Author response: Arabidopsis 14-3-3 epsilon members contribute to polarity of PIN auxin carrier and auxin transport-related development
2017-02-08

Full text Figures and data Side by side Abstract Introduction Results and discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which – epsilon – is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development. https://doi.org/10.7554/eLife.24336.001 Introduction Members of the eukaryotic 14-3-3 family are highly conserved eukaryotic proteins that have been implicated in the regulation of distinct cellular processes by protein-protein interactions. Notably, 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner and are required to change the activity state of the respective target protein. They enforce conformational changes, act as intermolecular bridge or modify the subcellular localization of their clients (Mackintosh, 2004). Plant 14-3-3s have been demonstrated to be of utmost importance for the regulation of enzymes that are crucial for nutrient uptake and processing, such as the plasma membrane (PM) H+-ATPase and nitrate reductase. Yet, another key aspect of 14-3-3 activity is the modification of transcriptional regulators involved in phytohormone action, such as BRASSINAZOLE-RESISTANT1, or in developmental signaling, such as the floral mediator (de Boer et al., 2013). While the individual studies focusing on particular 14-3-3 client proteins have generated substantial insight into the function of plant 14-3-3s, the question of functional diversity among 14-3-3 isoforms is still not fully resolved. Arabidopsis thaliana expresses thirteen 14-3-3 isoforms which can be divided into two major phylogenetic groups, the non-epsilon group (eight members) and the ancestral epsilon group (five members) (Figure 1A). Members of both groups are present in all plant genomes sequenced so far. The phylogenetic relationship of 14-3-3 isoforms from six plant species (A. thaliana, Solanum lycopersicum, Medicago truncatula, Populus trichocarpa, Oryza sativa, Physcomitrella patens) and their expression patterns (based on publicly accessible RNA-seq or microarray data) are depicted in Figure 1—figure supplement 1 and Figure 1—source data 1, respectively. Except for the moss, P. patens, the total transcript level of all non-epsilon members in a given plant species is generally higher than that of the epsilon group isoforms. However, whether this relates to lower amounts of 14-3-3 epsilon proteins in individual cells or tissues remains unclear. The ancestral epsilon group may mediate fundamental cellular features while non-epsilon members may perform organism-specific functions (Ferl et al., 2002). In this regard, it has recently been shown that the Arabidopsis 14-3-3 isoform kappa (non-epsilon) is required for PHOTOTROPIN2-mediated blue-light-induced stomatal opening (Tseng et al., 2012). However, with the exception of upsilon, the knockout mutant of which flowers late under long day conditions (Mayfield et al., 2012), several single and double T-DNA-induced loss-of-function alleles of non-epsilon isoforms are indistinguishable from wild type under normal growth conditions and even quadruple mutants showed only a mild reduction of primary root length (van Kleeff et al., 2014). Nevertheless, under given abiotic stress conditions, a certain specificity of defined double mutants was uncovered (van Kleeff et al., 2014). Taken together, functional overlap seems to be a predominant issue among non-epsilon 14-3-3 members. So, what about the ancient epsilon group in Arabidopsis? Figure 1 with 2 supplements see all Download asset Open asset Phylogenetic tree of the Arabidopsis 14-3-3 isoforms and expression of genomic 14-3-3:GFP fusions under the control of the endogenous promoter. (A) Unrooted phylogenetic tree of the Arabidopsis 14-3-3 protein family. Maximum parsimony analyses were performed using PAUP 4.0b10 (Altivec) with the bootstrap-algorithm and 1000 replica. (B, C) Expression of iota-GFP is restricted to pollen. (D) to (G) Pi-GFP is exclusively expressed in the chalazal cyst of the seed (D, E) and the connective tissue of the anthers (F, G). (H) to (O) The expression of nu-GFP is ubiquitous. Expression in seeds (H), seedlings (I), root tips (J), roots (K), hypocotyl (L), leaf epidermis (M) and floral organs (N, O) is shown. (P) to (S) Mu-GFP (P, R) and merge with FM4-64 (Q, S) in epidermal root cells (elongation zone) of seedlings (4d) treated with CHX (50 μM) for 60 min followed by treatment with CHX (P, Q) or CHX and 50 μM BFA (R, S) for 60 min. Note that mu-GFP localizes to both nucleus and cytoplasm but accumulates in BFA bodies upon inhibitor treatment. Arrowheads indicate nuclei, arrows mark BFA bodies. https://doi.org/10.7554/eLife.24336.002 Figure 1—source data 1 Expression patterns of 14-3-3 isoforms in various tissues of six plant species based on publicly accessible RNA-seq or microarray data. Normalized data for the expression of 14-3-3 isoforms (absolute signal intensities) were obtained from the eFP Browser of the Bio-Analytic Resource for Plant Biology (BAR) (http://bar.utoronto.ca) (microarray data: A. thaliana, M. truncatula, P. trichocarpa, O. sativa, P. patens) or the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi) (RNA-seq data: S. lycopersicum). Expression of ubiquitin is depicted for comparison. Individual Excel sheets have been created for the different plant species. Each Excel sheet lists detailed information of 14-3-3s, such as gene ID and phylogenetic subgroup as well as the origin of expression data including references. https://doi.org/10.7554/eLife.24336.003 Download elife-24336-fig1-data1-v1.xlsx With the exception of omicron, no single T-DNA-induced knockout mutant of epsilon group members has been described so far. Omicron seems to participate in the regulation of iron acquisition (Yang et al., 2013), while a knockdown allele of mu exhibited exaggerated Pi starvation responses (Cao et al., 2007). The same mu allele was later shown to have shorter roots under constant light (Mayfield et al., 2012) or water stress (He et al., 2015). In summary, the in vivo role of the epsilon group is virtually unknown. Furthermore, the important question of whether the ancestral epsilon group members indeed fulfill rather basal cellular functions remains largely unresolved. Here, we tackle this question by conditional knockdown of the entire 14-3-3 epsilon group in Arabidopsis seedlings (three of the five isoforms are expressed in seedlings). Interference with their function led to pronounced defects in PIN polar distribution and associated auxin-mediated plant development. At the cellular level, in vivo visualization of endocytic cargo revealed defects in defined post-Golgi trafficking processes. 14-3-3 proteins co-immunoprecipitated with several factors crucial for membrane trafficking, suggesting that lack of these interactions might underlie the mutant phenotype. Results and discussion Expression of 14-3-3 isoforms To determine the expression pattern of Arabidopsis 14-3-3 isoforms (Figure 1A), translational fusions between their genomic sequences, comprising the respective promoter, and the reporter green fluorescent protein (GFP) were constructed. The analysis of transgenic Arabidopsis revealed a strict tissue-specific expression pattern for two of the epsilon-group members. Expression of iota (AT1G26480) is restricted to pollen, while pi (AT1G78220) is exclusively expressed in anthers and the chalazal cyst of the seed (Figure 1B–G). Yet another epsilon group member, omicron (AT1G34760), is expressed in anthers too, but also moderately in roots and cotyledons. Overall, however, the expression of the remaining 10 non-epsilon and epsilon isoforms occurs in neither a tissue-specific nor a developmentally regulated manner (isoform nu shown in Figure 1H–O). Thus, in Arabidopsis, differences in the expression patterns of 14-3-3 isoforms are not assumed to contribute to possible isoform specificity. The same applies to the majority of isoforms expressed in S. lycopersicum, M. truncatula, P. trichocarpa, O. sativa and P. patens (Figure 1—source data 1). RNAi and amiRNA of 14-3-3 epsilon members interferes with plant growth and development Unfortunately, we did not identify T-DNA-induced null alleles for most of the epsilon group members. It has previously been reported that a knockdown of the 14-3-3 isoform mu (SALK_004455) caused significantly shorter roots in constant light (Mayfield et al., 2012). The homozygous SALK line, however, did not show a striking phenotype in our hands (Figure 1—figure supplement 2; see also: [Cao et al., 2007; He et al., 2015]). Thus, we designed ethanol-inducible RNA interference (RNAi) (Figure 2, Figure 2—figure supplement 1) and artificial microRNA (amiRNA) (Figure 2—figure supplement 1) constructs to suppress the function of those epsilon group members that are rather ubiquitously expressed, namely epsilon (AT1G22300), mu (AT2G42590) and omicron (AT1G34760). We analyzed independent homozygous transgenic lines that are referred to as emo-RNAi (three lines) and amiRNA-(em)o (one line) plants, respectively. Figure 2 with 3 supplements see all Download asset Open asset Ethanol-inducible emo-RNAi causes growth retardation phenotypes. (A) to (F) Seedlings (wildtype and three independent emo-RNAi lines) grown either for 6 days in the light (A, B) or for 4 days in the dark (C–F) on noninductive (A, C, E) or inductive (0.1% (v/v) EtOH) medium (B, D, F), optionally supplemented with ACC (10 μM) (E, F). (G) Semiquantitative RT-PCR analysis of the transcript level of selected 14-3-3 isoforms in light-grown seedlings. (H) Hypocotyl length of etiolated seedlings (n = 30). (I) Primary root growth of seedlings grown for 4 days in the absence of ethanol followed by transfer to non-inductive (−) or inductive (+) medium for the indicated times (n = 16). https://doi.org/10.7554/eLife.24336.006 Semiquantitative RT-PCR confirmed that the expression of the targeted 14-3-3 isoforms was efficiently reduced upon ethanol treatment (Figure 2G, Figure 2—figure supplement 1C). Notably, transcript levels of all non-epsilon isoforms were unaffected (kappa, chi, upsilon shown in Figure 2G). Considering that 14-3-3 isoforms are highly homologous, this clearly suggests that the risk of potential off-targets of produced siRNA is marginal. Under inductive conditions (0.1%(v/v) ethanol), emo-RNAi (Figure 2B) and amiRNA-(em)o (Figure 2—figure supplement 1B) seedlings were strongly defective in growth, with highly impaired root elongation and nonexpanded, epinastic cotyledons. This is in striking contrast to the mild phenotypes reported even for quadruple knockout mutants of 14-3-3 non-epsilon isoforms (van Kleeff et al., 2014) and might highlight a particular importance of the ancestral epsilon group. Etiolated emo-RNAi seedlings germinated on inductive medium showed a severe defect in elongation of both roots and hypocotyls (Figure 2D). Ethylene overproduction is not the underlying cause since the biosynthesis inhibitor aminovinylglycine (AVG) does not rescue the phenotype (Figure 2H). In addition, the hypocotyls are hookless even in the presence of the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylate (ACC) (Figure 2F). Both apical hook development and ethylene-induced hook exaggeration have been most extensively connected with auxin (Stepanova et al., 2008; Vandenbussche et al., 2010; Zádníková et al., 2010). Taking into account that light grown emo-RNAi/amiRNA(em)o seedlings exhibit a wavy root phenotype and do not form lateral roots, the phenotypic features might be related to alterations in auxin-mediated processes. Since the independent emo-RNAi/amiRNA-(em)o-lines reacted in a qualitatively and quantitatively comparable manner, we focused on the characterization of emo1-RNAi. RNAi of 14-3-3 epsilon members interferes with auxin transport-dependent processes Auxin-induced gene expression was not severely affected in roots of the mutant following ethanol treatment (Figure 2—figure supplement 2), indicating that auxin perception and the subsequent degradation of Aux/IAA repressors were not compromised. We therefore tested whether interference with 14-3-3 function impacts auxin-transport dependent processes, such as lateral root formation and response to gravity. Upon ethanol treatment (3 days) in the presence of the transportable synthetic auxin naphthalene acetic acid (NAA) (48 hr), discrete lateral root primordia formed along the main root of control seedlings (Figure 3B). By contrast, emo1-RNAi seedlings displayed highly disorganized proliferation of pericycle cells (Figure 3E). This resembles wild-type roots treated with the non-transportable auxin analogue 2,4-dichlorphenoxyacetic acid (2,4D) (Figure 3C) (Geldner et al., 2004). In addition, emo1-RNAi roots failed to orient their growth with respect to the gravity vector (Figure 2—figure supplement 3A–D) and moreover, etiolated seedlings had agravitropic hypocotyls (Figure 2—figure supplement 3F,G). Yet, another process that depends on polar auxin transport in the shoot is the formation of adventitious roots as a consequence of surgical removal of the primary root (Geldner et al., 2004). As expected, wildtype seedlings spontaneously regenerated a root from the hypocotyl stump, while emo1-RNAi seedlings were unable to form adventitious roots (Figure 2—figure supplement 3M–P). Together, these data show that the function of 14-3-3 epsilon group members is required for auxin-transport-dependent development in both roots and shoots. Figure 3 with 1 supplement see all Download asset Open asset Disorganized lateral root primordia and failure in the establishment of auxin response gradients caused by emo-RNAi induction. Seedlings grown for 4 days in the absence of ethanol were transferred to inductive medium for 24 hr followed by treatment with exogenous auxin.(A) to (F) Lateral root primordia of wildtype (A–C) and emo1-RNAi (D–F) after 48 hr of treatment with either mock solution (A, D), 1 μM NAA (B, E) or 1 μM 2,4D (C, F) in the presence of ethanol. (G) to (L) Auxin response gradients visualized by DR5::GUS in wild type (G–I) and emo1-RNAi (J–L) after treatment with either mock solution (G, J) or 0.1 μM NAA for either 24 hr (H, K) or 48 hr (I, L) in the presence of ethanol. https://doi.org/10.7554/eLife.24336.010 RNAi of 14-3-3 epsilon members interferes with auxin distribution Because gravitropic growth and formation of both lateral and adventitious roots depend on dynamic auxin redistribution (Geldner et al., 2004; Luschnig et al., 1998; Benková et al., 2003), we investigated the effect of RNAi induction on auxin distribution with the auxin reporter DR5::GUS (Ulmasov et al., 1997; Sabatini et al., 1999) as well as DR5rev::GFP (Friml et al., 2003). In wild-type root meristems, DR5 was highly active in the columella and in stele tissues (Figure 3G, Figure 3—figure supplement 1A). By contrast, induction of emo1-RNAi caused an ectopic accumulation of GUS activity (Figure 3J) / expression of GFP (Figure 3—figure supplement 1B) in the entire lateral root cap. As reported previously, roots have homeostatic mechanisms that maintain the endogenous auxin gradients in the presence of exogenously applied auxin (Geldner et al., 2004; Friml et al., 2002). Exposure to low concentrations of the transportable auxin NAA did hence not lead to strong alterations in DR5::GUS staining in control roots (Figure 3H,I), whereas GUS activity expanded across the entire root of emo1-RNAi seedlings (Figure 3K,L). Apparently, emo roots have a reduced capacity to maintain auxin gradients in the presence of exogenous auxin. After gravistimulation of the wild type, the DR5rev::GFP signal relocated asymmetrically to the gravistimulated side (Figure 3—figure supplement 1E,G), indicating a shift in auxin distribution which is essential for the differential growth response (Ottenschläger et al., 2003). By contrast, the ectopic lateral expression pattern of DR5rev::GFP in the root meristem of emo1-RNAi persisted, independent of gravistimulation (Figure 3—figure supplement 1F,G). Taken together, these data suggest that interference with 14-3-3 epsilon group members affects auxin distribution. Therefore, the effect of reduced 14-3-3 expression on auxin transport toward the root apex was analyzed. As compared with the non-induced emo1-RNAi control, auxin transport to the root tip decreased upon ethanol induction (Figure 2—figure supplements 3E, 24 hr: 22%, 48 hr: 42%, n = 10) and, furthermore, the gradual decrease in auxin response gradients within the root tip could be visualized by using the DR5rev::GFP reporter (Figure 3—figure supplement 1B–D). However, another striking aspect of the RNAi-mutant upon transfer to ethanol-containing medium is an epinasty cotyledon phenotype (Figure 2B), characteristic of auxin overproducers (Zhao et al., 2001; Boerjan et al., 1995). Consequently, in contrast to the situation in roots, the auxin level might be elevated in emo hypocotyls and cotyledons. Accordingly, the mutant showed a severe increase in DR5::GUS reporter activity in the cotyledons upon induction (Figure 2—figure supplement 3L). Intriguingly, treatment of the wild type with the auxin transport inhibitor NPA resulted in agravitropic hypocotyls (Figure 2—figure supplement 3H), an increase in GUS activity in cotyledons (Figure 2—figure supplement 3J) and the formation of epinastic leaves (Geldner et al., 2004). Taken together, this strongly suggests that suppression of 14-3-3 expression impairs auxin transport in both roots and aerial tissues, resulting in depletion of auxin in the root tip and an elevated auxin level in the apical part. 14-3-3 epsilon group members: master regulators of the H+-ATPase? The phenotypic analysis of emo mutants revealed an impressive number of auxin-related, in particular auxin transport-related, phenotypes. This, however, does not necessarily imply that 14-3-3 members regulate auxin transport directly. In fact, several mutants affected in rather fundamental eukaryotic processes, such as vesicular trafficking events, exhibit auxin-related phenotypes (Geldner et al., 2003; Jaillais et al., 2006; Kitakura et al., 2011; Mravec et al., 2011). What might be the major 14-3-3 regulated cellular process or 14-3-3 target protein(s) causing the phenotype? According to the chemiosmotic hypothesis for polar auxin transport, the activity of the PM localized H+-ATPase is crucial for auxin uptake into the cell and, consequently, directed cellular export mediated by auxin efflux facilitators. Regulation of this P-type proton pump is one of the most highly explored functions of plant 14-3-3 proteins. In brief, phosphorylation-dependent association of 14-3-3 with the penultimate residue results in a displacement of the C-terminal autoinhibitor and hence activation of the pump (Speth et al., 2010). Dexamethasone (Dex)-dependent expression of one of the major H+-ATPase isoforms, AHA2, deleted of its C-terminal autoinhibitor (94 amino acids, 1-887, AHA2Δ94) (Pacheco-Villalobos et al., 2016) and a active of the from did not rescue the emo1-RNAi phenotype (Figure Notably, the in wild type upon treatment absence of an apical hook and are indeed the consequence of an activation of the H+-ATPase since can be by a of the et al., (Figure Therefore, of the H+-ATPase is not the underlying cause of the emo1-RNAi phenotype. Figure 4 with 1 supplement see all Download asset Open asset expression of a active of does not rescue the emo-RNAi phenotype. (A) to and emo1-RNAi seedlings were grown for 4 days in the dark in the absence or presence of ethanol and (10 Semiquantitative RT-PCR analysis of the transcript level of the deleted (A, lower and of the H+-ATPase in (B, arrows indicate the of the confirmed expression of the growth (C, and of the hypocotyl length (E, n = is shown. (F) to (H) with an of the the in transgenic wild type upon shorter absence of an apical hook and growth (G, and of the hypocotyl length (F, n = in the absence or presence of is shown. thought to might 14-3-3 proteins with auxin efflux carriers such as PIN The PIN are by a localized in the cytoplasm and et al., 2010; et al., 2010; et al., 2014). of 14-3-3 epsilon and the was not by and in and 2012) (Figure supplement 1). This, however, is no for the lack of direct association of 14-3-3 with PIN proteins. RNAi of 14-3-3 epsilon members interferes with PIN expression and polar localization that RNAi of the 14-3-3 epsilon group members interferes with auxin transport, we analyzed expression and localization of members of the PIN of auxin efflux in Under our conditions, et al., was the basal side of stele and cells of the wild type with expression in cells (Figure and is expressed in epidermis and cells apical and basal (Figure Interference with 14-3-3 function resulted in of the two auxin efflux was in cells with an comparable to that in cells and also moderately in the epidermis (Figure showed in the epidermis and cells and in the entire lateral root as well (Figure of PIN gene expression has been described in mutants or with auxin transport et al., and the defects in emo1-RNAi roots are the consequence of the interference with auxin Figure Download asset Open asset Ethanol-inducible emo-RNAi causes of PIN proteins in root (A) to (H) Expression of and (G, in wildtype (A, and emo1-RNAi (C, D, seedlings grown for 4 days on inductive The of GFP on the basal to that on the lateral side of the plasma membrane is shown (E, n roots, n 10 Notably, the subcellular polar localization of proteins was reduced as a consequence of RNAi induction. In the signal was not restricted to the basal side of those cells In fact, was in the lateral of cells and did not show pronounced polarity in cells (Figure Moreover, by contrast with the wild type, is in the lateral of cells upon interference with 14-3-3 function (Figure RNAi of 14-3-3 epsilon members interferes with endocytic membrane trafficking PIN proteins between and the PM trafficking for the establishment and of polarity (Geldner et al., 2004; et al., 2007). Furthermore, PIN proteins are to the late bodies resulting in protein degradation and of auxin et al., Accordingly, treatment with trafficking affects PIN causes PIN accumulation in BFA of and that by and the et al., We that the accumulation was pronounced in root epidermis cells of emo1-RNAi as compared to the wild type (Figure of the presence of the protein inhibitor (Figure comparable has been described for et al., 2012) and et al., 2012) which are affected in and PIN to the plasma respectively. however, does not to be significantly altered in emo1-RNAi as by uptake of the endocytic FM4-64 (Figure supplement the mutant displayed BFA even treated with 10 μM BFA (Figure a which and the interference with trafficking et al., 2010; et al., 2012). This suggests that might be affected in emo1-RNAi. Therefore, a after BFA treatment was While the BFA efficiently in wild-type root epidermal cells n in emo1-RNAi n (Figure Taken together, reduced expression of the 14-3-3 epsilon members interferes with to the Figure 6 Download asset Open asset Ethanol-inducible emo-RNAi membrane trafficking processes. (A) to (F) in epidermal root cells of wild-type (A–C) and emo1-RNAi (D–F) seedlings grown for 4 days on inductive medium and treated with BFA for 10 min (A, D), min (B, E) or 60 min (C, F). The BFA is given in (G) to in epidermal root cells of (4d) wild-type (G, and emo1-RNAi (I, J) seedlings treated with 50 μM) for 60 min followed by treatment with CHX and 50 μM BFA (G, and subsequent of BFA (H, J) for 60 min respectively. L) of in BFA bodies and the PM BFA and 1 PM n n endocytic transport to the was analyzed. The a of the but accumulates the et al., 2014) that in the presence of et al., While striking differences could not be in the absence of the of was reduced in emo1-RNAi as compared to the wild type (Figure a phenotype has – to our – not been described so far. of however, characteristic of mutants such as et al., et al., and et al., which are affected in diverse post-Golgi trafficking processes but all or with Accordingly, we revealed by FM4-64 and of and BFA that trafficking to the membrane is significantly in emo1-RNAi in to the wild type (Figure The trafficking of FM4-64 and of PM in to the is also in the absence of BFA (Figure supplement with the on trafficking, emo1-RNAi an