinogalactan-proteins (FLA) in wheat (*Triticum aestivum*) and rice (*Oryza sativa*): Identification and bioinformatic analyses. *Mol. Genet Genom.* **2006**, *276*, 478-494. [CrossRef] [PubMed]

52. Tan, L.; Qiu, F.; Lamport, D.T.; Kieliszewski, M.J. Structure of a hydroxyproline (Hyp)-arabinogalactan polysaccharide from repetitive Ala-Hyp expressed in transgenic *Nicotiana tabacum*. *J. Biol. Chem.* **2004**, *279*, 13156-13165. [CrossRef] [PubMed]

53. Tryfona, T.; Liang, H.C.; Kotake, T.; Tsumuraya, Y.; Stephens, E.; Dupree, P. Structural characterization of Arabidopsis leaf arabinogalactan polysaccharides. *Plant Physiol.* **2012**, *160*, 653-666. [CrossRef] [PubMed]

54. Tryfona, T.; Theys, T.E.; Wagner, T.; Stott, K.; Keegstra, K.; Dupree, P. Characterisation of FUT4 and FUT6 α-(1→2)-fucosyltransferases reveals that absence of root arabinogalactan fucosylation increases Arabidopsis root growth salt sensitivity. *PLoS ONE* **2014**, *9*, e93291. [CrossRef] [PubMed]

55. Seifert, G.J.; Roberts, K. The biology of arabinogalactan proteins. *Ann. Rev. Plant Biol.* **2007**, *58*, 137-161. [CrossRef] [PubMed]

56. Tan, L.; Showalter, A.M.; Egelund, J.; Hernandez-Sanchez, A.; Doblin, M.S.; Bacic, A. Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans. *Front. Plant Sci.* **2012**, *3*, 140. [CrossRef] [PubMed]

57. Tan, L.; Eberhard, S.; Pattathil, S.; Warder, C.; Glushka, J.; Yuan, C.; Hao, Z.; Zhu, X.; Avci, U.; Miller, J.S.; et al. An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein. *Plant Cell* **2013**, *25*, 270-287. [CrossRef] [PubMed]

58. Moller, I.; Sorensen, I.; Bernal, A.J.; Blaukopf, C.; Lee, K.; Obro, J.; Pettolino, F.; Roberts, A.; Mikkelsen, J.D.; Knox, J.P.; et al. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. *Plant J.* **2007**, *50*, 1118-1128. [CrossRef] [PubMed]
59. Tan, L.; Tees, D.; Qian, J.; Kareem, S.; Kieliszewski, M.J. Intermolecular interactions between glycomodules of plant cell wall arabinogalactan-proteins and extensins. *Cell Surf.* **2018**, *1*, 25–33. [CrossRef]

60. Huang, Y.; Wang, Y.; Tan, L.; Sun, L.; Petrosino, J.; Cui, M.Z.; Hao, F.; Zhang, M. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy. *Proc. Natl. Acad. Sci. USA* **2016**, *113*, E3193-3202. [CrossRef] [PubMed]

61. Kii, I.; Ito, H. Periostin and its interacting proteins in the construction of extracellular architectures. *Cell Mol. Life Sci.* **2017**, *74*, 4269–4277. [CrossRef] [PubMed]

62. Park, S.Y.; Jung, M.Y.; Lee, S.J.; Kang, K.B.; Gratchev, A.; Riabov, V.; Kzhyshkowska, J.; Kim, I.S. Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages. *J. Cell Sci.* **2009**, *122*, 3365–3373. [CrossRef] [PubMed]

63. Harris, E.N.; Parry, S.; Sutton-Smith, M.; Pandey, M.S.; Panico, M.; Morris, H.R.; Haslam, S.M.; Dell, A.; Weigel, P.H. N-glycans on the link domain of human HARE/Stabilin-2 are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan. *Glycobiology* **2010**, *20*, 991–1001. [CrossRef] [PubMed]

64. Ohno, S.; Noshiro, M.; Makihira, S.; Kawamoto, T.; Shen, M.; Yan, W.; Kawashima-Ohya, Y.; Fujimoto, K.; Tanne, K.; Kato, Y. RGD-CAP (βig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin α1β1. *Biochim. Biophys. Acta* **1999**, *1451*, 196–205. [CrossRef]

65. Kim, H.J.; Kim, I.S. Transforming growth factor-β-induced gene product, as a novel ligand of integrin αMβ2, promotes monocytes adhesion, migration and chemotaxis. *Int. J. Biochem. Cell Biol.* **2008**, *40*, 991–1004. [CrossRef] [PubMed]

66. Hoersch, S.; Andrade-Navarro, M.A. Periostin shows increased evolutionary plasticity in its alternatively spliced region. *BMC Evol. Biol.* **2010**, *10*, 30. [CrossRef] [PubMed]

67. Kudo, A. Introductory review: Periostin-gene and protein structure. *Cell. Mol. Life Sci.* **2017**, *74*, 4259–4268. [CrossRef] [PubMed]

68. Kii, I.; Nishiyama, T.; Li, M.; Matsumoto, K.; Saito, M.; Amizuka, N.; Kudo, A. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture. *J. Biol. Chem.* **2010**, *285*, 2028–2039. [CrossRef] [PubMed]

69. Liu, H.; Shi, R.; Wang, X.; Pan, Y.; Li, Z.; Yang, X.; Zhang, G.; Ma, Z. Characterization and expression analysis of a fiber differentially expressed fasciclin-like arabinogalactan protein gene in sea island cotton fibers. *PLoS ONE* **2013**, *8*, e70185. [CrossRef] [PubMed]

70. Huang, G.Q.; Xu, W.L.; Gong, S.Y.; Li, B.; Wang, X.L.; Xu, D.; Li, X.B. Characterization of 19 novel cotton *FLA* genes and their expression profiling in fiber development and in response to phytohormones and salt stress. *Physiol. Plant.* **2008**, *134*, 348–359. [CrossRef] [PubMed]

71. MacMillan, C.P.; Taylor, L.; Bi, Y.; Southerton, S.G.; Evans, R.; Spokevicius, A. The fasciclin-like arabinogalactan protein family of *Eucalyptus grandis* contains members that impact wood biology and biomechanics. *New Phytol.* **2015**, *206*, 1314–1327. [CrossRef] [PubMed]

72. Guerriero, G.; Mangeot-Peter, L.; Legay, S.; Behr, M.; Lutts, S.; Siddiqui, K.S.; Hausman, J.F. Identification of fasciclin-like arabinogalactan proteins in textile hemp (*Cannabis sativa* L.): In silico analyses and gene expression patterns in different tissues. *BMC Genom.* **2017**, *18*, 741. [CrossRef] [PubMed]

73. Li, J.; Wu, W.G. Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (*Brassica rapa* L.). *Mol. Biol. Rep.* **2012**, *39*, 1573–4978.

74. Ma, H.; Zhao, J. Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (*Oryza sativa* L.). *J. Exp. Bot.* **2010**, *61*, 2647–2668. [CrossRef] [PubMed]

75. Zang, L.; Zheng, T.; Chu, Y.; Ding, C.; Zhang, W.; Huang, Q.; Su, X. Genome-wide analysis of the fasciclin-like arabinogalactan protein gene family reveals differential expression patterns, localization, and salt stress response in *Populus*. *Front. Plant Sci.* **2015**, *6*, 1140. [CrossRef] [PubMed]

76. Bonnet, N.; Garnero, P.; Ferrari, S. Periostin action in bone. *Mol. Cell. Endocrinol.* **2016**, *432*, 75–82. [CrossRef] [PubMed]

77. Walker, J.T.; McLeod, K.; Kim, S.; Conway, S.J.; Hamilton, D.W. Periostin as a multifunctional modulator of the wound healing response. *Cell Tissue Res.* **2016**, *365*, 453–465. [CrossRef] [PubMed]

78. Kzhyshkowska, J.; Gratchev, A.; Goerdt, S. Stabilin-1, a homeostatic scavenger receptor with multiple functions. *J. Cell. Mol. Med.* **2006**, *10*, 635–649. [CrossRef] [PubMed]
79. Kzhyshkowska, J. Multifunctional receptor stabilin-1 in homeostasis and disease. *Sci. World J.* **2010**, *10*, 2039–2053. [CrossRef] [PubMed]

80. Conway, S.J.; Izuhara, K.; Kudo, Y.; Litvin, J.; Markwald, R.; Ouyang, G.; Arron, J.R.; Holweg, C.T.; Kudo, A. The role of periostin in tissue remodeling across health and disease. *Cell. Mol. Life Sci.* **2014**, *71*, 1279–1288. [CrossRef] [PubMed]

81. Kim, J.E.; Kim, E.H.; Han, E.H.; Park, R.W.; Park, I.H.; Jun, S.H.; Kim, J.C.; Young, M.F.; Kim, I.S. A TGF-β-inducible cell adhesion molecule, βig-h3, is downregulated in melorheostosis and involved in osteogenesis. *J. Cell. Biochem.* **2000**, *77*, 169–178. [CrossRef]

82. Klamer, S.; Voermans, C. The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment. *Cell. Adh. Migr.* **2014**, *8*, 563–577. [CrossRef] [PubMed]

83. Klamer, S.E.; Kuijk, C.G.; Hordijk, P.L.; van der Schoot, C.E.; von Lindern, M.; van Hennik, P.B.; Voermans, C. BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells. *Cell. Adh. Migr.* **2013**, *7*, 434–449. [CrossRef] [PubMed]

84. Han, K.E.; Choi, S.I.; Kim, T.I.; Maeng, Y.S.; Stulting, R.D.; Ji, Y.W.; Kim, E.K. Pathogenesis and treatments of TGFBI corneal dystrophies. *Prog. Retin. Eye Res.* **2016**, *50*, 67–88. [CrossRef] [PubMed]

85. Murugan, E.; Venkatraman, A.; Lei, Z.; Mouvet, V.; Rui Yi Lim, R.; Muruganantham, N.; Goh, E.; Swee Lim Peh, G.; Beuerman, R.W.; Chaurasia, S.S.; et al. pH induced conformational transitions in the transforming growth factor β-induced protein (TGFβIp) associated corneal dystrophy mutants. *Sci. Rep.* **2016**, *6*, 23836. [CrossRef] [PubMed]

86. Poulsen, E.T.; Runager, K.; Nielsen, N.S.; Lukassen, M.V.; Thomsen, K.; Snider, P.; Simmons, O.; Vorum, H.; Conway, S.J.; Enghild, J.J. Proteomic profiling of TGFBI-null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI-linked corneal dystrophies. *FEBS J.* **2017**, *285*, 101–114. [CrossRef] [PubMed]

87. Rios, H.; Koushik, S.V.; Wang, H.; Wang, J.; Zhou, H.M.; Lindsley, A.; Rogers, R.; Chen, Z.; Maeda, M.; Kruyynska-Freitag, A.; et al. Periostin null mice exhibit dwarfism, incisor enamel defects, and an early-onset periodontal disease-like phenotype. *Mol. Cell. Biol.* **2005**, *25*, 11131–11144. [CrossRef] [PubMed]

88. Bonnet, N.; Conway, S.J.; Ferrari, S.L. Regulation of β catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin. *Proc. Natl. Acad. Sci. USA* **2012**, *109*, 15048–15053. [CrossRef] [PubMed]

89. Guo, Y.S.; Zhao, R.; Ma, J.; Cui, W.; Sun, Z.; Gao, B.; He, S.; Han, Y.H.; Fan, J.; Yang, L.; et al. βig-h3 promotes human osteosarcoma cells metastasis by interacting with integrin α2β1 and activating PI3K signaling pathway. *PLoS ONE* **2014**, *9*, e90220. [CrossRef] [PubMed]

90. Bae, J.S.; Lee, S.H.; Kim, J.E.; Choi, J.Y.; Park, R.W.; Yong Park, J.; Park, H.S.; Sohn, Y.S.; Lee, D.S.; Bae Lee, E.; et al. βig-h3 supports keratinocyte adhesion, migration, and proliferation through α3β1 integrin. *Biochem. Biophys. Res. Commun.* **2002**, *294*, 940–948. [CrossRef]

91. Park, S.W.; Bae, J.S.; Kim, K.S.; Park, S.H.; Lee, B.H.; Choi, J.Y.; Park, J.Y.; Ha, S.W.; Kim, Y.L.; Kwon, T.H.; et al. βig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with α3β1 integrin. *Exp. Mol. Med.* **2004**, *36*, 211–219. [CrossRef] [PubMed]

92. Maeng, Y.S.; Choi, Y.J.; Kim, E.K. TGFB1p regulates differentiation of EPC (CD133⁺ c-kit⁺ lin⁻ cells) to EC through activation of the notch signaling pathway. *Stem Cells* **2015**, *33*, 2052–2062. [CrossRef] [PubMed]

93. Utispan, K.; Sonongbua, J.; Thuwajit, P.; Chau-In, S.; Pairojkul, C.; Wongkham, S.; Thuwajit, C. Periostin activates integrin α5β1 through a PI3K/AKT-dependent pathway in invasion of cholangiocarcinoma. *Int. J. Oncol.* **2012**, *41*, 1110–1118. [CrossRef] [PubMed]

94. Kim, M.O.; Yun, S.J.; Kim, I.S.; Sohn, S.; Lee, E.H. Transforming growth factor-β-inducible gene-h3 (βig-h3) promotes cell adhesion of human astrocytoma cells in vitro: Implication of α6β4 integrin. *Neurosci. Lett.* **2003**, *336*, 93–96. [CrossRef]

95. Lu, Y.; Liu, X.; Jiao, Y.; Xiong, X.; Wang, E.; Wang, X.; Zhang, Z.; Zhang, H.; Pan, L.; Guan, Y.; et al. Periostin promotes liver steatosis and hypertriglyceridemia through downregulation of PPARα. *J. Clin. Investig.* **2014**, *124*, 3501–3513. [CrossRef] [PubMed]

96. Ferguson, J.W.; Thoma, B.S.; Mikesh, M.F.; Kramer, R.H.; Bennett, K.L.; Purchio, A.; Bellard, B.J.; LeBaron, R.G. The extracellular matrix protein βig-h3 is expressed at myotendinous junctions and supports muscle cell adhesion. *Cell Tissue Res.* **2003**, *313*, 93–105. [CrossRef] [PubMed]
97. Nam, J.O.; Kim, J.E.; Jeong, H.W.; Lee, S.J.; Lee, B.H.; Choi, J.Y.; Park, R.W.; Park, J.Y.; Kim, I.S. Identification of the αvβ3 integrin-interacting motif of βig-h3 and its anti-angiogenic effect. *J. Biol. Chem.* **2003**, *278*, 25902–25909. [CrossRef] [PubMed]

98. Thapa, N.; Kang, K.B.; Kim, I.S. βig-h3 mediates osteoblast adhesion and inhibits differentiation. *Bone* **2005**, *36*, 232–242. [CrossRef] [PubMed]

99. Choi, S.I.; Maeng, Y.S.; Kim, T.I.; Lee, Y.; Kim, Y.S.; Kim, E.K. Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis. *PLoS ONE* **2015**, *10*, e0119561. [CrossRef] [PubMed]

100. Gillan, L.; Matei, D.; Fishman, D.A.; Gerbin, C.S.; Karlan, B.Y.; Chang, D.D. Periostin secreted by epithelial ovarian carcinoma is a ligand for αvβ3 and αvβ5 integrins and promotes cell motility. *Cancer Res.* **2002**, *62*, 5358–5364. [PubMed]

101. Li, G.; Jin, R.; Norris, R.A.; Zhang, L.; Yu, S.; Wu, F.; Markwald, R.R.; Nanda, A.; Conway, S.J.; Smyth, S.S.; et al. Periostin mediates vascular smooth muscle cell migration through the integrins αvβ3 and αvβ5 and focal adhesion kinase (FAK) pathway. *Atherosclerosis* **2010**, *208*, 358–365. [CrossRef] [PubMed]

102. Lee, Y.J.; Kim, I.S.; Park, S.A.; Kim, Y.; Lee, J.E.; Noh, D.Y.; Kim, K.T.; Ryu, S.H.; Suh, P.G. Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis. *Mol. Ther.* **2013**, *21*, 1004–1013. [CrossRef] [PubMed]

103. Matsuzawa, M.; Arai, C.; Nomura, Y.; Murata, T.; Yamakoshi, Y.; Oida, S.; Hanada, N.; Nakamura, Y. Periostin of human periodontal ligament fibroblasts promotes migration of human mesenchymal stem cell through the αvβ3 integrin/FAK/PI3K/Akt pathway. *J. Period. Res.* **2015**, *50*, 855–863. [CrossRef] [PubMed]

104. Chuanyu, S.; Yuqing, Z.; Chong, X.; Guowei, X.; Xiaojun, Z. Periostin promotes migration and invasion of renal cell carcinoma through the integrin/focal adhesion kinase/c-Jun N-terminal kinase pathway. *Tumour Biol.* **2017**, *39*, [CrossRef] [PubMed]

105. Zhou, W.; Ke, S.Q.; Huang, Z.; Flavahan, W.; Fang, X.; Paul, J.; Wu, L.; Sloan, A.E.; McLendon, R.E.; Li, X.; et al. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth. *Nat. Cell Biol.* **2015**, *17*, 170–182. [CrossRef] [PubMed]

106. Lee, B.H.; Bae, J.S.; Park, R.W.; Kim, J.E.; Park, J.Y.; Kim, I.S. βig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through αvβ5 integrin. *Exp. Mol. Med.* **2006**, *38*, 153–161. [CrossRef] [PubMed]

107. Kim, S.; Park, S.Y.; Kim, S.Y.; Bae, D.J.; Pyo, J.H.; Hong, M.; Kim, I.S. Cross talk between engulfment receptors stabilin-2 and integrin αvβ5 orchestrates engulfment of phosphatidylserine-exposed erythrocytes. *Mol. Cell. Biol.* **2012**, *32*, 2698–2708. [CrossRef] [PubMed]

108. Ghatak, S.; Misra, S.; Norris, R.A.; Moreno-Rodriguez, R.A.; Hoffman, S.; Levine, R.A.; Hascall, V.C.; Markwald, R.R. Periostin induces intracellular cross-talk between kinases and hyaluronan in atrioventricular valvulogenesis. *J. Biol. Chem.* **2014**, *289*, 8545–8561. [CrossRef] [PubMed]

109. Johansson, M.W.; Annis, D.S.; Mosher, D.F. αMβ2 integrin-mediated adhesion and motility of IL-5-stimulated eosinophils on periostin. *Am. J. Respir. Cell. Mol. Biol.* **2013**, *48*, 503–510. [CrossRef] [PubMed]

110. Jung, M.Y.; Park, S.Y.; Kim, I.S. Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with αMβ2 integrin. *J. Leukoc. Biol.* **2007**, *82*, 1156–1165. [CrossRef] [PubMed]

111. Hashimoto, K.; Noshiro, M.; Ohno, S.; Kawamoto, T.; Satakedai, H.; Akagawa, Y.; Nakashima, K.; Okimura, A.; Ishida, H.; Okamoto, T.; et al. Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/βig-h3) that binds to collagen. *Biochim. Biophys. Acta* **1997**, *1355*, 303–314. [CrossRef]

112. Kii, I.; Nishiyama, T.; Kudo, A. Periostin promotes secretion of fibronectin from the endoplasmic reticulum. *Biochem. Biophys. Res. Commun.* **2016**, *470*, 888–893. [CrossRef] [PubMed]

113. Hwang, E.Y.; Jeong, M.S.; Park, E.K.; Kim, J.H.; Jang, S.B. Structural characterization and interaction of periostin and bone morphogenetic protein for regulation of collagen cross-linking. *Biochem. Biophys. Res. Commun.* **2014**, *449*, 425–431. [CrossRef] [PubMed]

114. Maruhashi, T.; Kii, I.; Saito, M.; Kudo, A. Interaction between periostin and BMP-1 promotes proteolytic activation of lysyl oxidase. *J. Biol. Chem.* **2010**, *285*, 13294–13303. [CrossRef] [PubMed]

115. Kzhyshkowska, J.; Gratchev, A.; Brundiers, H.; Mamidi, S.; Krusell, L.; Goerdt, S. Phosphatidylinositide 3-kinase activity is required for stabilin-1-mediated endosomal transport of acLDL. *Immunobiology* **2005**, *210*, 161–173. [CrossRef] [PubMed]
116. Khyshkowska, J.; Workman, G.; Cardo-Vila, M.; Arap, W.; Pasqualini, R.; Gratchev, A.; Krusell, L.; Goerdt, S.; Sage, E.H. Novel function of alternatively activated macrophages: Stabilin-1-mediated clearance of SPARC. *J. Immunol.* **2006**, *176*, 5825–5832. [CrossRef] [PubMed]

117. Workman, G.; Sage, E.H. Identification of a sequence in the matricellular protein SPARC that interacts with the scavenger receptor stabilin-1. *J. Cell. Biochem.* **2011**, *112*, 1003–1008. [CrossRef] [PubMed]

118. Khyshkowska, J.; Gratchev, A.; Schmuttermaier, C.; Brundiers, H.; Krusell, L.; Mamidi, S.; Zhang, J.; Workman, G.; Sage, E.H.; Anderle, C.; et al. Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis. *J. Immunol.* **2008**, *180*, 3028–3037. [CrossRef] [PubMed]

119. Khyshkowska, J.; Mamidi, S.; Gratchev, A.; Kremmer, E.; Schmuttermaier, C.; Krusell, L.; Haus, G.; Utikal, J.; Schledzewski, K.; Scholtze, J.; et al. Novel stabilin-1 interacting chitinase-like protein (SI-CLP) is up-regulated in alternatively activated macrophages and secreted via lysosomal pathway. *Blood* **2006**, *107*, 3221–3228. [CrossRef] [PubMed]

120. Meng, G.; Zhao, Y.; Bai, X.; Liu, Y.; Green, T.J.; Luo, M.; Zheng, X. Structure of human stabilin-1 interacting chitinase-like protein (SI-CLP) reveals a saccharide-binding cleft with lower sugar-binding selectivity. *J. Biol. Chem.* **2010**, *285*, 39898–39904. [CrossRef] [PubMed]

121. Harris, E.N.; Weigel, J.A.; Weigel, P.H. The human hyaluronan receptor for endocytosis (HARE/stabilin-2) is a systemic clearance receptor for heparin. *J. Biol. Chem.* **2008**, *283*, 17341–17350. [CrossRef] [PubMed]

122. Pandey, M.S.; Weigel, P.H. A hyaluronan receptor for endocytosis (HARE) link domain N-glycan is required for extracellular signal-regulated kinase (ERK) and nuclear factor-κB (Nf-κB) signaling in response to the uptake of hyaluronan but not heparin, dermatan sulfate, or acetylated low density lipoprotein (LDL). *J. Biol. Chem.* **2014**, *289*, 21807–21817. [PubMed]

123. Penberthy, K.K.; Ravichandran, K.S. Apoptotic cell recognition receptors and scavenger receptors. *Immunol. Rev.* **2016**, *269*, 44–59. [CrossRef] [PubMed]

124. Park, S.Y.; Yun, Y.; Lim, J.S.; Kim, M.J.; Kim, S.Y.; Kim, J.E.; Kim, I.S. Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration. *Nat. Commun.* **2016**, *7*, 10871. [CrossRef] [PubMed]

125. Kim, G.W.; Park, S.Y.; Kim, I.S. Novel function of stabilin-2 in myoblast fusion: The recognition of extracellular phosphatidylyserine as a "fuse-me" signal. *BMB Rep.* **2016**, *49*, 303–304. [CrossRef] [PubMed]

126. Park, S.Y.; Jung, M.Y.; Kim, I.S. Stabilin-2 mediates homophilic cell–cell interactions via its FAS1 domains. *FEBS Lett.* **2009**, *583*, 1375–1380. [CrossRef] [PubMed]

127. Zhu, J.K. Salt and drought stress signal transduction in plants. *Annu. Rev. Plant Biol.* **2002**, *53*, 247–273. [CrossRef] [PubMed]

128. Xu, S.L.; Rahman, A.; Baskin, T.I.; Kieber, J.J. Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis. *Plant Cell* **2008**, *20*, 3065–3079. [CrossRef] [PubMed]

129. Hauser, M.T.; Morikami, A.; Benfey, P.N. Conditional root expansion mutants of Arabidopsis. *Development* **1995**, *121*, 1237–1252. [PubMed]

130. Seifert, G.J.; Xue, H.; Acet, T. The *Arabidopsis thaliana* fasciclin like arabinogalactan protein 4 gene acts synergistically with abscisic acid signalling to control root growth. *Ann. Bot.* **2014**, *114*, 1125–1133. [CrossRef] [PubMed]

131. Harpaz-Saad, S.; McFarlane, H.E.; Xu, S.; Divi, U.K.; Forward, B.; Western, T.L.; Kieber, J.J. Cellulose synthesis via the FEI2 rlk/Sos5 pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis. *Plant J.* **2011**, *68*, 941–953. [CrossRef] [PubMed]

132. Harpaz-Saad, S.; Western, T.L.; Kieber, J.J. The FEI2-SOS5 pathway and CELLULOSE SYNTHASE 5 are required for cellulose biosynthesis in the Arabidopsis seed coat and affect pectin mucilage structure. *Plant Signal. Behav.* **2012**, *7*, 285–288. [CrossRef] [PubMed]

133. Griffiths, J.S.; Crepeau, M.J.; Ralet, M.C.; Seifert, G.J.; North, H.M. Dissecting seed mucilage adherence mediated by FEI2 and Sos5. *Front. Plant Sci.* **2016**, *7*, 1073. [CrossRef] [PubMed]

134. Griffiths, J.S.; Tsai, A.Y.; Xue, H.; Voiniciuc, C.; Sola, K.; Seifert, G.J.; Mansfield, S.D.; Haughn, G.W. Salt-overly sensitive5 mediates Arabidopsis seed coat mucilage adherence and organization through pectins. *Plant Physiol.* **2014**, *165*, 991–1004. [CrossRef] [PubMed]
135. Tsang, D.L.; Edmond, C.; Harrington, J.L.; Nuhse, T.S. Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway. *Plant Physiol.* **2011**, *156*, 596–604. [CrossRef] [PubMed]

136. Xue, H.; Seifert, G.J. Fasciclin like arabinogalactan protein 4 and respiratory burst oxidase homolog d and F independently modulate abscisic acid signaling. *Plant Signal. Behav.* **2015**, *10*, e989064. [CrossRef] [PubMed]

137. Wormit, A.; Butt, S.M.; Chairam, I.; McKenna, J.F.; Nunes-Nesi, A.; Kjaer, L.; O'Donnelly, K.; Fernie, A.R.; Woscholski, R.; Barter, M.C.; et al. Osmosensitive changes of carbohydrate metabolism in response to cellulose biosynthesis inhibition. *Plant Physiol.* **2012**, *159*, 105–117. [CrossRef] [PubMed]

138. Steinwand, B.J.; Xu, S.; Polko, J.K.; Doctor, S.M.; Westafer, M.; Kieber, J.J. Alterations in auxin homeostasis suppress defects in cell wall function. *PLoS ONE* **2014**, *9*, e98193. [CrossRef] [PubMed]

139. North, H.M.; Berger, A.; Saez-Aguayo, S.; Ralet, M.C. Understanding polysaccharide production and properties using seed coat mutants: Future perspectives for the exploitation of natural variants. *Ann. Bot.* **2014**, *114*, 1251–1263. [CrossRef] [PubMed]

140. Voiniciuc, C.; Yang, B.; Schmidt, M.H.; Gunl, M.; Usadel, B. Starting to gel: How Arabidopsis seed coat epidermal cells produce specialized secondary cell walls. *Int. J. Mol. Sci.* **2015**, *16*, 3452–3473. [CrossRef] [PubMed]

141. Ralet, M.C.; Crepeau, M.J.; Vigouroux, J.; Tran, J.; Berger, A.; Salle, C.; Granier, F.; Botran, L.; North, H.M. Xylans provide the structural driving force for mucilage adhesion to the Arabidopsis seed coat. *Plant Physiol.* **2016**, *171*, 165–178. [CrossRef] [PubMed]

142. Voiniciuc, C.; Gunl, M.; Schmidt, M.H.; Usadel, B. Highly branched xylan made by IRX14 and MUC121 links mucilage to Arabidopsis seeds. *Plant Physiol.* **2015**, *169*, 2481–2495. [CrossRef] [PubMed]

143. Dean, G.H.; Zheng, H.; Tewari, J.; Huang, J.; Young, D.S.; Hwang, Y.T.; Western, T.L.; Carpita, N.C.; McCann, M.C.; Mansfield, S.D.; et al. The Arabidopsis *Mum2* gene encodes a β-galactosidase required for the production of seed coat mucilage with correct hydration properties. *Plant Cell* **2007**, *19*, 4007–4021. [CrossRef] [PubMed]

144. Voiniciuc, C.; Dean, G.H.; Griffiths, J.S.; Kirchsteiger, K.; Hwang, Y.T.; Gillett, A.; Dow, G.; Western, T.L.; Estelle, M.; Haughn, G.W. Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage. *Plant Cell* **2013**, *25*, 944–959. [CrossRef] [PubMed]

145. Tsai, A.Y.; Kunieda, T.; Rogalski, J.; Foster, L.J.; Ellis, B.E.; Haughn, G.W. Identification and characterization of Arabidopsis seed coat mucilage proteins. *Plant Physiol.* **2017**, *173*, 1059–1074. [CrossRef] [PubMed]

146. Basu, D.; Tian, L.; Debrosse, T.; Poirier, E.; Emch, K.; Herock, H.; Travers, A.; Showalter, A.M. Glycosylation of a fasciclin-like arabinogalactan-protein (SOS5) mediates root growth and seed mucilage adherence via a cell wall receptor-like kinase (FEI1/FEI2) pathway in Arabidopsis. *PLoS ONE* **2016**, *11*, e0145092. [CrossRef] [PubMed]

147. Basu, D.; Wang, W.; Ma, S.; DeBrosse, T.; Poirier, E.; Emch, K.; Soukup, E.; Tian, L.; Showalter, A.M. Two hydroxyproline galactosyltransferases, Galt5 and Galt2, function in arabinogalactan-protein glycosylation, growth and development in Arabidopsis. *PLoS ONE* **2015**, *10*, e0125624. [CrossRef] [PubMed]

148. Brown, D.M.; Zeef, L.A.; Ellis, J.; Goodacre, R.; Turner, S.R. Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. *Plant Cell* **2005**, *17*, 2281–2295. [CrossRef] [PubMed]

149. Persson, S.; Wei, H.; Milne, J.; Page, G.P.; Somerville, C.R. Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets. *Proc. Natl. Acad. Sci. USA* **2005**, *102*, 8633–8638. [CrossRef] [PubMed]

150. Ito, S.; Suzuki, Y.; Miyamoto, K.; Ueda, J.; Yamaguchi, I. AtFLA11, a fasciclin-like arabinogalactan-protein, specifically localized in sclerenchyma cells. *Biosci. Biotechnol. Biochem.* **2005**, *69*, 1963–1969. [CrossRef] [PubMed]

151. Dahiya, P.; Findlay, K.; Roberts, K.; McCann, M.C. A fasciclin-domain containing gene, *ZeFLA11*, is expressed exclusively in xylem elements that have reticulate wall thickenings in the stem vascular system of *Zinnia elegans* cv Envy. *Planta* **2006**, *223*, 1281–1291. [CrossRef] [PubMed]

152. Lafarguette, F.; Leplé, J.-C.; Déjardin, A.; Laurans, F.; Costa, G.; Lesage-Descauses, M.-C.; Pilate, G. Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood. *New Phytol.* **2004**, *164*, 107–121. [CrossRef]
153. Gritsch, C.; Wan, Y.; Mitchell, R.A.; Shewry, P.R.; Hanley, S.J.; Karp, A. G-fibre cell wall development in willow stems during tension wood induction. *J. Exp. Bot.* **2015**, *66*, 6447–6459. [CrossRef] [PubMed]

154. Qiu, D.; Wilson, I.W.; Gan, S.; Washusen, R.; Moran, G.F.; Southerton, S.G. Gene expression in eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers. *New Phytol.* **2008**, *179*, 94–103. [CrossRef] [PubMed]

155. Hobson, N.; Deyholos, M.K. LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (*Linum usitatissimum*). *Plant Cell Rep.* **2013**, *32*, 517–528. [CrossRef] [PubMed]

156. MacMillan, C.P.; Mansfield, S.D.; Stachurski, Z.H.; Evans, R.; Southerton, S.G. Fasciclin-like arabinogalactan proteins: Specialization for stem biomechanics and cell wall architecture in Arabidopsis and Eucalyptus. *Plant J.* **2010**, *62*, 689–703. [CrossRef] [PubMed]

157. Wang, H.; Jiang, C.; Wang, C.; Yang, Y.; Yang, L.; Gao, X.; Zhang, H. Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in *Populus* inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees. *J. Exp. Bot.* **2015**, *66*, 1291–1302. [CrossRef] [PubMed]

158. Wang, H.; Jin, Y.; Wang, C.; Li, B.; Jiang, C.; Sun, Z.; Zhang, Z.; Kong, F.; Zhang, H. Fasciclin-like arabinogalactan proteins, PtFLAs, play important roles in GA-mediated tension wood formation in *Populus*. *Sci. Rep.* **2017**, *7*, 6182. [CrossRef] [PubMed]

159. Huang, G.Q.; Gong, S.Y.; Xu, W.L.; Li, W.; Li, P.; Zhang, C.J.; Li, D.D.; Zheng, Y.; Li, F.G.; Li, X.B. A fasciclin-like arabinogalactan protein, GhFLA1, is involved in fiber initiation and elongation of cotton. *Plant Physiol.* **2013**, *161*, 1278–1290. [CrossRef] [PubMed]

160. Li, Y.; Liu, D.; Tu, L.; Zhang, X.; Wang, L.; Zhu, L.; Tan, J.; Deng, F. Suppression of GhAgp4 gene expression repressed the initiation and elongation of cotton fiber. *Plant Cell Rep.* **2010**, *29*, 193–202. [CrossRef] [PubMed]

161. Motose, H.; Sugiyama, M.; Fukuda, H. A proteoglycan mediates inductive interaction during plant vascular development. *Nature* **2004**, *429*, 873–878. [CrossRef] [PubMed]

162. Li, A.; Wang, R.; Li, X.; Liu, M.; Fan, J.; Guo, K.; Luo, B.; Chen, T.; Feng, S.; Wang, Y.; et al. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers. *Sci. Rep.* **2016**, *6*, 26356. [CrossRef] [PubMed]

163. Wu, X.N.; Sanchez Rodriguez, C.; Pertl-Obermeyer, H.; Obermeyer, G.; Schulze, W.X. Sucrose-induced receptor kinase Sirk1 regulates a plasma membrane aquaporin in Arabidopsis. *Mol. Cell. Proteom.* **2013**, *12*, 2856–2873. [CrossRef] [PubMed]

164. Klepikova, A.V.; Logacheva, M.D.; Dmitriev, S.E.; Penin, A.A. RNA-seq analysis of an apical meristem time series reveals a critical point in *Arabidopsis thaliana* flower initiation. *BMC Genom.* **2015**, *16*, 466. [CrossRef] [PubMed]

165. Klepikova, A.V.; Kasianov, A.S.; Gerasimov, E.S.; Logacheva, M.D.; Penin, A.A. A high resolution map of the *Arabidopsis thaliana* developmental transcriptome based on RNA-seq profiling. *Plant J.* **2016**, *88*, 1058–1070. [CrossRef] [PubMed]

166. Li, J.; Yu, M.; Geng, L.L.; Zhao, J. The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis. *Plant J.* **2010**, *64*, 482–497. [CrossRef] [PubMed]

167. Johnson, K.L.; Kibble, N.A.; Bacic, A.; Schultz, C.J. A fasciclin-like arabinogalactan-protein (FLA) mutant of *Arabidopsis thaliana*, FLA1, shows defects in shoot regeneration. *PLoS ONE* **2011**, *6*, e25154. [CrossRef] [PubMed]

168. Kirchner, T.W.; Niehaus, M.; Debener, T.; Schenk, M.K.; Herde, M. Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of *Brassica carinata*. *PLoS ONE* **2017**, *12*, e0185429. [CrossRef] [PubMed]

169. Cagnola, J.I.; de Chassart, G.J.D.; Ibarra, S.E.; Chimenti, C.; Ricardi, M.M.; Delzer, B.; Ghiglione, H.; Zhu, T.; Otegui, M.E.; Estevez, J.M.; et al. Reduced expression of selected fasciclin-like arabinogalactan protein genes associates with the abortion of kernels in field crops of *Zea mays* (maize) and of Arabidopsis seeds. *Plant Cell Environ.* **2018**. [CrossRef] [PubMed]

170. Nirmal, R.C.; Furtado, A.; Rangan, P.; Henry, R.J. Fasciclin-like arabinogalactan protein gene expression is associated with yield of flour in the milling of wheat. *Sci. Rep.* **2017**, *7*, 12539. [CrossRef] [PubMed]

171. Wiker, H.G. MPB70 and MPB83—Major antigens of *Mycobacterium bovis*. *Scand. J. Immunol.* **2009**, *69*, 492–499. [CrossRef] [PubMed]
172. Rahman, S.A.; Singh, Y.; Kohli, S.; Ahmad, J.; Ehtesham, N.Z.; Tyagi, A.K.; Hasnain, S.E. Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of *Mycobacterium tuberculosis*. *MBio* **2014**, *5*, e02020. [CrossRef] [PubMed]

173. Elkins, T.; Hortsch, M.; Bieber, A.J.; Snow, P.M.; Goodman, C.S. *Drosophila* fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting. *J. Cell Biol.* **1990**, *110*, 1825-1832. [CrossRef] [PubMed]

174. Chen, S.T.; Li, J.Y.; Zhang, Y.; Gao, X.; Cai, H. Recombinant MPT83 derived from *Mycobacterium tuberculosis* induces cytokine production and upregulates the function of mouse macrophages through TLR2. *J. Immunol.* **2012**, *188*, 668-677. [CrossRef] [PubMed]

175. Shiu, S.H.; Bleecker, A.B. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. *Proc. Natl. Acad. Sci. USA* **2001**, *98*, 10763-10768. [CrossRef] [PubMed]

176. Oke, V.; Long, S.R. Bacteroid formation in the *Rhizobium*-legume symbiosis. *Curr. Opin. Microbiol.* **1999**, *2*, 641-646. [CrossRef]

177. Oke, V.; Long, S.R. Bacterial genes induced within the nodule during the *Rhizobium*-legume symbiosis. *Mol. Microbiol.* **1999**, *32*, 837-849. [CrossRef] [PubMed]

178. Paulsrud, P.; Lindblad, P. Fasciclin domain proteins are present in *Nostoc* symbionts of lichens. *Appl. Environ. Microb.* **2002**, *68*, 2036-2039. [CrossRef]

179. Delmotte, N.; Knief, C.; Chaffron, S.; Innerebner, G.; Roschitzki, B.; Schlapbach, R.; von Mering, C.; Vorholt, J.A. Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. *Proc. Natl. Acad. Sci. USA* **2009**, *106*, 16428-16433. [CrossRef] [PubMed]

180. Knief, C.; Ramette, A.; Frances, L.; Alonso-Blanco, C.; Vorholt, J.A. Site and plant species are important determinants of the methylobacterium community composition in the plant phyllosphere. *ISME J.* **2010**, *4*, 719-728. [CrossRef] [PubMed]

181. Tejerizo, G.T.; Kim, Y.S.; Maus, I.; Wibberg, D.; Winkler, A.; Off, S.; Puhler, A.; Scherer, P.; Schluter, A. Genome sequence of *Methanobacterium congolense* strain buetzberg, a hydrogenotrophic, methanogenic archaeon, isolated from a mesophilic industrial-scale biogas plant utilizing bio-waste. *J. Biotechnol.* **2017**, *247*, 1-5. [CrossRef] [PubMed]

182. Bornstein, P. Diversity of function is inherent in matricellular proteins: An appraisal of thrombospondin 1. *J. Cell Biol.* **1995**, *130*, 503-506. [CrossRef] [PubMed]

183. Alberts, B.; Johnson, A.; Lewis, J.; Morgan, D.; Raff, M.; Roberts, K.; Walter, P. *Molecular Biology of the Cell*, 6th ed.; Garland Science: New York, NY, USA, 2015.

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© 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
<figure><img src="image_92.png" /></figure>

Article

# Evolution Analysis of the Fasciclin-Like Arabinogalactan Proteins in Plants Shows Variable Fasciclin-AGP Domain Constitutions

Jiadai He<fnref n="1" />,†, Hua Zhao<fnref n="1" />,†, Zhilu Cheng<fnref n="2" />, Yuwei Ke<fnref n="3" />, Jiaxi Liu<fnref n="1" /> and Haoli Ma<fnref n="1" />,*

Received: 22 February 2019; Accepted: 19 April 2019; Published: 20 April 2019

**Abstract:** The fasciclin-like arabinogalactan proteins (FLAs) play important roles in plant development and adaptation to the environment. FLAs contain both fasciclin domains and arabinogalactan protein (AGP) regions, which have been identified in several plants. The evolutionary history of this gene family in plants is still undiscovered. In this study, we identified the *FLA* gene family in 13 plant species covering major lineages of plants using bioinformatics methods. A total of 246 *FLA* genes are identified with gene copy numbers ranging from one (*Chondrus crispus*) to 49 (*Populus trichocarpa*). These FLAs are classified into seven groups, mainly based on the phylogenetic analysis of plant FLAs. All FLAs in land plants contain one or two fasciclin domains, while in algae, several FLAs contain four or six fasciclin domains. It has been proposed that there was a divergence event, represented by the reduced number of fasciclin domains from algae to land plants in evolutionary history. Furthermore, introns in *FLA* genes are lost during plant evolution, especially from green algae to land plants. Moreover, it is found that gene duplication events, including segmental and tandem duplications are essential for the expansion of *FLA* gene families. The duplicated gene pairs in *FLA* gene family mainly evolve under purifying selection. Our findings give insight into the origin and expansion of the *FLA* gene family and help us understand their functions during the process of evolution.

**Keywords:** fasciclin-like AGP; FLA; evolution; phylogeny

## 1. Introduction

The cell wall plays an important role in plant growth and development by providing structural support and protection, and acting as a filtering mechanism. Although cell wall proteins account for less than 10% of the cell wall mass, they are predominantly involved in the wall structure, support, signaling, and interactions with other wall components and with the plasma membrane [1,2]. Hydroxyproline-rich glycoproteins (HRGs) are a major group of cell wall glycoproteins that play important roles in plant growth and development [3].

HRGs are characterized by a protein backbone rich in hydroxyproline (Hyp). The HRGs superfamily can be divided into three main subfamilies based on the varying degrees of O-glycosylation: Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs) [4–6]. The protein backbones of AGPs are rich in hydroxyproline/proline (Hyp/Pro), alanine (Ala), serine (Ser), and threonine (Thr), and these amino acids are regularly arranged as Ala-Pro, Ser-Pro, and Thr-Pro, which were introduced as arabinogalactan (AG) glycomodules [7–9]. The carbohydrate side chains of

<footnote n="1">¹ College of Agronomy, Northwest A&F University, Xianyang 712100, Shaanxi, China; 2015014922@nwsuaf.edu.cn (J.H.); zhaohua362@nwsuaf.edu.cn (H.Z.); 2015014872@nwsuaf.edu.cn (J.L.)</footnote>

<footnote n="2">² College of Landscape Architecture and Arts, Northwest A&F University, Xianyang 712100, Shaanxi, China; czl980525@163.com</footnote>

<footnote n="3">³ College of Life Sciences, Northwest A&F University, Xianyang 712100, Shaanxi, China; keyuwei98@163.com</footnote>

<footnote>* Correspondence: mahaoli@nwsuaf.edu.cn; Tel.: +86-29-87081551</footnote>

<footnote>† These authors contribute equally to this work.</footnote>
AGPs are attached to Hyp and enriched in arabinose and galactose [10]. Based on the variable protein backbones [6], AGPs can be classified into classical AGPs, chimeric AGPs, and AGP-EXT hybrids. The chimeric AGPs can be further categorized into three subclasses based on different conserved domains: Fasciclin-like AGPs (FLAs) [11–13], phytocyanin-like AGPs (PAGs) [14,15], and xylogen-like AGPs (XYLPs) [16,17]. As one subclass of the chimeric AGPs, FLAs consist of both fasciclin domains and AGP regions. In most plant species, FLAs contain one or two fasciclin domains. The fasciclin domains contain two highly conserved motifs (H1 and H2) of about 10 amino acids long each and a conserved central YH motif [18]. Proteins with fasciclin domains were first identified in grasshoppers [19] and as adhesion factors were first identified in fruit flies [20]. Since then, more and more fasciclin domains have been identified in animal, yeast, bacteria and plant proteins [18]. The majority of plant fasciclin-like proteins are FLAs and the functions of FLAs are related to many important processes in development and stress responses, such as contributing to biophysical properties (e.g., swelling and interpolymer connectivity), affecting secondary cell wall formation and structure, acting in male gametophyte development, influencing organ formation, and sensing salt stress in roots [18].

To date, FLAs have been identified in several plants, including *Arabidopsis* (*Arabidopsis thaliana*) [21], rice (*Oryza sativa*) [12,22], wheat (*Triticum aestivum*) [22], poplar (*Populus trichocarpa*) [23,24], zinnia (*Zinnia elegans*) [25], cotton (*Gossypium raimondii*) [26], sea island cotton (*Gossypium barbadense*) [27], Chinese cabbage (*Brassica rapa*) [28], eucalyptus (*Eucalyptus grandis*) [13], and textile hemp (*Cannabis sativa*) [29]. The analysis of HRGPs from 1000 plant transcriptomes has provided new insights into the evolution of HRGPs across major evolutionary milestones and reveals the origin and diversity of Glycosylphosphatidylinositol (GPI)-anchored AGPs [3]. However, the evolutionary history of the FLA family in plants is little known. In a previous study, it was proposed that a conserved group of FLAs with a single fasciclin domain was specific to the evolution of flowering plant secondary cell wall formation and properties through phylogenetic analysis of >100 FLA mature proteins [30]. In this study, we identify 246 FLAs from 13 plant species belonging to algae, liverworts, mosses, lycophytes, gymnosperms, dicots, and monocots. Moreover, bioinformatics methods are adopted to reveal the evolutionary mechanisms of the FLA family. In order to understand the functions of the FLAs, the evolutionary history of FLAs is investigated in this study. It is found that the *FLA* genes are abundant in most investigated green plants, but only in one red alga. Additionally, our study shows that there is a reduction in the number of fasciclin domains in FLAs from algae to land plants, which indicates that the reduced number of fasciclin domains plays a crucial role in land plant evolution.

## 2. Results and Discussions

### 2.1. Identification of the FLA Family in Plants

FLAs contain both fasciclin domains and AGP regions [6]. We first used the HMM profile of fasciclin downloaded from Pfam (available online: http://pfam.xfam.org/family/PF02469) to identify the proteins with fasciclin domains from 13 plant species (C. crispus, *Chlamydomonas reinhardtii*, *Chara braunii*, *Marchantia polymorpha*, *Physcomitrella patens*, *Selaginella moellendorffii*, *Picea abies*, *Amborella trichopoda*, *Brachypodium distachyon*, *O. sativa*, *A. thaliana*, *E. grandis*, and *P. trichocarpa*) [31–43]. Then, the obtained proteins were examined by using Batch CD-search tool in the NCBI conserved domain database (available online: http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). After that, the AGP regions were identified from these fasciclin proteins by using Finding-AGP program [7]. The proteins that contained both AGP regions and fasciclin domains were identified as FLAs. A total of 235 *FLA* genes were identified by the HMMER-Finding-AGP program method.

However, the number of *FLA* genes found in some plants was different from those described in former studies. In *A. thaliana*, *FLA20* (*AT5G40940*) and *FLA21* (*AT5G06920*) [21] were not identified, while a new putative *FLA* gene, *AT5G16920*, was identified. In *E. grandis*, *Eucgr.A01741* and *Eucgr.K02662* were missing [13], and *Eucgr.K00086* was a newly identified *FLA* gene. In *P. trichopoda*, 46 *FLA* genes were identified compared with the 50 *FLA* genes analyzed in a previous
study [24]: *Potri.013G152200*, *Potri.T130300*, *Potri.001G440800*, *Potri.018G005100*, *Potri.008G127500*, *Potri.008G128200*, and *Potri.005G079500* were not identified, whereas *Potri.019G049600*, *Potri.T118500* and *Potri.012G006200* were new putative *FLA* genes identified in this study. In *O. sativa*, two *FLA* genes found in a previous study (*LOC_Os02g49420* and *LOC_Os02g26290*) [12] were not identified, while a putative new *FLA* gene (*LOC_Os12g13160*) was identified in our work. Among 13 *FLA* genes that were not identified by the HMMER-Finding-AGP program method, it was found that *Potri.T130300*, *Potri.018G005100*, *LOC_Os02g49420*, and *LOC_Os02g26290* did not contain a fasciclin domain by using Batch CD-Search tool. Besides, because the AGP regions of Eucgr.K02662, *Potri.008G127500*, and *Potri.008G128200* were found in the fasciclin domain, they were not identified as FLAs in this study. Then, the remaining six FLAs (AT5G40940, AT5G06920, Eucgr.A01741, *Potri.013G152200*, *Potri.001G440800*, and *Potri.005G079500*) were included in this study and also used as queries to perform BLAST searches to identify their homologous FLAs in other plant species: Phpat.003G041000 in *P. patens*, *MA_89859g0010* and *MA_10360g0010* in *P. abies*, scaffold00024.69 in *A. trichopoda*, and Eucgr.H00590.1 in *E. grandis*. As a result, 246 *FLA* genes were identified.

The number of *FLA* genes ranged from 1 to 49 across the different plant species; in most species, the number of *FLA* genes was between 11 and 26. *C. crispus* had only one *FLA* gene, while *P. trichocarpa* contained the highest number of *FLA* genes (49), almost double the number of the second one, *O. sativa* (26). It was found that the number of *FLA* genes and genome size were uncorrelated. *P. abies*, for instance, which had the largest genome size (19,600 Mb) among these 13 plant species, had only 24 *FLA* genes compared with *P. trichocarpa* which had 49 *FLA* genes with a much smaller genome size (434.29 Mb) (Table 1). The number of *FLA* genes was also uncorrelated with the number of predicted genes in plant species. For example, *E. grandis* contained more genes (45,226) than *O. sativa*, while *O. sativa* had more *FLA* genes (26) than *E. grandis* (18) (Table 1). Overall, higher plants contained the highest number of *FLA* genes and the number of *FLA* genes increased from lower plants to higher plants. For example, the number of *FLA* genes was doubled from lycophytes to gymnosperm.

Moreover, the intron-exon structures of 246 *FLA* genes were retrieved from the OrcAE website (available online: https://bioinformatics.psb.ugent.be/orca/overview/Chbra), Phytozome website (Version 12; available online: https://phytozome.jgi.doe.gov/pz/portal.html), and ConGenIE website (available online: http://congenie.org/) and were displayed by GSDS 2.0 (available online: http://gsds.cbi.pku.edu.cn/) [44]. Green algae *FLA* genes contained a large number of introns, while most land plants *FLA* genes contained one intron or even had no intron (Table S1). It seemed that introns in *FLA* genes were lost during plant evolution, especially from green algae to land plants.

<table><caption>**Table 1.** Information about genome size and fasciclin-like arabinogalactan protein (FLA) gene number in the plants of interest for this study.</caption><thead><tr><th>Lineage</th><th>Organism</th><th>Genome Size (Mb)</th><th>No. of Predicted Genes</th><th>No. of FLA Genes</th><th>Reference</th></tr></thead><tbody><tr><td>Red algae</td><td>Chondrus crispus</td><td>104.98</td><td>9843</td><td>1</td><td>This study</td></tr><tr><td rowspan="2">Green algae</td><td>Chlamydomonas reinhardtii</td><td>120.405</td><td>14,488</td><td>11</td><td>This study</td></tr><tr><td>Chara braunii</td><td>1751.21</td><td>35,424</td><td>24</td><td>This study</td></tr><tr><td>Liverworts</td><td>Marchantia polymorpha</td><td>215.739</td><td>19,287</td><td>14</td><td>This study</td></tr><tr><td>Mosses</td><td>Physcomitrella patens</td><td>472.081</td><td>23,733</td><td>12</td><td>This study</td></tr><tr><td>Lycophytes</td><td>Selaginella moellendorffii</td><td>212.315</td><td>34,782</td><td>9</td><td>This study</td></tr><tr><td>Gymnosperm</td><td>Picea abies</td><td>19,600</td><td>28,354</td><td>24</td><td>This study</td></tr><tr><td rowspan="2">Amborellales</td><td>Amborella trichopoda</td><td>706.495</td><td>19,354</td><td>12</td><td>This study</td></tr><tr><td>Arabidopsis thaliana</td><td>119.148</td><td>38,093</td><td>22</td><td>Schultz et al. [21]</td></tr><tr><td rowspan="3">Eudicots</td><td>Eucalyptus grandis</td><td>691.43</td><td>45,226</td><td>19</td><td>MacMillan et al. [13]</td></tr><tr><td>Populus trichocarpa</td><td>434.29</td><td>37,197</td><td>49</td><td>Showalter et al. [24]</td></tr><tr><td>Brachypodium distachyon</td><td>218.345</td><td>34,310</td><td>23</td><td>This study</td></tr><tr><td>Monocots</td><td>Oryza sativa</td><td>374.423</td><td>33,185</td><td>26</td><td>Ma and Zhao [12]</td></tr></tbody></table>
## 2.2. Phylogenetic Analysis and Classification of FLAs

In order to understand the relationships between FLAs with different numbers of fasciclin domains, evolutionary analysis was performed based on multiple sequence alignments of FLAs. First, all the FLA protein sequences were filtered by BLAST+ [45] with a ~5 expect (E) threshold. The sequences (CreFLA2, CreFLA3, CreFLA4, CreFLA5, CreFLA6, and CreFLA7 in C. reinhardtii, CbrFLA5, CbrFLA6, CbrFLA8, CbrFLA10, CbrFLA12, CbrFLA13, CbrFLA14, CbrFLA17, CbrFLA18, and CbrFLA21 in C. braunii) with low similarity to other plant species were removed, and classified into Group F (Table S1). Next, after removing sequences of signal peptides and GPI anchor addition signals, the filtered 230 FLA sequences were aligned by Clustal Omega 1.2.2, and the HMM profile of fasciclin domains was used as a guide [46,47]. Then, the fasciclin domains could be divided into two types (Type 1 and Type 2) based alignment results (Figure 1 and Figure S1). The FLA sequences with Type 1 and Type 2 fasciclin domains were further aligned, respectively (Figures S2 and S3). Interestingly, for some algae FLA sequences that contained more than two fasciclin domains, only one or two fasciclin domains had hits in other FLA sequences: The first and the fourth fasciclin domains in CreFLA11, the second fasciclin domain in CreFLA10. It was likely that the other fasciclin domains with low similarity to those in higher plants were lost in the course of evolution from algae to land plants.

<figure><img src="image_93.png" /><figcaption>**Figure 1.** Multiple sequence alignment of representative FLA sequences. Fasciclin domains were divided into two types (Type 1 and Type 2). Residues with high similarity (80%, 60%) were highlighted in dark pink and light pink, respectively.</figcaption></figure>

The phylogenetic tree of filtered 230 FLA sequences could not be built because the identity of alignment was very low (<30%). Once the identity was above 30%, the accuracy of alignment was acceptable [48–50]. The accuracy of the FLA alignment results was tested by computing the overall mean distance with the P-distance method in Mega 7 [49,51]. As P-distance equals 1 minus
the identity of amino acids, the identities of Type 1 and Type 2 fasciclin domains were 31.7% and 30.4%, respectively. The accuracy results of Type 1 and Type 2 were 0.683 and 0.696, respectively. These indicators made it suitable for building the phylogenetic trees. The Maximum Likelihood (ML) trees for each type were built using the best models: Le_Gascuel_2008 model [52] + Gamma distribution + evolutionarily invariable (LG + G + I) for Type 1, Le_Gascuel_2008 model + Gamma distribution (LG + G) for Type 2, with 85% partial deletion by Mega 7. Bootstrap analyses with 1000 replicates were performed for support estimation. Confidence values below 50% were cut off, and confidence values higher than 70% were shown on nodes (Figures 2 and 3). Although the similarity between full-length sequences of FLAs are quite low, the fasciclin domains exhibited two highly conserved motifs (H1 and H2) and a conserved central YH motif [18]. MEME web server (available online: http://meme-suite.org/tools/meme) [53] was used to find the conserved motif (H1, H2, and YH motifs) of Type 1 and Type 2 sequences. The H1 and YH motif were similar between Type 1 and Type 2 sequences, while the H2 region was quite different. In Type 1 sequences, the H2 motif was characterized by [Gly/Ile/Val/Leu/Phe]-X-[Ile/Val/Cys]-His-Gly-[Ile/Val/Leu]-X-X-[Leu/Val/Pro/Ile]-[Leu/Met/Ile] sequence. In Type 2 sequences, the H2 motif was characterized by [Val/Ile/Met/Leu]-[Tyr/His/Phe/Gln]-X-[Val/Ile/Leu]-X-X-[Val/Leu]-Leu-[Leu/Phe/Val]-Pro sequence (X represents any amino acid) (Figures 2 and 3). Interestingly, most FLAs with single fasciclin domain was of Type 2, while only a few FLAs with single fasciclin domain was of Type 1.

Based on the sequence similarity, phylogenetic analysis, and previous study [11], we have classified FLAs into seven groups: Group A (including FLA6, FLA7, FLA 9, FLA 11-13 from *A. thaliana*), Group B (FLA 15-18 from *A. thaliana*), Group C (including FLA 1-3, FLA 5, FLA8, FLA10, FLA14 from *A. thaliana*), Group D1 (including FLA 19-22 from *A. thaliana*), Group D2 (including FLA4 from *A. thaliana*), Group E, and Group F (Table S1). Group F sequences were all algae FLAs which were not included in building phylogenetic trees. The remaining algae FLAs were all in Group D1 and Group E, which meant that Group D1 and Group E might be traced back to the origin of the FLA family in plants. Moreover, *FLA3*, *5*, *14*, *20*, *21*, and 22 were specifically expressed in anthers at different stages of floral development [18,54,55]. *FLA3* was involved in microspore development, and its knock-down plants showed reduced female fertility [56]. There was a probability that Group C and Group D1 FLAs were mainly related to male gametophyte development. Group C and Group D1 FLAs were also related to the growth regulator. For instance, *FLA1* and *FLA2* might play an important role in root development [57,58]. Interestingly, in Group A, all FLAs were with single fasciclin domain. A previous study proposed that Group A FLAs were specific to the evolution of flowering plant secondary cell wall formation and properties [30]. For example, *FLA11*, *FLA12*, and *ZeFLA11* are highly expressed in vascular tissue and double mutants of *FLA11* and *12* showed defects in secondary cell wall thickening [25,30]. *EgrFLA1*, 2, and 3 were also highly expressed in stems. *EgrFLA2* was involved in altering fiber cellulose deposition in woody tissue, and *EgrFLA3* influenced flexural strength [13]. In *Eucalyptus nitens*, *EniFLA1*, 2, and 3, which were closely related to *FLA11* and 12, as well as highly similar to *EgrFLA1* and 2, could affect stem biomechanics [30]. These Group A FLAs and their homologs in other plants (poplar, zinnia) were also involved in secondary cell wall biosynthesis [23,25]. In addition, *FLA9* in Group A was also related to seed development. It had been shown that the stress-induced reductions of *FLA9* gene expression enhanced the abortion of fertilized ovaries [59].

In addition, the variable fasciclin number of FLAs had a tight relationship to the phylogenetic tree. All the FLAs with multiple fasciclin domains (>2) were in Group D1 and Group E. As these FLAs were only identified in algae, they might be the most original FLAs in the course of evolutionary history. In Group A, all the FLAs were with single fasciclin domain and belonged to seed plants. Group A FLAs were the latest FLAs generated in the course of evolutionary history. From Group E to Group A, the number of fasciclin domains reduced over the course of evolutionary history. Except for Group A FLAs, the structures of FLAs were quite diverse, especially for Group E FLAs, which included the most
original FLAs. Moreover, Group E FLA genes contained more introns than other groups. The number of introns also reduced over the course of evolutionary history.

<figure><img src="image_94.png" /></figure>

**Figure 2.** Phylogenetic relationships between Type 1 fasciclin domains in plant species. The amino acid sequences of fasciclin domains in FLAs were aligned by Clustal Omega 1.2.2 with the guide of HMM profile of fasciclin domains, and the phylogenetic trees were built by Mega 7 using the Maximum Likelihood (ML) method with 85% partial deletion. Bootstrap analyses with 1,000 replicates were performed for support estimation. The confidence values below 50% were cut off, and the confidence values higher than 70% are shown on nodes. The tree was divided into four major clades: Group B, Group C, Group D1, and Group D2. Plant species from different lineages are shown in different shape. FLAs from *A. thaliana* are indicated for each clade. The order of fasciclin domains was designated from the N-terminus to the C-terminus (e.g., FLA4.1, FLA4.2, and so on). The conserved motifs (H1, H2, and YH motifs) shown below the tree were found using the MEME web server.
<figure><img src="image_95.png" /></figure>

**Figure 3.** Phylogenetic relationships between Type 2 fasciclin domains in plant species. The amino acid sequences of fasciclin domains in FLAs were aligned by Clustal Omega 1.2.2 with the guide of HMM profile of fasciclin domains, and the phylogenetic trees were built by Mega 7 using the Maximum Likelihood (ML) method with 85% partial deletion. Bootstrap analyses with 1000 replicates were performed for support estimation. The confidence values below 50% were cut off, and the confidence values higher than 70% are shown on nodes. The tree was divided into six major clades: Group A, Group B, Group C, Group D1, Group D2, and Group E. Plant species from different lineages are shown in different shape. FLAs from *A. thaliana* are indicated for each clade. The domain closest to the N-terminus is indicated by .1 and the second by .2. The conserved motifs (H1, H2, and YH motifs) shown below the tree were found using the MEME web server.

Moreover, to understand the relationship between FLAs with single fasciclin domain, a phylogenetic tree of FLAs with single fasciclin domain from nine plant species (*C. reinhardtii*, *C. crispus*, *M. polymorpha*, *P. patens*, *S. moellendorffii*, *P. abies*, *A. trichopoda*, *B. distachyon*, and *A. thaliana*) was built by the Maximum Likelihood (ML) method under the LG + G model with 85% partial deletion. Bootstrap analyses with 1000 replicates were performed for support estimation; confidence values higher than 50% were shown on nodes. The structure displays of these FLAs were generated by GSDS 2.0 (available online: http://gsds.cbi.pku.edu.cn/) [44] (Figure 4). The structure of Group A FLA genes was very similar. Except for *PabFLA12*, *PabFLA14*, and *AtrFLA6*, the remaining Group A FLA genes did not contain introns, and most of their fasciclin domains were flanked by two AGP regions. The structures of FLAs with single fasciclin domains in Group E were quite diverse. By contrast, the phylogenetic relationship of FLAs with single fasciclin domain was similar to the phylogenetic relationships of Type 2 (Figure 3). The main type of fasciclin domain in these FLAs was Type 2 fasciclin domain. Most of Group D1 FLAs contained Type 1 fasciclin domains. It is likely that the Type 1 fasciclin domain was lost mainly in FLAs with single fasciclin domain over the course of evolutionary history. Different from phylogenetic relationships of Type 1 and Type 2 fasciclin domains (Figures 2 and 3), Group C appeared to be divergent (Figure 4). Some Group C FLAs were close to Group D2, while others were close to Group B. Moreover,
the structure of these diverged Group C was different. The fasciclin domains of FLAs tailed with AGP regions belonged to Group C, which were close to Group B. For FLAs from Group C which was close to Group D2, their fasciclin domains were covered by two AGP regions.

<figure><img src="image_96.png" /></figure>

**Figure 4.** Phylogenetic relationships and structure display of FLAs with single fasciclin domain in nine plant species (C. reinhardtii, C. crispus, M. polymorpha, P. patens, S. moellendorffii, P. abies, A. trichopoda, B. distachyon, and A. thaliana). Plant species from different lineages are shown in different shapes. The phylogenetic trees were built by Mega 7 using the Maximum Likelihood (ML) method under LG+G model with 85% partial deletion. Bootstrap analyses with 1000 replicates were performed for support estimation, the confidence values higher than 50% are shown on nodes. The tree was divided into six groups according to the classifications based on two types fasciclin domains (Figures 2 and 3): Group A, Group B, Group C, Group D1, Group D2 and Group E. The structure displays were generated by GSDS 2.0. Black lines represent introns, gray rectangles the CDS regions, red rectangles the AGP regions, blue rectangles the fasciclin domains, green rectangles signal peptides, and yellow wedges GPI-anchor modification sites. The framed FLAs denote functionally characterized FLAs (FLA3, FLA9, FLA11, and FLA12).
2.3. *Structural and Evolutionary Analysis of FLAs*

The amino acid sequences of 246 FLAs identified in our work were shown in Figure S4. One hundred seventy-six of them contained a single fasciclin domain, and 66 of them contained two fasciclin domains. Only four FLAs with more than two fasciclin domains were found in algae, one in red algae and three in green algae. Moreover, FLAs with a single fasciclin domain, as well as with two domains first appeared in green algae (Figure 5). It was likely that divergence happened in green algae. From green algae to land plants, the number of fasciclin domains in FLAs was reduced. It had been proven that FLAs with a single fasciclin domain had conserved roles in secondary cell wall biology and properties [13]. Besides, there was an example of the functional roles of different fasciclin domains in one FLA protein. The C-proximal fasciclin domain of FLA4 was responsible for its genetic functions, while the N-proximal fasciclin domain was required for stabilization of plasma membrane localization [60,61]. It was likely that the number of fasciclin domains was related to the functions of FLAs.

FLAs were classified into seven groups based on the sequence similarity, phylogenetic analysis, and previous study [11]. Different from the previous study [11], Group D was divided into Group D1 and Group D2 because of their difference in phylogenetic analysis. Moreover, Group E and Group F present in non-seed plants are the groups newly proposed in this work. The evolutionary history of FLA family was shown in Figure 5. FLAs evolved very early during plant evolution. Group E first appeared in the plant kingdom, then Group F, Group D1, Group C, Group D2, Group B, Group A appeared successively. The Group E FLA from red algae was the most original FLA. Group F was largely dissimilar to the other groups and only existed in green algae. Group D1 and Group C evolved early during green plant evolution. The divergence of FLAs occurred in green algae; Group D1 and Group C remained, while Group F was lost after the separation between green algae and land plants. Group B and Group D2 evolved after plants conquered the land. Group A, the latest group appeared, evolved during seed plant evolution. By contrast, Group E, the earliest appeared group, was lost in seed plants.

<figure><img src="image_97.png" /><figcaption>**Figure 5.** Evolutionary model of the FLA family in plants. The green letters display the appearance of different groups of FLAs. The red letters display the disappearance of Group E and Group F FLAs. The cubes display the number of fasciclin domains in FLAs.</figcaption></figure>
## 2.4. Analysis of *FLA* Duplication Patterns during the Process of Evolution

The evolution of genomes and genetic systems is mainly driven by gene duplications [62]. The three elementary gene expansion patterns are tandem duplication, segmental duplication, and transposition events [63,64]. In the plant kingdom, tandem duplication and segmental duplication are the main processes of gene family expansion compared with transposition events [65,66]. We investigated these two duplication events to understand the *FLA* genes' expansion patterns in the plant kingdom. The paralogous genes that exist in the same chromosome within a 50 kb physical distance are examples of tandem duplication [65]. First, in order to find the chromosomal locations, the annotation information for the *FLA* genes was downloaded from OrcAE (available online: https://bioinformatics.psb.ugent.be/orcae/overview/Chbra), Phytozome (available online: https://phytozome.jgi.doe.gov/pz/portal.html) and ConGenIE (available online: http://congenie.org/). Then, the distances between *FLA* genes' locations were compared in the same chromosome. The locus search tool on PGDD (available online: http://chibba.agtec.uga.edu/duplication/index/locus) and MCSCAN were used to find the segmental duplications (Table S2). The duplications in *FLA* genes were related to whole-genome duplication events (Figure 6). The higher plants exhibited more duplications than lower plants. *P. trichocarpa* had the highest number of duplicated *FLA* genes, which made it have more *FLA* genes than other plant species. Although most duplicated pairs shared the same structure type, some duplicated genes had different structure types. For example, in *C. reinhardtii*, *Cre16.g687742* containing two fasciclin domains and *Cre16.g687854* containing single fasciclin domain most probably result from tandem duplication. It seemed that some *FLA* genes with single fasciclin domain evolved from *FLA* genes with two fasciclin domains. FLAs with single fasciclin domain evolved from FLAs with multiple fasciclin domains, and the number of fasciclin domains was reduced in evolutionary history.

<figure><img src="image_98.png" /><figcaption>**Figure 6.** Duplication events of *FLA* genes in the plant kingdom. The phylogenetic tree on the left was built based on the Tree of Life Web project (available online: http://www.tolweb.org/Green_plants) and whole-genome duplication events in PGDD (available online: http://chibba.pgml.uga.edu/duplication/index/home). The number next to the tree is the number of *FLA* genes resulting from segmental duplication, tandem duplication, and total *FLA* genes in the species. Seg: Segmental duplication (pairs); Tan: Tandem duplication (pairs); Total: Total number of *FLA* genes in the species.</figcaption></figure>

In order to understand the evolution processes of the *FLA* gene family in the plant kingdom, duplicated gene pairs among *FLAs* were used to estimate the molecular evolutionary rates by calculating their Ka/Ks value (Table S2). The Ka/Ks values of all the duplicated gene pairs except the *Mapoly0075s0013.1/Mapoly0075s0013.2* gene pair were lower than 1. It was assumed that *FLA* duplicated gene pairs evolved under purifying selection, indicating that the functions of the *FLAs* gene family were crucial to plant development and functional mutations in *FLA* genes might have negative impacts on plants. The Ka/Ks ratio of *Mapoly0075s0013.1/Mapoly0075s0013.2* gene pair was 2.3512, showing that this gene pair underwent positive selection pressure during evolution. However, plants could not escape from their environment in order to adapt to changes, so positive selection,
which could lead to beneficial functional changes, was also important during plant evolution [67]. The *Mapoly0075s0013.1/Mapoly0075s0013.2* gene pair, which was found to experience positive selection, might have improved the adaptation of the plant to new environments.

# 3. Materials and Methods

## 3.1. Bioinformatics Identification of FLAs

Multiple searches were carried out in order to identify FLA genes in 13 plant species (*C. crispus*, *C. reinhardtii*, *C. crispus*, *M. polymorpha*, *P. patens*, *S. moellendorffii*, *P. abies*, *A. trichopoda*, *B. distachyon*, *O. sativa*, *A. thaliana*, *E. grandis*, and *P. trichocarpa*) [31–43]. The predicted proteomes of *C. crispus* was downloaded from NCBI, that of *C. braunii* were from the OrcAE website (available online: https://bioinformatics.psb.ugent.be/orcae/overview/Chbra), that of *P. abies* were from the ConGenIE website (available online: http://congenie.org/), and that of other species from the Phytozome website (Version 12; available online: https://phytozome.jgi.doe.gov/pz/portal.html). Except for *P. abies* [35], the statistics of genome size overall number of predicted genes were from the NCBI Genome database (available online: https://www.ncbi.nlm.nih.gov/genome).

Then, the Hidden Markov Model (HMM) profile built for fasciclin domains was downloaded from Pfam (available online: http://pfam.xfam.org/family/PF02469) [68], and HMMER 3.0 [69] was used to search proteins with fasciclin domains from the selected plants. Then the presence of fasciclin domains corresponding to the obtained proteins was examined by the NCBI conserved domain database (available online: http://www.ncbi.nlm.nih.gov/cdd). Next, the Finding-AGP program [7] was used to identify AGP regions from proteins with fasciclin domains. Finally, proteins with both fasciclin domains and AGP regions were identified as FLAs. Also, the omitted FLA sequences that were identified in former studies (AT5G40940, AT5G06920, Eucgr.A01741, Potri.013G152200, Potri.001G440800, and Potri.005G079500) were used as queries to perform BLAST searches with a –3 expect (E) threshold to find FLAs that could not be identified by HMMER 3.0.

Moreover, most FLAs have a predicted signal peptide and GPI-anchor. Therefore, SignalP 4.1 Server (available online: http://www.cbs.dtu.dk/services/SignalP/) was used to predict signal peptides [70] and big-PI Plant Predictor (available online: http://mendel.imp.ac.at/gpi/plant_server.html) was used to predict GPI modification sites [71]. The intron of red algae *FLA* was detected by the GSDS website (available online: http://gsds.cbi.pku.edu.cn/) [44], and the intron of other *FLAs* were found from the OrcAE website (available online: https://bioinformatics.psb.ugent.be/orcae/overview/Chbra), the Phytozome website (Version 12; available online: https://phytozome.jgi.doe.gov/pz/portal.html), and the ConGenIE website (available online: http://congenie.org/). The amino acid sequences and the presence of AGP regions, signal peptides, fasciclin domains, and GPI-anchor signals are given in Table S1.

## 3.2. Multiple Sequence Alignment and Phylogenetic Analysis

All of the *FLA* protein sequences were searched against each other by BLAST+ with a –5 expect (E) threshold [45]. The sequences with low similarity were removed. Then, signal peptides and GPI modification sites were removed from filtered *FLA* sequences. These sequences were aligned by Clustal Omega 1.2.2 with HMM of the fasciclin domain as a guide in the alignment [46,47]. The fasciclin domains were designated as Type 1 and Type 2 and were also aligned by Clustal Omega 1.2.2 with the HMM of the fasciclin domain as a guide in the alignment [46,47]. GeneDoc [72] was used to display multiple sequence alignments.

The reliability of alignment results was tested by computing overall mean distance with the P-distance method by Mega 7 [49,51]. The alignments of Type 1, Type 2, and FLAs with a single fasciclin domain was then used to build phylogenetic trees with the Maximum Likelihood (ML) method. The best models for ML trees were found by Mega 7 [51,73]. Then, ML trees were built under the best
model with 85% partial deletion by Mega 7. Bootstrap analyses with 1,000 replicates were performed for support estimation [51,52].

### 3.3. Motif Prediction

In order to identify the conserved domains and motifs of Type 1 and Type 2 fasciclin domains, MEME web server (available online: http://meme-suite.org/tools/meme) [53] was used to identify the conserved motifs (H1 and H2 regions, YH motif). The following parameters were used when running the MEME: (1) The motif sites in sequences were distributed by 0 or 1 occurrence per sequence; (2) the maximum of motifs was set to be 10 for the H1 and H2 regions, and 3 for the YH motif; and (3) a 0-order model of sequences was used as the background model.

### 3.4. Gene Duplication and Molecular Evolution

The annotation information of the *FLA* genes on the phytozome website (available online: https://phytozome.jgi.doe.gov/pz/portal.html), the OrcAE website (available online: https://bioinformatics.psb.ugent.be/orcae/overview/Chbra), and the ConGenIE website (available online: http://congenie.org/) was used to find the chromosomal locations. The paralogous genes that exit in the same chromosome within a 50-kb physical distance was defined as tandem duplication [64]. The segmental duplications of 10 plants (C. reinhardtii, P. patens, S. moellendorffii, P. abies, A. trichopoda, B. distachyon, O. sativa, A. thaliana, E. grandis, and P. trichocarpa) were found by the PGDD locus search tool (available online: http://chibba.agtec.uga.edu/duplication/index/locus). Because *M. polymorpha* and *C. crispus* data were absent in PGDD, Multiple Collinearity Scan (MCSCAN) [74–77] was used to find the segmental duplications in *M. polymorpha*.

To calculate the molecular evolutionary rates between *FLAs* duplicated gene pairs, pairwise alignment was performed among these gene pairs by ClustalW (codons) in MEGA7 [51]. Then, the MYN (Modified YN) model in KaKs_Calculator 2.0 was used to estimate the nonsynonymous substitution rate (Ka), the synonymous substitution rate (Ks) and the Ka/Ks value of these duplicated gene pairs [78].

## 4. Conclusions

FLAs play an important role in plant development and adaption to the environment. Two hundred forty-six *FLA* genes in 13 plant species were identified in this study. It was found that FLAs first appeared in algae. Based on the sequence similarity and phylogenetic analysis, FLAs could be classified into seven groups: Group A, Group B, Group C, Group D1, Group D2, Group E, and Group F. Group E FLAs were the earliest to appear in evolutionary history and disappeared in seed plants, while Group A FLAs were the latest and only existed in seed plants. FLAs with multiple fasciclin domain (>2) were possibly the first *FLA* type to appear in Archaeplastida because they only existed in algae. FLAs with single fasciclin domain and with two fasciclin domains were dominant in green plants. The number of fasciclin domains in FLAs varied in green algae and was reduced to one or two in land plants. In addition, introns in *FLA* genes were lost during plant evolution, especially from green algae to land plants. Moreover, tandem and segmental duplications contributed to the expansion of the *FLA* gene family, and duplicated gene pairs in *FLAs* mainly evolved under purifying selection.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/8/1945/s1.

**Author Contributions:** H.M. conceived of and designed the research plans; J.H. and H.Z. performed most of the experiments and analyzed the data; Z.C., Y.K., and J.L. provided technical assistance to J.H. and H.Z.; J.H. and H.Z. wrote the article with contributions from all the authors; H.M. supervised and supported the writing.

**Acknowledgments:** This research was supported by the National Natural Science Foundation of China (31500212), the Natural Science Foundation of Shaanxi Province (2015JQ3090), and the Undergraduate Innovation Foundation of Northwest A&F University (No. 1201710712099).

**Conflicts of Interest:** The authors declare no conflict of interest.
Abbreviations

<table><tr><td>FLA</td><td>Fasciclin-like arabinogalactan protein</td></tr><tr><td>AGP</td><td>Arabinogalactan protein</td></tr><tr><td>GPI</td><td>Glycosylphosphatidylinositol</td></tr><tr><td>PAST%</td><td>The percentage of Pro, Ala, Ser, and Thr residues in a protein amino-acid sequence</td></tr><tr><td>Ccr</td><td>Chondrus crispus</td></tr><tr><td>Cre</td><td>Chlamydomonas reinhardtii</td></tr><tr><td>Mpo</td><td>Marchantia polymorpha</td></tr><tr><td>Smo</td><td>Selaginella moellendorffii</td></tr><tr><td>Pab</td><td>Picea abies</td></tr><tr><td>Atr</td><td>Amborella trichopoda</td></tr><tr><td>Egr</td><td>Eucalyptus grandis</td></tr><tr><td>Pt</td><td>Populus trichocarpa</td></tr><tr><td>Bdi</td><td>Brachypodium distachyon</td></tr><tr><td>Os</td><td>Oryza sativa</td></tr><tr><td>Ka</td><td>Nonsynonymous substitution rate</td></tr><tr><td>Ks</td><td>Synonymous substitution rate</td></tr><tr><td>PGDD</td><td>Plant Genome Duplication Database</td></tr><tr><td>NCBI</td><td>National Center for Biotechnology Information</td></tr><tr><td>ConGenIE</td><td>Conifer Genome Integrative Explorer</td></tr><tr><td>HMM</td><td>Hidden Markov Model</td></tr><tr><td>BLASTP</td><td>Protein Basic Local Alignment Search Tool</td></tr><tr><td>MYN</td><td>Modified Yang-Nielsen Algorithm</td></tr><tr><td>MCSCAN</td><td>Multiple Collinearity Scan</td></tr><tr><td>MEME</td><td>Multiple Expectation maximization for Motif Elicitation</td></tr><tr><td>LG</td><td>Le_Gascuel_2008 model</td></tr><tr><td>G</td><td>Gamma distribution</td></tr><tr><td>I</td><td>Evolutionarily invariable</td></tr><tr><td>GSDS</td><td>Gene Structure Display Server</td></tr><tr><td>OrcAE</td><td>Online Resource for Community Annotation of Eukaryotes</td></tr></table>

References

1. Nothnagel, E.A. Proteoglycans and related components in plant cells. *Int. Rev. Cytol.* **1997**, *174*, 195–291. [PubMed]

2. Chivasa, S.; Ndimba, B.K.; Simon, W.J.; Robertson, D.; Yu, X.L.; Knox, J.P.; Bolwell, P.; Slabas, A.R. Proteomic analysis of the *Arabidopsis thaliana* cell wall. *Electrophoresis* **2002**, *23*, 1754–1765. [CrossRef]

3. Johnson, K.L.; Cassin, A.M.; Lonsdale, A.; Wong, G.K.-S.; Soltis, D.E.; Miles, N.W.; Melkonian, M.; Melkonian, B.; Deyholos, M.K.; Leebens-Mack, J.; et al. Insights into the Evolution of Hydroxyproline-rich Glycoproteins from 1000 plant Transcriptomes. *Plant Physiol.* **2017**, *174*, 904–921. [CrossRef]

4. Jamet, E.; Albenne, C.; Boudart, G.; Irshad, M.; Canut, H.; Pont-Lezica, R. Recent advances in plant cell wall proteomics. *Proteomics* **2008**, *8*, 893–908. [CrossRef] [PubMed]

5. Nothnagel, E.A.; Bacic, A.; Clarke, A.E. *Cell and Developmental Biology of Arabinogalactan-Proteins*; Springer Science Business Media: New York, NY, USA, 2000.

6. Showalter, A.M. Arabinogalactan-proteins: Structure, expression and function. *Cell. Mol. Life Sci.* **2001**, *58*, 1399–1417. [CrossRef]

7. Ma, Y.; Yan, C.; Li, H.; Wu, W.; Liu, Y.; Wang, Y.; Chen, Q.; Ma, H. Bioinformatics Prediction and Evolution Analysis of Arabinogalactan Proteins in the Plant Kingdom. *Front. Plant Sci.* **2017**, *8*, 66. [CrossRef]

8. Ellis, M.; Egelund, J.; Schultz, C.J.; Bacic, A. Arabinogalactan-proteins: Key Regulators at the Cell Surface? *Plant Physiol.* **2010**, *153*, 403–419. [CrossRef]

9. Shpak, E.; Barbar, E.; Leykam, J.F.; Kieliszewski, M.J. Contiguous Hydroxyproline Residues Direct Hydroxyproline Arabinosylation in *Nicotiana tabacum*. *J. Biol. Chem.* **2001**, *276*, 11272–11278. [CrossRef] [PubMed]
10. Showalter, A.M.; Basu, D. Extensin and Arabinogalactan-Protein Biosynthesis: Glycosyltransferases, Research Challenges, and Biosensors. *Front. Plant Sci* **2016**, *7*, 814. [CrossRef] [PubMed]

11. Johnson, K.L.; Jones, B.J.; Bacic, A.; Schultz, C.J. The fasciclin-like arabinogalactan proteins of Arabidopsis. *A multigene family of putative cell adhesion molecules*. *Plant Physiol.* **2003**, *133*, 1911–1925. [CrossRef] [PubMed]

12. Ma, H.; Zhao, J. Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (*Oryza sativa* L.). *J. Exp. Bot.* **2010**, *61*, 2647–2668. [CrossRef]

13. MacMillan, C.P.; Taylor, L.; Bi, Y.; Southerton, S.G.; Evans, R.; Spokevicius, A. The fasciclin-like arabinogalactan protein family of *Eucalyptus grandis* contains members that impact wood biology and biomechanics. *New Phytol.* **2015**, *206*, 1314–1327. [CrossRef] [PubMed]

14. Mashiguchi, K.; Asami, T.; Suzuki, Y. Genome-wide identification, structure and expression studies, and mutant collection of 22 early nodulin-like protein genes in Arabidopsis. *Biosci. Biotechnol. Biochem.* **2009**, *73*, 2452–2459. [CrossRef]

15. Ma, H.; Zhao, H.; Liu, Z.; Zhao, J. The phytocyanin gene family in rice (*Oryza sativa* L.): genome-wide identification, classification and transcriptional analysis. *PLoS ONE* **2011**, *6*, e25184. [CrossRef]

16. Motose, H.; Sugiyama, M.; Fukuda, H. A proteoglycan mediates inductive interaction during plant vascular development. *Nature* **2004**, *429*, 873–878. [CrossRef] [PubMed]

17. Kobayashi, Y.; Motose, H.; Iwamoto, K.; Fukuda, H. Expression and genome-wide analysis of the xylogen-type gene family. *Plant Cell Physiol.* **2011**, *52*, 1095–1106. [CrossRef] [PubMed]

18. Seifert, G.J. Fascinating Fasciclin: A Surprisingly Widespread Family of Proteins that Mediate Interactions between the Cell Exterior and the Cell Surface. *Int. J. Mol. Sci.* **2018**, *19*, 1628. [CrossRef]

19. Bastiani, M.J.; Harrelson, A.L.; Snow, P.M.; Goodman, C.S. Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. *Cell* **1987**, *48*, 745–755. [CrossRef]

20. Elkins, T.; Zinn, K.; McAllister, L.; Hoffmann, F.M.; Goodman, C.S. Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations. *Cell* **1990**, *60*, 565–575. [CrossRef]

21. Schultz, C.J.; Rumsewicz, M.P.; Johnson, K.L.; Jones, B.J.; Gaspar, Y.M.; Bacic, A. Using Genomic Resources to Guide Research Directions. The Arabinogalactan Protein Gene Family as a Test Case. *Plant Physiol.* **2002**, *129*, 1448–1463. [CrossRef]

22. Faik, A.; Abouzouhair, J.; Sarhan, F. Putative fasciclin-like arabinogalactan-proteins (FLA) in wheat (*Triticum aestivum*) and rice (*Oryza sativa*): identification and bioinformatic analyses. *Mol. Genet. Genom.* **2006**, *276*, 478–494. [CrossRef]

23. Lafarguette, F.; Leplé, J.C.; Déjardin, A.; Laurans, F.; Costa, G.; Lesage-Descauses, M.C.; Pilate, G. Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood. *New Phyto.* **2004**, *164*, 107–121. [CrossRef]

24. Showalter, A.M.; Keppler, B.D.; Liu, X.; Lichtenberg, J.; Welch, L.R. Bioinformatic Identification and Analysis of Hydroxyproline-Rich Glycoproteins in *Populus trichocarpa*. *Bmc Plant Biol.* **2016**, *16*, 229. [CrossRef] [PubMed]

25. Dahiya, P.; Findlay, K.; Roberts, K.; McCann, M.C. A fasciclin-domain containing gene, ZeFLA11, is expressed exclusively in xylem elements that have reticulate wall thickenings in the stem vascular system of *Zinnia elegans* cv Envy. *Planta* **2006**, *223*, 1281–1291. [CrossRef] [PubMed]

26. Huang, G.Q.; Xu, W.L.; Gong, S.Y.; Li, B.; Wang, X.L.; Xu, D.; Li, X.B. Characterization of 19 novel cotton *FLA* genes and their expression profiling in fiber development and in response to phytohormones and salt stress. *Physiol. Plant* **2008**, *134*, 348–359. [CrossRef] [PubMed]

27. Liu, H.; Shi, R.; Wang, X.; Pan, Y.; Li, Z.; Yang, X.; Zhang, G.; Ma, Z. Characterization and expression analysis of a fiber differentially expressed Fasciclin-like arabinogalactan protein gene in sea island cotton fibers. *PLoS ONE* **2013**, *8*, e70185. [CrossRef]

28. Jun, L.; Xiaoming, W. Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (*Brassica rapa* L.). *Mol. Biol. Rep.* **2012**, *39*, 10541–10555. [CrossRef]
29. Guerriero, G.; Mangeot-Peter, L.; Legay, S.; Behr, M.; Lutts, S.; Siddiqui, K.S.; Hausman, J.F. Identification of fasciclin-like arabinogalactan proteins in textile hemp (*Cannabis sativa* L.): in silico analyses and gene expression patterns in different tissues. *Bmc Genom.* **2017**, *18*, 741. [CrossRef]

30. MacMillan, C.P.; Mansfield, S.D.; Stachurski, Z.H.; Evans, R.; Southerton, S.G. Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in Arabidopsis and *Eucalyptus*. *Plant J.* **2010**, *62*, 689–703. [CrossRef] [PubMed]

31. Collen, J.; Porcel, B.; Carre, W.; Ball, S.G.; Chaparro, C.; Tonon, T.; Barbeyron, T.; Michel, G.; Noel, B.; Valentin, K.; et al. Genome structure and metabolic features in the red seaweed *Chondrus crispus* shed light on evolution of the Archaeplastida. *Proc. Natl. Acad. Sci. USA* **2013**, *110*, 5247–5252. [CrossRef] [PubMed]

32. Bowman, J.L.; Kohchi, T.; Yamato, K.T.; Jenkins, J.; Shu, S.; Ishizaki, K.; Yamaoka, S.; Nishihama, R.; Nakamura, Y.; Berger, F.; et al. Insights into Land Plant Evolution Garnered from the *Marchantia polymorpha* Genome. *Cell* **2017**, *171*, 287–304. [CrossRef] [PubMed]

33. Lang, D.; Ullrich, K.K.; Murat, F.; Fuchs, J.; Jenkins, J.; Haas, F.B.; Piednoel, M.; Gundlach, H.; Van Bel, M.; Meyberg, R.; et al. The *Physcomitrella patens* chromosome-scale assembly reveals moss genome structure and evolution. *Plant J.* **2018**, *93*, 515–533. [CrossRef] [PubMed]

34. Banks, J.A.; Nishiyama, T.; Hasebe, M.; Bowman, J.L.; Gribskov, M.; dePamphilis, C.; Albert, V.A.; Aono, N.; Aoyama, T.; Ambrose, B.A.; et al. The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants. *Science* **2011**, *332*, 960–963. [CrossRef] [PubMed]

35. Nystedt, B.; Street, N.R.; Wetterbom, A.; Zuccolo, A.; Lin, Y.-C.; Scofield, D.G.; Vezzi, F.; Delhomme, N.; Giacomello, S.; Alexeyenko, A.; et al. The Norway spruce genome sequence and conifer genome evolution. *Nature* **2013**, *497*, 579–584. [CrossRef] [PubMed]

36. *Ambrella* Genome Project. The *Ambrella* Genome and the Evolution of Flowering Plants. *Science* **2013**, *342*, 1241089. [CrossRef] [PubMed]

37. The International Brachypodium Initiative. Genome sequencing and analysis of the model grass *Brachypodium distachyon*. *Nature* **2010**, *463*, 763–768. [CrossRef]

38. Ouyang, S.; Zhu, W.; Hamilton, J.; Lin, H.; Campbell, M.; Childs, K.; Thibaud-Nissen, F.; Malek, R.L.; Lee, Y.; Zheng, L.; et al. The TIGR rice Genome Annotation Resource: improvements and new features. *Nucleic Acids Res.* **2007**, *35*, D883–D887. [CrossRef]

39. Merchant, S.S.; Prochnik, S.E.; Vallon, O.; Harris, E.H.; Karpowicz, S.J.; Witman, G.B.; Terry, A.; Salamov, A.; Fritz-Laylin, L.K.; Marechal-Drouard, L.; et al. The *Chlamydomonas* Genome Reveals the Evolution of Key Animal and Plant Functions. *Science* **2007**, *318*, 245–250. [CrossRef] [PubMed]

40. Lamesch, P.; Berardini, T.Z.; Li, D.; Swarbreck, D.; Wilks, C.; Sasidharan, R.; Muller, R.; Dreher, K.; Alexander, D.L.; Garcia-Hernandez, M.; et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. *Nucleic Acids Res.* **2012**, *40*, D1202–D1210. [CrossRef]

41. Myburg, A.A.; Grattapaglia, D.; Tuskan, G.A.; Hellsten, U.; Hayes, R.D.; Grimwood, J.; Jenkins, J.; Lindquist, E.; Tice, H.; Bauer, D.; et al. The genome of *Eucalyptus grandis*. *Nature* **2014**, *510*, 356–362. [CrossRef]

42. Tuskan, G.A.; Difazio, S.; Jansson, S.; Bohlmann, J.; Grigoriev, I.; Hellsten, U.; Putnam, N.; Ralph, S.; Rombauts, S.; Salamov, A.; et al. The Genome of Black Cottonwood, *Populus trichocarpa* (Torr. & Gray). *Science* **2006**, *313*, 1596–1604.

43. Nishiyama, T.; Sakayama, H.; de Vries, J.; Buschmann, H.; Saint-Marcoux, D.; Ullrich, K.K.; Haas, F.B.; Vanderstraeten, L.; Becker, D.; Lang, D.; et al. The *Chara* Genome: Secondary Complexity and Implications for Plant Terrestrialization. *Cell* **2018**, *174*, 448–464. [CrossRef]

44. Hu, B.; Jin, J.; Guo, A.-Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: an upgraded gene feature visualization server. *Bioinformatics* **2015**, *31*, 1296–1297. [CrossRef]

45. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: architecture and applications. *Bmc Bioinform.* **2009**, *10*, 421. [CrossRef] [PubMed]

46. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Soding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. *Mol. Syst. Biol.* **2011**, *7*, 539. [CrossRef]

47. Sievers, F.; Higgins, D.G. Clustal Omega for making accurate alignments of many protein sequences. *Protein Sci.* **2018**, *27*, 135–145. [CrossRef]

48. Thompson, J.D.; Plewniak, F.; Poch, O. A comprehensive comparison of multiple sequence alignment programs. *Nucleic Acids Res.* **1999**, *27*, 2682–2690. [CrossRef] [PubMed]
49. Hall, B.G. *Phylogenetic Trees Made Easy: A How-To Manual*, 4th ed.; Oxford University Press: Cary, NC, USA, 2011.

50. Ogden, T.H.; Rosenberg, M.S. Multiple Sequence Alignment Accuracy and Phylogenetic Inference. *Syst. Biol.* **2006**, *55*, 314–328. [CrossRef] [PubMed]

51. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. *Mol. Biol. Evol.* **2016**, *33*, 1870–1874. [CrossRef]

52. Le, S.Q.; Gascuel, O. An Improved General Amino Acid Replacement Matrix. *Mol. Biol. Evol.* **2008**, *25*, 1307–1320. [CrossRef]

53. Bailey, T.L.; Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. *Proc. Int. Conf. Intell. Syst. Mol. Biol.* **1994**, *2*, 28–36. [PubMed]

54. Klepikova, A.V.; Logacheva, M.D.; Dmitriev, S.E.; Penin, A.A. RNA-seq analysis of an apical meristem time series reveals a critical point in *Arabidopsis thaliana* flower initiation. *Bmc Genom.* **2015**, *16*, 466. [CrossRef] [PubMed]

55. Klepikova, A.V.; Kasianov, A.S.; Gerasimov, E.S.; Logacheva, M.D.; Penin, A.A. A high resolution map of the *Arabidopsis thaliana* developmental transcriptome based on RNA-seq profiling. *Plant J.* **2016**, *88*, 1058–1070. [CrossRef]

56. Li, J.; Yu, M.; Geng, L.L.; Zhao, J. The fasciclin-like arabinogalactan protein gene, *FLA3*, is involved in microspore development of Arabidopsis. *Plant J.* **2010**, *64*, 482–497. [CrossRef] [PubMed]

57. Basu, D.; Tian, L.; Debrosse, T.; Poirier, E.; Emch, K.; Herock, H.; Travers, A.; Showalter, A.M. Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence Via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis. *PLoS ONE* **2016**, *11*, e0145092. [CrossRef]

58. Johnson, K.L.; Kibble, N.A.; Bacic, A.; Schultz, C.J. A Fasciclin-Like Arabinogalactan-Protein (FLA) Mutant of *Arabidopsis thaliana*, *FLA1*, Shows Defects in Shoot Regeneration. *PLoS ONE* **2011**, *6*, e25154. [CrossRef]

59. Cagnola, J.I.; Dumont de Chassart, G.J.; Ibarra, S.E.; Chimenti, C.; Ricardi, M.M.; Delzer, B.; Ghiglione, H.; Zhu, T.; Otegui, M.E.; Estevez, J.M.; et al. Reduced expression of selected *FASCICLIN-LIKE ARABINOGALACTAN PROTEIN* genes associates with the abortion of kernels in field crops of *Zea mays* (maize) and of Arabidopsis seeds. *Plant Cell Env.* **2018**, *41*, 661–674. [CrossRef] [PubMed]

60. Turupcu, A.; Almohamed, W.; Oostenbrink, C.; Seifert, G.J. A speculation on the tandem fasciclin 1 repeat of *FLA4* proteins in angiosperms. *Plant Signal. Behav.* **2018**, *13*, e1507403. [CrossRef]

61. Xue, H.; Veit, C.; Abas, L.; Tryfona, T.; Maresch, D.; Ricardi, M.M.; Estevez, J.M.; Strasser, R.; Seifert, G.J. *Arabidopsis thaliana* *FLA4* functions as a glycan-stabilized soluble factor via its carboxy-proximal fasciclin 1 domain. *Plant J.* **2017**, *91*, 613–630. [CrossRef]

62. Moore, R.C.; Purugganan, M.D. The early stages of duplicate gene evolution. *Proc. Natl. Acad. Sci. USA* **2003**, *100*, 15682–15687. [CrossRef] [PubMed]

63. Cao, J.; Li, X. Identification and phylogenetic analysis of late embryogenesis abundant proteins family in tomato (*Solanum lycopersicum*). *Planta* **2015**, *241*, 757–772. [CrossRef]

64. Kong, H.; Landherr, L.L.; Frohlich, M.W.; Leebens-Mack, J.; Ma, H.; dePamphilis, C.W. Patterns of gene duplication in the plant *SKP1* gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth. *Plant J.* **2007**, *50*, 873–885. [CrossRef] [PubMed]

65. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in *Arabidopsis thaliana*. *Bmc Plant Biol.* **2004**, *4*, 10. [CrossRef] [PubMed]

66. Zhu, Y.; Wu, N.; Song, W.; Yin, G.; Qin, Y.; Yan, Y.; Hu, Y. Soybean (*Glycine max*) expansin gene superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies. *Bmc Plant Biol.* **2014**, *14*, 93. [CrossRef] [PubMed]

67. Cao, J.; Li, X.; Lv, Y.; Ding, L. Comparative analysis of the phytocyanin gene family in 10 plant species: A focus on *Zea mays*. *Front. Plant Sci.* **2015**, *6*, 515. [CrossRef] [PubMed]

68. Finn, R.D.; Coggill, P.; Eberhardt, R.Y.; Eddy, S.R.; Mistry, J.; Mitchell, A.L.; Potter, S.C.; Punta, M.; Qureshi, M.; Sangrador-Vegas, A.; et al. The Pfam protein families database: towards a more sustainable future. *Nucleic Acids Res.* **2016**, *44*, D279–D285. [CrossRef]

69. Eddy, S.R. Accelerated profile HMM searches. *Plos Comput. Biol.* **2011**, *7*, e1002195. [CrossRef]
70. Petersen, T.N.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. *Nat. Methods* **2011**, *8*, 785–786. [CrossRef] [PubMed]

71. Eisenhaber, B.; Wildpaner, M.; Schultz, C.J.; Borner, G.H.; Dupree, P.; Eisenhaber, F. Glycosylphosphatidylinositol Lipid Anchoring of Plant Proteins. Sensitive Prediction from Sequence- and Genome-wide Studies for Arabidopsis and Rice. *Plant Physiol.* **2003**, *133*, 1691–1701. [CrossRef] [PubMed]

72. Nicholas, K.B.; Nicholas, H.B., Jr.; Deerfield, D.W., II. GeneDoc: Analysis and visualization of genetic variation. *Embnew* **1997**, *4*, 1–4.

73. Nei, M.; Kumar, S. *Molecular Evolution and Phylogenetics*; Oxford University Press: New York, NY, USA, 2000.

74. Lee, T.H.; Tang, H.; Wang, X.; Paterson, A.H. PGDD: a database of gene and genome duplication in plants. *Nucleic Acids Res.* **2013**, *41*, D1152–D1158. [CrossRef]

75. Tang, H.; Bowers, J.E.; Wang, X.; Ming, R.; Alam, M.; Paterson, A.H. Synteny and Collinearity in Plant Genomes. *Science* **2008**, *320*, 486–488. [CrossRef] [PubMed]

76. Tang, H.; Wang, X.; Bowers, J.E.; Ming, R.; Alam, M.; Paterson, A.H. Unraveling ancient hexaploidy through multiply-aligned angiosperm gene maps. *Genome Res.* **2008**, *18*, 1944–1954. [CrossRef] [PubMed]

77. Tang, H.; Bowers, J.E.; Wang, X.; Paterson, A.H. Angiosperm genome comparisons reveal early polyploidy in the monocot lineage. *Proc. Natl. Acad. Sci. USA* **2010**, *107*, 472–477. [CrossRef] [PubMed]

78. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A Toolkit Incorporating Gamma-Series Methods and Sliding Window Strategies. *Genom. Proteom. Bioinform.* **2010**, *8*, 77–80. [CrossRef]

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Review

# Membrane-Bound Class III Peroxidases: Unexpected Enzymes with Exciting Functions

Sabine Lüthje <fnref n="1" />,* and Teresa Martinez-Cortes <fnref n="2" />

Received: 6 August 2018; Accepted: 17 September 2018; Published: 21 September 2018

**Abstract:** Class III peroxidases are heme-containing proteins of the secretory pathway with a high redundancy and versatile functions. Many soluble peroxidases have been characterized in great detail, whereas only a few studies exist on membrane-bound isoenzymes. Membrane localization of class III peroxidases has been demonstrated for tonoplast, plasma membrane and detergent resistant membrane fractions of different plant species. In silico analysis revealed transmembrane domains for about half of the class III peroxidases that are encoded by the maize (*Zea mays*) genome. Similar results have been found for other species like thale-cress (*Arabidopsis thaliana*), barrel medic (*Medicago truncatula*) and rice (*Oryza sativa*). Besides this, soluble peroxidases interact with tonoplast and plasma membranes by protein-protein interaction. The topology, spatiotemporal organization, molecular and biological functions of membrane-bound class III peroxidases are discussed. Besides a function in membrane protection and/or membrane repair, additional functions have been supported by experimental data and phylogenetics.

**Keywords:** *Arabidopsis thaliana*; Class III peroxidase; *Medicago truncatula*; microdomains; phylogenetics; plasma membrane; protein-protein interaction; *Oryza sativa*; tonoplast; *Zea mays*

## 1. Introduction

Classical secretory plant peroxidases (EC 1.11.1.7; class III peroxidases; donor: H₂O₂ oxidoreductases) are heme-containing enzymes that belong to the peroxidase-catalase superfamily [1,2]. According to PeroxiBase, as at August 2018 [3], at least 158 class III peroxidases have been identified in the maize (*Zea mays* L.) genome, 155 isoenzymes in the rice (*Oryza sativa* L.) genome, 103 isoenzymes in the barrel medic (*Medicago truncatula* GAERTN. (Gärtner, Joseph)) genome, and 75 isoenzymes in the thale-cress (*Arabidopsis thaliana* (L.) HEYNH (Heynhold, Gustav)) genome. Differences result partially from some unique peroxidase clusters in monocotyledonous plants that were not found in dicotyledonous plants [4]. Additional isoenzymes can be produced by post-transcriptional and post-translational modifications of peroxidase transcripts [5,6].

As high as the number of peroxidases in plant cells is, so are their implications in different functions various [7–9]: peroxidases are involved in cell wall modifications like lignification, suberisation, and cross-linking of hydroxyprolin-rich glycoproteins and polysaccharides. They are involved in phytohormone metabolism, senescence, and in several biosynthetic pathways, and fulfill important functions in stress-related processes [8,10–12]. Since peroxidases exhibit an almost 1000-fold higher affinity for hydrogen peroxide as catalases and their activities can be modified in the presence of different stress factors, these enzymes play a key role in the detoxification of reactive oxygen species (ROS) [13].

<footnote n="1">¹ Oxidative Stress and Plant Proteomics Group, Institute for Plant Science and Microbiology, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany</footnote>

<footnote n="2">² Dpto de Biología Animal, Biología Vegetal y Ecología (Lab. Fisiología Vegetal), Facultad de Ciencias—Universidade da Coruña, A Zapateira s/n, 15071 A Coruña, Spain; teresa.mcortes@udc.es</footnote>

<footnote>* Correspondence: sabine.luethje@uni-hamburg.de Tel.: +49-(0)40-42816-340</footnote>
## 2. Membrane-Bound Class III Peroxidases

Evidence for membrane-bound class III peroxidases has been presented for different plant species and tissues on the protein level [14]. Guaiacol peroxidase activities have been detected at thylakoid and peroxisomal membranes, tonoplast and plasma membranes [15–20]. Class III peroxidases from tonoplast and plasma membrane (PM) have been partially purified and characterized in more detail [20–22]. Further membrane-bound peroxidases have been identified by genomic and proteomic approaches, but still lack biochemical characterization.

A membrane-bound class III peroxidase has been identified in tonoplast of Madagascar periwinkle (*Catharantus roseus* (L.) D.DON) leaves [23]. Green fluorescence protein (GFP)-fusion constructs verified localization of CroPrx01 (CrPrx1) at the inner surface of the tonoplast [21].

At the same time, class III peroxidases have been identified in highly enriched PM preparations of maize roots. At least four PM-bound peroxidases (ZmPrx01, ZmPrx66, ZmPrx70 and pmPOX2a) have been partially purified and characterized [22,24]. Depending on the state of development and oxidative stress, further peroxidases have been identified in PM of maize roots [25].

Proteomic and genomic approaches identified PM-bound class III peroxidases in different plant species and tissues; OsPrx95 has been identified in root PM of a salt sensitive rice cultivar [26]. PsPrx13 has been identified in PM of iron deficient pea (*Pisum sativum* L.) roots [27]. AtPrx64 has been shown to interact with Casparian strip formation at the PM [28,29]. AtPrx64 showed high sequence similarity to AtPrx66 as did AtPrx47 [30]. It was shown that AtPrx66 and AtPrx47 were associated with lignification of vessels, whereas AtPrx64 was associated with lignification of sclerenchyma [30,31]. A recent study demonstrated that aluminum tolerance of tobacco (*Nicotiana tabacum* L.) plants was improved by overexpression of *atprx64* [32].

A localization of class III peroxidases in detergent resistant membranes (DRM) has been demonstrated for PM of barrel medic, maize and sugar beet (*Beta vulgaris* L.) roots [14,33,34]. MtPrx02 was identified in DRM of barrel medic [33]. A precursor of BvPrx12 has been identified in DRM of sugar beet under iron deficiency [34].

## 3. Structure

The structure of class III peroxidases is well conserved [6]. The proteins contain N-terminal signal peptides, binding-sites for heme and calcium, and four conserved disulfide bridges (Figure 1). Transmembrane domains were predicted for ZmPrx01, OsPrx95, AtPrx47 and MtPrx02, whereas for CroPrx01 and AtPrx64 transmembrane helices (TMH) appear unlikely.

A hypothetical model of CroPrx01 has been published [35]. Besides an N-terminal propeptide (34 amino acids) that directed the GFP-fusion construct of CroPrx01 to the endoplasmic reticulum (ER), a C-terminal extension of 23 to 25 amino acids directed the GFP-fusion constructs of the protein to the vacuole [35]. C-terminal extensions have been found for ZmPrx01 and OsPrx95 (Figure 1). In contrast to CroPrx01, these proteins were identified in highly enriched PM preparations [21,25]. Additionally, prediction of N-glycosylation sites (Table 1) suggested a localization of these enzymes at the apoplastic side.

In accordance with earlier predictions, structures of membrane-bound peroxidases have 13-21 α-helices and between two and 11 β-sheets [14,21]. Exceptions were AtPrx47 and AtPrx64, whose hypothetical structures contained no β-sheets (Figure 2). For AtPrx64, the second calcium binding-site was not conserved (Figure 1). The structures of these peroxidases may need further elucidation.

In comparison to horseradish peroxidase (1HCH1A, HRP) [36], active sites of AtPrx47, AtPrx64, MtPrx02 and ZmPrx01 were well conserved (Figure 1). In contrast, the distal His-42 of HRP was replaced by Val-70 in OsPrx95 (Figure 1). This His facilitates formation of the initial iron-peroxide complex by deprotonating the peroxide and subsequently promote cleavage of the oxygen-oxygen bond by protonating the distal oxygen [37]. In comparison to the wild type HRP, guaiacol peroxidase activity of the H42V mutant was much lower. This observation was in accordance with a ~106-fold lower formation of compound I by the mutant in comparison to the wildtype [38]. As a consequence,
reduction of compound II has been more rapid than reduction of compound I. So far, biochemical characterization of OsPrx95 is lacking.

<figure><img src="image_102.png" /><figcaption>**Figure 1.** Active sites of horseradish peroxidase (HRP) and OsPrx95 and multiple sequence alignment of class III peroxidases. Superposition of active sites of HRP (blue) and OsPrx95 (yellow) was prepared by Phyton-enhanced molecular graphic tool (PyMOL): (a) site view, (b) top view; in HRP Arg-38 and His-42 build the distal active-site structure [39]. The heme group (grey), with δ-meso edge (red), is fixed by His-170 in the active site. (c) Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used for multiple sequence alignment of HRP [40], membrane-bound peroxidases and soluble peroxidases that were used as templates for modelling of structures shown in Figure 2 [36,41–43]. Active sites (yellow), calcium binding-sites (grey), conserved cysteine residues for formation of disulfide bridges (blue), transmembrane domains (bold and underlined), signal peptides (italic).</figcaption></figure>
<table><caption>**Table 1.** Properties and physiological functions of membrane-bound class III peroxidases of arabidopsis, barrel medic, rice and maize. After identification of peroxidase sequences with trans-membrane domains [44], signal peptides [45], post translational modifications [46] and localisation [47] of proteins have been further analysed by bioinformatic tools. In case of missing experimental data on biological functions these have been predicted by a Basic Local Alignment Search Tool (BLAST) (*) search against all peroxidases (inclusive soluble isoenzymes) with highest sequence similarity and known function. ER, endoplasmatic reticulum membrane; Golgi, golgi apparatus; MoM, mitochondrial outer membrane; MS, identified by mass spectrometry [14]; MW, molecular weight; pI, point isoelectric; PM, plasma membrane; PSORTI, protein subcellular localization prediction tool; Vac., vacuole. Predicted pyrrolidone carboxylic acid (PCA) modification (+).</caption><thead><tr><th>Species</th><th>Acc. No. 1</th><th>Protein 2</th><th>MW 3</th><th>pI 3</th><th>SignalP 4</th><th>N-Glyco 5</th><th>NetPhos 6</th><th>PCA 7</th><th>PSORTI 8</th><th>Response to</th></tr></thead><tbody><tr><td rowspan="10">A. thaliana</td><td>Q9IL1E5</td><td>AlfPrx04</td><td>34.28</td><td>7.87</td><td>1-19</td><td>1</td><td>39</td><td>+</td><td>PM</td><td>senescence</td></tr><tr><td>Q9FX85</td><td>AlfPrx10</td><td>38.03</td><td>6.17</td><td>—</td><td>2</td><td>29</td><td>—</td><td> Vac</td><td>hormones and stress</td></tr><tr><td>Q9Z593</td><td>AlfPrx13</td><td>36.88</td><td>5.15</td><td>—</td><td>1</td><td>23</td><td>+</td><td> ER</td><td>—</td></tr><tr><td>Q9S852</td><td>AlfPrx18</td><td>35.50</td><td>5.03</td><td>—</td><td>2</td><td>41</td><td>—</td><td> PM</td><td>floral organ development</td></tr><tr><td>Q8I829</td><td>AlfPrx25</td><td>37.45</td><td>8.48</td><td>—</td><td>2</td><td>38</td><td>—</td><td> PM</td><td>etiolation and drought</td></tr><tr><td>Q9S289</td><td>AlfPrx64<sup>MS</sup></td><td>35.97</td><td>8.57</td><td>—</td><td>1</td><td>30</td><td>—</td><td> PM</td><td>lignification of vessels</td></tr><tr><td>Q8I735</td><td>AlfPrx48</td><td>44.19</td><td>4.99</td><td>—</td><td>2</td><td>42</td><td>—</td><td> PM</td><td>—</td></tr><tr><td>P9I201</td><td>AlfPrx58</td><td>35.29</td><td>4.92</td><td>—</td><td>2</td><td>30</td><td>—</td><td> PM</td><td>—</td></tr><tr><td>Q43872</td><td>AlfPrx64<sup>MS</sup></td><td>34.71</td><td>9.05</td><td>—</td><td>1-23</td><td>3</td><td>30</td><td>—</td><td> PM</td><td>etiolation and drought</td></tr><tr><td>Q96511</td><td>AlfPrx69</td><td>35.68</td><td>9.47</td><td>—</td><td>1-23</td><td>3</td><td>29</td><td>—</td><td> ER</td><td>etiolation and cold stress</td></tr><tr><td>Q9FM17</td><td>AlfPrx70</td><td>36.00</td><td>6.97</td><td>—</td><td>1-23</td><td>2</td><td>25</td><td>—</td><td> PM</td><td>—</td></tr><tr><td rowspan="10">M. truncatula</td><td>G7K8K6</td><td>MifPrx02<sup>MS</sup></td><td>36.02</td><td>9.20</td><td>1-23</td><td>1</td><td>27</td><td>—</td><td> PM</td><td>nitrogen starvation, wounding and pathogen</td></tr><tr><td>A0A072UJ14</td><td>MifPrx03</td><td>35.99</td><td>5.35</td><td>1-25</td><td>3</td><td>28</td><td>—</td><td> Vac</td><td>rhizobium inoculation</td></tr><tr><td>G8A779</td><td>MifPrx04</td><td>35.50</td><td>8.32</td><td>1-25</td><td>5</td><td>34</td><td>—</td><td> PM</td><td>nitrogen starvation and pathogen</td></tr><tr><td>A0A072UQ9</td><td>MifPrx09</td><td>35.92</td><td>6.51</td><td>1-25</td><td>6</td><td>41</td><td>—</td><td> Goggi</td><td>phosphate starvation and pathogen</td></tr><tr><td>G7C123</td><td>MifPrx13</td><td>35.26</td><td>8.01</td><td>1-25</td><td>2</td><td>22</td><td>—</td><td> PM</td><td>nodulation and pathogen</td></tr><tr><td>G7KFM2</td><td>MifPrx29</td><td>35.79</td><td>9.41</td><td>1-28</td><td>1</td><td>27</td><td>+</td><td> PM</td><td>drought, wounding, nitrogen starvation, phosphate starvation, pathogen, methyl jasmonate and ultraviolet (UV) irradiation</td></tr><tr><td>A0A072UJ17</td><td>MifPrx32</td><td>36.88</td><td>8.62</td><td>1-23</td><td>1</td><td>35</td><td>—</td><td> PM</td><td>nitrogen starvation and pathogen</td></tr><tr><td>Q1RXM7</td><td>MifPrx34</td><td>34.38</td><td>9.11</td><td>1-22</td><td>3</td><td>32</td><td>—</td><td> Vac</td><td>drought, insect damage and pathogen</td></tr><tr><td>13S041</td><td>MifPrx37</td><td>34.90</td><td>7.15</td><td>1-22</td><td>1</td><td>30</td><td>—</td><td> Vac</td><td>drought, insect damage, phosphate starvation and pathogen</td></tr><tr><td>G7TH4</td><td>MifPrx39</td><td>36.19</td><td>6.5</td><td>1-25</td><td>5</td><td>31</td><td>—</td><td> PM</td><td>pathogen</td></tr><tr><td>A0A072UQ8</td><td>MifPrx40</td><td>37.48</td><td>4.73</td><td>1-28</td><td>2</td><td>33</td><td>+</td><td> PM</td><td>pathogen</td></tr><tr><td>A0A072TW11</td><td>MifPrx41</td><td>34.60</td><td>5.03</td><td>1-23</td><td>1</td><td>30</td><td>—</td><td> PM</td><td>phosphate starvation and pathogen</td></tr><tr><td>A0SC11</td><td>MifPrx42</td><td>36.08</td><td>9.09</td><td>1-25</td><td>1</td><td>27</td><td>+</td><td> ER</td><td>phosphate starvation and pathogen</td></tr><tr><td>A0A072UHRP</td><td>MifPrx49</td><td>35.30</td><td>9.72</td><td>—</td><td>—</td><td>31</td><td>—</td><td> Vac</td><td>nodulation and pathogen</td></tr><tr><td rowspan="10">M. truncatula</td><td>G7TKC1</td><td>MifPrx25</td><td>34.47</td><td>9.45</td><td>1-23</td><td>2</td><td>33</td><td>—</td><td> PM</td><td>nodulation and pathogen</td></tr><tr><td>G7K822</td><td>MifPrx38</td><td>36.06</td><td>6.87</td><td>1-25</td><td>2</td><td>31</td><td>+</td><td> ER</td><td>nodulation and pathogen</td></tr><tr><td>G7CJN8</td><td>MifPrx66</td><td>34.64</td><td>8.21</td><td>1-19</td><td>1</td><td>27</td><td>+</td><td> PM</td><td>nitrogen starvation and pathogen</td></tr><tr><td>A0A072UJ14</td><td>MifPrx74</td><td>38.49</td><td>9.28</td><td>1-29</td><td>2</td><td>28</td><td>—</td><td> PM</td><td>nodulation and pathogen</td></tr><tr><td>13T3K6</td><td>MifPrx79</td><td>36.47</td><td>8.82</td><td>—</td><td>2</td><td>48</td><td>—</td><td> PM</td><td>nodulation*</td></tr><tr><td>A0A072UJ14</td><td>MifPrx81</td><td>35.06</td><td>8.14</td><td>1-25</td><td>—</td><td>30</td><td>—</td><td> Vac</td><td>pathogen</td></tr><tr><td>G7J21</td><td>MifPrx83</td><td>41.78</td><td>5.78</td><td>1-25</td><td>—</td><td>29</td><td>—</td><td> ER</td><td>—</td></tr><tr><td>G7J952</td><td>MifPrx84</td><td>33.52</td><td>9.17</td><td>1-26</td><td>2</td><td>44</td><td>—</td><td> PM</td><td>—</td></tr><tr><td>Q5SAT8</td><td>MifPrx91</td><td>36.52</td><td>7.22</td><td>1-21</td><td>3</td><td>35</td><td>—</td><td> Vac</td><td>—</td></tr><tr><td>G7LB60</td><td>MifPrx92</td><td>34.76</td><td>8.81</td><td>1-25</td><td>1</td><td>40</td><td>—</td><td> MoM</td><td>nodulation and pathogen</td></tr><tr><td>G7J1A7</td><td>MifPrx93</td><td>35.84</td><td>5.79</td><td>1-23</td><td>2</td><td>36</td><td>+</td><td> ER</td><td>—</td></tr><tr><td>G7JKL3</td><td>MifPrx94</td><td>46.33</td><td>9.21</td><td>—</td><td>3</td><td>49</td><td>—</td><td> Vac</td><td>—</td></tr><tr><td>G7JKV1</td><td>MifPrx95</td><td>38.06</td><td>6.14</td><td>—</td><td>5</td><td>36</td><td>—</td><td> PM</td><td>—</td></tr><tr><td>G7J952</td><td>MifPrx98</td><td>33.99</td><td>8.06</td><td>1-26</td><td>3</td><td>48</td><td>+</td><td> PM</td><td>—</td></tr></tbody></table>