BACKGROUND
transfer cells  trans-differentiate from a range of existing cell types belonging to the major tissue systems of dermal , ground  and vascular .
once tc development is completed, a 10- to 20-fold amplification of their plasma membrane surface area, containing high densities of solute transporters, confer these cells with an extraordinarily high capacity for nutrient exchange between apo- and symplasmic compartments located at bottlenecks for long-distance transport of nutrients throughout the plant body  <cit> .
an intricate invaginated complex of cell wall ingrowths provides structural scaffolding on which the amplified plasma membrane is arrayed to enhance nutrient flows.
the tight coupling of this structure/function relationship is graphically illustrated by compromised seed filling of mutants in which tcs, located at the maternal/filial interfaces of both eudicots and monocots, exhibit an attenuated construction of their cell wall ingrowth complex  <cit> .
since a number of major crop species, including cereals and grain legumes, contain tcs positioned at the maternal/filial interfaces of their developing seeds  <cit> , the shrivelled seed phenotype exhibited by tc mutants underscores the important role these cells play in determining crop yields.
the cell wall ingrowth complex is organized into one of two architectural types – flange or reticulate  <cit> .
flange ingrowths form as ribs or bands of wall material while the more commonly occurring reticulate wall ingrowths arise as numerous wall papillae that develop at right angles to the original wall.
the extent of their reticulation varies from cylindrical papillae alone to ones in which papillae branch and fuse to generate fenestrated wall layers  <cit> .
immediately preceding construction of reticulate, but not flange wall ingrowths  <cit> , a structurally distinctive wall-layer, the so-called uniform wall, is rapidly laid down over the pre-existing primary wall of the trans-differentiating cell to a thickness that can extend up to 50% of that of the pre-existing primary wall  <cit> .
collectively, the uniform wall and reticulate wall ingrowths form the ingrowth wall  <cit> .
despite the central role the ingrowth wall, and particularly its wall ingrowth component, plays in underpinning transport function of tcs, little is known about the mechanisms responsible for inducing and then orchestrating its construction.
transcriptional analyses of cells committed to a developmental pathway leading to forming a tc-morphology have identified up-regulated expression of genes encoding components of signalling pathways for abscisic acid, auxin, ethylene, reactive oxygen species  and ca2+ in developing seeds  <cit>  and for auxin and ethylene in giant cells induced by nematode infection  <cit> .
events downstream from the inductive signals largely have been identified by transcriptome analyses of developing cereal seeds and, in particular, for flange wall ingrowths formed in basal endosperm tcs  of maize  <cit>  and barley  <cit> .
a more limited analysis has been reported for the formation of reticulate wall ingrowths in eudicots  <cit> .
while there are undoubtedly some shared features, given the disparate architectures of flange and reticulate wall ingrowths, we hypothesize that signalling pathways, cell wall biosynthesis and delivery of polysaccharides to the wall matrix will have features that are peculiar to each architectural type of ingrowth wall.
this characteristic likely extends to distinctive elements responsible for constructing the uniform wall and wall ingrowth papillae.
to this end, we used vicia faba cotyledons, in which on transfer to culture, their adaxial epidermal cells spontaneously undergo trans-differentiation to a tc-morphology  <cit> .
this experimental system provided the opportunity to undertake a transcriptome analysis of tc-specific gene networks  <cit> .
in addition, because of the temporal sequence for the deposition of the uniform wall followed by wall ingrowth papillae, gene cohorts associated with these two developmental events could be distinguished.
hereafter, wall ingrowth papillae, which represent the initial stage of wall ingrowth construction  <cit> , are referred to as wall ingrowths.
methods
plant growth conditions, cotyledon culture and collection of tissue samples for sequencing
v. faba l.  plants were raised under controlled environmental conditions  <cit> .
cotyledons of harvested pods were surgically excised and cultured aseptically on liquid ms medium for specified times  before being fixed in 75% ethanol and 25% acetic acid for 1 h at 4°c.
peels of the adaxial epidermis and blocks of storage parenchyma cells  were surgically removed from each fixed cotyledon.
collected tissues were immediately snap-frozen in liquid nitrogen and stored at -80°c until used for rna extraction.
selection of times to obtain tissue samples from cultured cotyledons was based on the temporal pattern of uniform wall deposition preceding that of wall ingrowths .
to this end, for the reference library, a representative balance of genes induced to regulate uniform wall and wall ingrowth construction was obtained by collecting 6 mg of epidermal peels from each of freshly harvested cotyledons  and cotyledons cultured for  <dig> and 3 h ,  <dig> and 9 h .
to identify expression of tc-specific genes regulating uniform wall and wall ingrowth construction, epidermal peels  and storage parenchyma tissue  were sampled from replicate batches of freshly harvested cotyledons and cotyledons cultured for 3 h  and 12 h .figure  <dig> tissue sampling times  informed by the sequential temporal deposition of the uniform wall followed by wall ingrowth papillae.
note that across the period sampled, wall ingrowth papillae continue to be deposited and have not commenced to form the first fenestrated layer  <cit> .
ec, epidermal cell.
to verify that each sampled population of cultured cotyledons was trans-differentiating, percentages of epidermal cells forming wall ingrowths of a sub-set of cotyledons at 12 h of culture were scored .
rna isolation, cdna-library construction and illumina sequencing
total rna was extracted using qiagen  rneasy plant mini kits.
contaminating genomic dna was removed using dnase i.
for preparation of an epidermal-specific tc transcriptome reference library, 1 μg of total rna was aliquoted from the total rna extracts of each specified harvested time point.
the rna aliquots were combined to generate a temporally mixed rna library .
to identify genes responsible for uniform wall and wall ingrowth formation, 2 μg aliquots of total rna were sampled from each biological replicate of epidermal and storage parenchyma rna extracts.
total rna quality was verified by determining the integrity of the 25s and 18s rna with an agilent  <dig> bioanalyzer .
the cdna libraries were prepared from poly-a mrna isolated from 1 μg of total rna using a truseq® rna v <dig> sample prep kit  according to manufacturer’s instructions.
cdna quality was evaluated by determining size and purity using an agilent  <dig> bioanalyzer .
cdna fragments, ranging from  <dig> – 700 bp, were selected by agarose gel purification.
for the reference library, selected cdna fragments were 100 bp pair-end sequenced in a single lane on an illumina hiseq  <dig> platform .
to characterize gene cohorts linked with uniform wall and wall ingrowth deposition, only biological replicates with the anticipated 70%  of their epidermal cells at 12 h of cotyledon culture containing detectable wall ingrowths  <cit>  were processed for illumina sequencing.
in three compliant biological replicates for each specified harvest time, cdna libraries  were prepared from total rna extracts of epidermal peels and storage parenchyma tissues as indicated above .
cdna fragments were purified separately from each sample and indexed with unique nucleic acid identifiers .
the indexed cdna libraries were diluted to an average concentration of  <dig> nm and pooled in equal volumes  to generate the final mixed cdna pool for sequencing.
the pool was then 100 bp pair-end sequenced in two lanes on an illumina hiseq  <dig> platform .
using illumina casava pipeline version  <dig> .
<dig>  raw reads were trimmed with adaptor filtering and a read length cut-off of 50%.
thereafter, filtered reads with over 20% of their nucleotides having a q score <  <dig>  or their sequences having a n reading over 5% were removed.
de novo assembly, annotation and go classification of transcriptome library
filtered reads from the cdna library were assembled de novo into contigs by beijing genomics institution  using trinity software release- <dig>  <cit>  with a k-mer of  <dig> and a minimum k-mer threshold abundance of  <dig> .
the reads were then mapped back to contigs to assemble unigenes using trinity butterfly that filtered out transcriptional artifacts, misassembled transcripts and poorly supported transcripts.
potential redundant sequences were grouped, using the tgi clustering tool set with a minimum 40% sequence overlap and over 80% sequence identity.
assembled unigene sequences were annotated by alignment to the following publically available databases: ncbi nr ; swissprot ; kegg ; cog , using blastx with an e-value threshold of 1e- <dig>  unigenes with no hit in blastx were predicted using estscan.
mapping reads to the reference library, determining differentially expressed genes and go-enrichment analysis
reads sequenced by the illumina hiseq  <dig> platform from mrna extracts of cotyledon epidermal peels and storage parenchyma tissues were analyzed by bgi.
raw reads were filtered as described for the reference transcriptome library.
clean reads were aligned to the reference sequences  using the soapallgner/soap <dig> pipeline.
no more than  <dig> mismatches per read were allowed to ensure high quality alignment.
sequence coverage of unigenes in each sample was calculated.
during alignment, soapallgner/soap <dig> reported the number of mapped reads per kilo base per million reads  as a measure of transcript abundance of each unigene.
transcripts with rpkms <  <dig>  were considered not to be expressed and were removed from the data sets.
differentially expressed genes  were determined using a computational algorithm based on digital gene expression profiles  <cit>  to perform pairwise differential expression analysis.
multiple testing of the comparisons was corrected using the fdr method  <cit> .
sequences with a corrected fdr p value of no more than  <dig>  were selected.
after this correction, genes exhibiting a differential expression of two-fold or more  were identified as degs.
subcellular localization of selected proteins encoded by unigenes was predicted using the wolf psort algorithm.
quantitative rt-pcr validation
a collection of  <dig> unigenes with different expression patterns  was selected.
cdna was converted from the same rna samples sent for illumina sequencing using quantitect reverse transcription kit .
primers were designed using primer  <dig> plus  and synthesized by sigma-aldrich australia .
for each qrt-pcr reaction a 15 μl system containing  <dig>  μl sybr green master mix ,  <dig>  μl of forward and reverse primers ,  <dig>  μl of nuclease free h2o and 5 μl cdna was set up.
the following pcr cycle was used: 95°c for 5 min, 95°c for 15 s, 60°c for 20s, 72°c for 30 s; steps  <dig> to  <dig> were repeated  <dig> times.
high-resolution melting curves  following the final pcr cycle checked the specificity of the pcr products.
for each cdna sample, technical duplicates in each of three biological replicates were tested.
four housekeeping gene candidates were assessed using genorm.
relative expression levels of each unigene were determined using the two standard curves method.
RESULTS
de novo assembled transcriptome library for ingrowth wall deposition
sequencing of reference library cdna fragments on an illumina hiseq  <dig> platform generated  <dig> , <dig> pair-end reads .
after filtering raw reads,  <dig> , <dig> high-quality reads, in which 97% of nucleotides have a phred quality score of ≥ q <dig> level   were de novo assembled into  <dig>  contigs  with a n <dig> value of 980 bp and an average length of 423 bp.
clustering yielded  <dig>  unigenes with  <dig>  consensus sequences grouped into  <dig>  distinct clusters and  <dig>  singletons .
this unigene population had a n <dig> value of 1723 bp and an average length of 1076 bp.
high integrity and accuracy of the assembly was indicated by  <dig> % of filtered reads mapping to the assembled transcriptome with  <dig> % of reads uniquely mapping to the transcriptome .
the uniquely mapped reads had an average coverage depth of  <dig>  .table  <dig>
raw, filtered and mapped reads used for the
de novo
assembly of the reference transcriptome library for adaxial epidermal cells of freshly harvested and cultured
v. faba
cotyledons undergoing
trans
-differentiation to a tc morphology
category
number
percentage
blastx searches of publically available protein databases, using the putative unigenes as query sequences, yielded matches for  <dig> % of the unigenes.
blastx-derived alignments predicted sequence orientation for  <dig>  of the unigenes and identified  <dig>  open reading frames.
taxonomic distribution of the annotated unigenes by blast2go demonstrated that over 85% unigenes had their best hits  with their homologs in other legume species .
the raw reads and assembled unigene sequences are available at european nucleotide archive, accession number: prjeb <dig>
global gene expression patterns during early phases of trans-differentiation to a tc-morphology
total rna was extracted from epidermal peels and storage tissue blocks at  <dig>   <dig> and 12 h of cotyledon culture and processed for rnaseq on an illumina platform .
between  <dig> –  <dig> million clean reads were generated for each biological replicate .
an average of 85% of the clean reads mapped to expressed unigenes.
of these reads, 60% mapped with a perfect match and 40% with a mismatch less than 5 bp .
total numbers of unigenes, detected within epidermal and storage parenchyma cells across cotyledon culture were temporally stable .
some 70% of the unigenes exhibited expression levels >  <dig>  rpkm within a cell type and these were dominated by unigenes with expression levels ranging from  <dig>  to  <dig> rpkm .
an estimated  <dig>  unigenes were expressed in adaxial epidermal cells of developing v. faba cotyledons .
of these epidermal cell unigenes,  <dig>  were shared with the underlying storage parenchyma cells leaving  <dig>  unigenes that were epidermal cell specific .
upon transiting to a tc fate, expression of  <dig>  unigenes in the precursor epidermal cells was switched off .
the combined transcriptomes of trans-differentiating tcs at  <dig> and 12 h of cotyledon culture totalling  <dig>  unigenes approximated those detected by rnaseq analyses of differentiating betcs in barley grains  and of nematode giant tcs in rice roots .
seventy-three % of the epidermal tc transcriptome was represented by ongoing expression of genes by their precursor epidermal cells whilst 27% were induced .
the percentage of induced genes was comparable to the proportion of up-regulated genes in nematode giant tcs  <cit> .
of the induced genes, 66% were tc specific and the remainder was shared with the underlying storage parenchyma cells .
ten % of the epidermal cell genes were silenced in transiting to an epidermal tc identity and these were replaced 3-fold by induced genes .
tc specific genes were inspected manually to remove duplicates and those with < 70% coverage to yield a total of  <dig>  unigenes .figure  <dig> numbers of unigenes expressed preferentially in, or shared between,  cell types and  uniform wall and wall ingrowth formation occurring within adaxial epidermal cells of cultured v. faba cotyledons.
only unigenes with a rpkm value >  <dig>  in all three biological replicates were defined as ‘expressed’ in one or more cell types/phases of ingrowth wall deposition.
for , only unigenes with a coverage of > 70% were listed for further analysis.
ec- adaxial epidermal cell; tc – epidermal tc trans-differentiated from adaxial epidermal cell; sp- storage parenchyma cell; uw - uniform wall; wi- wall ingrowth.
transcriptome networks specifically expressed during uniform wall or wall ingrowth formation
the temporal sequence of uniform wall followed by wall ingrowth deposition  provided the opportunity to separate the tc-specific cohort of expressed genes into groups linked with the two phases of ingrowth wall construction and those shared between these phases as follows.
the latter were identified by manual inspection of the 3- and 12-h gene expression profiles .
the remaining expressed genes were then specifically linked with either uniform wall or wall ingrowth formation and these were identified on the following grounds.
at 12 h of cotyledon culture, uniform wall construction had ceased and tc-specific gene expression is solely committed to deposition of wall ingrowths .
thus the expression profile at 12 h contained transcripts specifically related to constructing wall ingrowths alone .
in contrast, at 3 h of cotyledon culture, uniform wall construction is proceeding rapidly concurrent with a smaller population of cells starting to deposit wall ingrowths .
hence at 3 h of culture, genes involved in uniform wall and wall ingrowth construction would be co-expressed.
however, those exclusively directing deposition of wall ingrowths would be expressed at relatively lower levels.
thus subtracting the 12-h  from the 3-h  gene expression profile identified those genes specifically expressed during uniform wall construction .
the above exercise demonstrated that of the tc-specific expressed genes, 66% were selectively linked with uniform wall  or wall ingrowth  formation with the remainder  being expressed throughout ingrowth wall formation .
this latter cohort of genes was categorised according to their relative expression levels in the two phases of ingrowth wall formation, that is, no change and up-regulated during uniform wall or wall ingrowth deposition.
genes whose expression levels did not change accounted for 60% of the common cohort .table  <dig>
numbers of annotated expressed genes ascribed to specified functional categories for genes switched off in epidermal cells transiting to a tc fate and for those specifically expressed in epidermal cells undergoing
trans
-differentiation to a tc morphology
functional category
epidermal switched-off genes
transfer cell specific expressed genes
uw/wi no change
uw up-regulated
uw specific
wi up-regulated
wi specific
total number of expressed genes
total number of annotated genes
dna synthesis and modelling
transcription/translation
signalling
ros/ca2+
intracellular organization
cell wall enzymes and structural proteins
membrane transporters
defense
flavonoid synthesis and compartmentation
tc specific genes separated into those genes expressed throughout uniform wall  and wall ingrowth  formation and those that are specific to each of these wall-building phases.
genes expressed throughout ingrowth wall formation are separated into groups depending on their differential expression patterns of no change, up-regulated during uw or wi formation .
blastx searches, at a stringency of < e- <dig>  established that 41% of the tc-specific genes and 34% of epidermal cell genes switched off shared homology with known genes listed in public databases .
the 59/ <dig> % of un-known transcripts equates with a similar value reported for the transcriptome of developing barley betcs  <cit> .
table  <dig> provides a summary of the changes in expression of selected functional categories of tc-specific genes that are known to be central to tc development and function.
for all categories, numbers of epidermal cell genes switched off in transiting to a tc fate were replaced by increased numbers of tc-specific transcripts between 3- to 12-fold.
amongst these, expression of transcription/translation, receptor kinase/kinases, cytoskeleton/vesicle trafficking, matrix polysaccharides and defense genes contributed to the two-fold greater number of genes expressed during uniform wall compared to wall ingrowth construction .
in addition, a portion of uniform wall associated expressed genes were linked with a co-occurring burst in cell division/expansion .
genes encoding the entire flavonol pathway were switched on during tc development .
a more detailed analysis was undertaken of the temporal expression of tc-specific genes contained in functional groups that are at the heart of regulating development of the tc’s structural specialization of a polarized ingrowth wall that realises their functional capacity to support high rates of nutrient transport across their plasma membranes  <cit> .
these categories included hormonal and ros/ca2+ signal generation and their signalling pathways, cytoskeleton/vesicle trafficking and lipid biosynthetic enzymes related to microdomain formation, cell wall biosynthetic and remodelling enzymes along with cell wall structural proteins and membrane transporters .
these data are supplemented with estimates of their annotation and relative expression levels at specified stages of ingrowth wall construction along with noting whether their homologues have been detected in transcriptomes of betcs and nematode giant tcs .
temporal patterns of expression of selected genes from the targeted functional categories determined by qrt-pcr were consistent with those derived from illumina sequencing , suggesting that the illumina data are relatively reliable .
discussion
the ability to obtain peels of cotyledon epidermal cells permitted cell-specific transcriptomes of these cells to be identified as they trans-differentiated to a tc-morphology  <cit> .
in addition, developmental stage-specific transcriptomes were isolated  by taking advantage of the temporal disjunction between uniform wall and wall ingrowth construction .
we interrogated these tc-specific transcriptomes to discover genes encoding proteins of functional categories considered to contribute to developing a transport capable tc .
transcriptional cascades are initiated by auxin and ethylene  <cit>  along with ros and ca2+ serving as secondary messengers and positional signals  <cit> .
the substantial numbers of expressed receptor kinases/kinases and transcription factors, point to these forming a significant component of the signalling network .
re-organization of the cytoskeleton and vesicle trafficking together with membrane microdomains, under control of the inductive signalling network, would be expected to direct targeted delivery of cargos containing cell wall biosynthetic enzymes and cell wall components to construct the polarized uniform wall and localized wall ingrowths .
the latter provide a scaffold to support an amplified plasma membrane surface area to which membrane transporters are targeted through a polarized endomembrane secretory system to confer enhanced tc transport function .
in addition, targeted positioning of transporters is responsible for generating the polarized extracellular ros  <cit>  and cytosolic ca2+  <cit>  signals and possibly altering positioning of auxin transporters that may contribute to generating the auxin maximum .
the following discussion focuses on expressed genes belonging to these three functional categories namely, signals and signalling pathways, intracellular organization and transport function .
the discussion starts with seeking out candidates responsible for generating the developmental signals and downstream signalling pathways.
in all cases, statements regarding gene identity and function should be considered as putative.figure  <dig> regulatory interrelationships  between the functional categories of expressed genes that contribute to developing a functional transfer cell.
signals and signalling pathways
hormonal signals, acting in series with ros and ca2+  <cit> , likely activate expression of receptor kinases/kinases and transcription factors to induce tc trans-differentiation .
auxin, ethylene and abscisic acid form a complex of primary signals putatively regulating tc development
consistent with previous findings for epidermal tcs  <cit>  and those reported for nematode giant tcs , induction of trans-differentiation was accompanied by changes in auxin activity  increasing five-fold between uniform wall and wall ingrowth formation driven by an auxin maxima generated by enhanced auxin biosynthesis and disrupted polar transport .
for instance, auxin biosynthesis could be elevated by expression of indole-3-acetate o-methyltransferase iaa, that catalyzes synthesis of a biologically more active iaa, methyl-indole-3-acetic acid  <cit>  and indole-3-glycerol phosphate synthase that starts the tryptophan independent iaa biosynthetic pathway  <cit> .
polar auxin transport was likely disrupted by de-localized distribution of expressed auxin efflux and influx carriers by co-expression of kan and hd-zipiii  <cit> .
as a point of distinction between mechanisms regulating flange and reticulate wall ingrowths, in contrast to the latter  <cit> , differentially expressed auxin-related genes were not detected in developing betcs  <cit> .
initiation of epidermal tc development is linked with a substantive burst in ethylene biosynthesis, regulated by an auxin-induced expression of acc synthases  <cit> , driving expression of ethylene response factors   <cit>  and particularly so for uniform wall formation .
for example, erf <dig>  and shine expression could invoke cell wall biosynthesis through respectively up-regulating callose and cellulose biosynthesis .
strong expression of ethylene signalling genes during uniform wall formation corresponded with that reported for early phases of wall ingrowth construction in barley betcs  <cit>  and nematode giant tcs  <cit> .
a switch to a specific aba signalling pathway throughout ingrowth wall formation was suggested by expression of pyl <dig>  an aba receptor  <cit> , lec <dig>  tightly connected with aba signalling regulating ab <dig> and ab <dig>  <cit>  and erf rap <dig>  that acts through an ab14-mediated signalling pathway .
aba also acts during wall ingrowth formation .
aba action throughout ingrowth wall formation in epidermal tcs contrasts with that for barley betcs where aba induces a maturation program once wall ingrowth deposition ceases  <cit> .
three gibberellin biosynthetic genes with the biological activities of their products being modulated by two gibberellin 2-oxidases and short internode related during uniform wall formation are expressed .
these gibberellins likely regulate cell division and expansion .
an absence of gibberellin activity during wall ingrowth construction was suggested by co-expression of short internode related and gibberellin 2-beta-dioxygenase 8-like .
residual cytokinin levels in the developing epidermal tcs would be depleted through degradation by the expressed cytokinin dehydrogenase  <cit>  and by conjugation to an inactive form catalyzed by the five cytokinin-o-glucosyltransferases .
no components of the salicylic acid or brassinosteroid signalling pathways were detected in the tc-specific transcriptome  indicating that these pathways likely did not play a role in trans-differentiation to a tc state.
reactive oxygen species and ca2+ serve as secondary messengers directing ingrowth wall construction
extracellular ros, produced by ethylene-induced respiratory burst oxidases , regulate biosynthesis and polarized deposition of the uniform wall in epidermal tcs  <cit> .
similarly, rboh expression co-occurs with wall ingrowth formation in barley betcs  <cit> .
in contrast, no differentially expressed rbohs were detected in nematode giant tcs  <cit> .
interestingly, ros-related gene expression was dominated by those involved in extra- and intracellular ros homeostasis  suggesting that ingrowth wall formation depends upon a spatio/temporal fine-tuning of ros.
for instance, antioxidant capacity could be down regulated by ascorbic acid oxidation catalyzed by an extracellular l-ascorbate oxidase  whilst concurrent ros degradation by peroxidation  <cit>  could be mediated by carboxymethylenebutenolidase-like protein , secreted to the cell wall  <cit> .
in contrast to epidermal tcs, differentially expressed peroxidases were not detected in barley betcs  <cit>  and were strongly down-regulated in nematode giant tcs  <cit> .
loci at which wall ingrowths are deposited are defined by a cytosolic ca2+ signal spatially organized into narrow plumes shaped by co-operative activities of plasma membrane ca2+-permeable channels organized into clusters surrounded by ca2+-atpases  <cit> .
however, unlike the unknown dhp receptor ca2+-permeable channels responsible for generating the ca2+ signal  <cit> , ion conductances of the expressed skor, annexin and cyclic nucleotide gated channels are not inhibited by verapamil or nifedipine .
this, together with channel expression coinciding with uniform wall construction , points to an additional role for ca2+ signalling in transiting precursor epidermal cells to a tc fate.
a conclusion that accommodates ca2+ signalling genes being expressed in both flange  <cit>  and reticulate forms  <cit>  of developing tcs.
ca2+ signalling would appear to be mediated through ca2+-dependent protein kinases during uniform wall formation while a calmodulin signalling route could operate when wall ingrowths are being deposited as suggested by expression of three iq-domain containing proteins  that bind calmodulins to facilitate their function.
receptor kinases/kinases represent a significant component of the signalling network
tc-specific receptor kinases/kinases represented a major component of the signalling networks  expressed within the epidermal tcs .
while their functions are unknown, similar to nematode giant tcs  <cit> , members of a two component signalling system present in barley betcs  <cit> , was not detected in the epidermal tc cohort of receptor kinases/kinases.
thus, the presence/absence of a two component signalling system could underpin the distinction between flange  <cit>  and reticulate  tc wall architectures.
intracellular organization
polarized accumulation of extracellular ros regulating uniform wall synthesis  <cit>  and cytosolic ca2+-plumes determining loci for wall ingrowth formation  <cit>  respectively depend upon a finely-tuned intracellular distribution of rbohs and ca2+-permeable channels.
this requirement is undoubtedly met through modifying cytoskeleton and vesicle trafficking combined with re-configuring microdomains in the plasma membrane abutting sites of ingrowth wall deposition .
tc-specific expression of cytoskeleton and vesicle trafficking confers tc polarity
microtubule and actin-associated genes switched off on transiting to a tc fate were predominantly linked to cell division  that declined rapidly from three to six hours .
consistent with polarized cell wall deposition  <cit> , tc- specific genes indicative of remodelling cytoskeletal elements  <cit>  were expressed either across ingrowth wall deposition  or only during one phase of the process .
uniform wall specific gene expression  indicated substantial polymerization/reorganization of actin filaments  and polarization of transport vesicles and organelles   <cit>  consistent with ros-regulated polarization of the uniform wall  <cit> .
wall ingrowth specific expression of 65-kda microtubule-associated protein 3-like and villin- <dig> respectively suggests establishing a stable microtubule  <cit>  and actin  <cit>  network to orchestrate deposition of wall ingrowths at loci.
in root hair growth of arabidopsis, atvln <dig> regulated actin organization is ca2+-dependent  <cit>  pointing to an interaction between actin and ca2+plumes that define wall ingrowth loci  <cit> .
rapid polarized/localized cell wall deposition during formation of the ingrowth wall  is dependent upon exo/endocytotic activity  <cit> .
regulation of cycling of er-derived proteins through assembly and directed transport of coat protein complex ii  and arfs  across ingrowth wall deposition is consistent with asymmetrical vesicle trafficking to effect polarity  <cit> .
expression of uniform wall- and wall ingrowth-specific exocyst complex components and v- and t-snares , to direct targeted vesicle delivery to the plasma membrane  <cit> , confirms the specialized nature of each phase of ingrowth wall deposition.
two of four genes encoding proteins involved in membrane trafficking  are potential regulators of localized endocytosis supporting wall ingrowth deposition.
these two dynamin genes represent two subfamilies of drps that interact and assemble with clathrin at discrete foci in the plasma membrane of arabidopsis cells to regulate endocytosis  <cit>  and cellulose deposition  <cit> .
this profile of cytoskeleton and vesicle trafficking genes is comparable to that reported for betcs  <cit>  and nematode giant tcs  <cit> .
formation of membrane microdomains may sub-compartmentalize the plasma membrane polar domain
sphingolipids and sterols form major components of membrane microdomains  <cit> .
indeed, genes encoding enzymes in their biosynthetic pathways are expressed during development of flange wall ingrowths in barley betcs  <cit> .
sphingolipids consist of a polar head group linked to an amino alcohol long-chain base  with the amine group acylated with a fatty acid  with c <dig> of lcb being  or not being  hydroxylated.
bax inhibitor 1-like  post-translationally activates the c <dig> hydrolase  <cit>  thus promoting flux through this route for acylation by very long-chain fatty acids .
significantly, synthesis of vlcfas may be enhanced by expression of four 3-ketoacyl-coa synthase isoforms, a elongation of fatty acid protein and a long-chain-fatty-acid-coa ligase  that form part of the elongase complex  <cit> .
an increased precursor flow into the vlcfa pathway is supported by up-regulated expression of biotin carboxyl carrier protein of acetyl-coa carboxylase , a sub-component of acetyl-coa carboxylase catalyzing the first carboxylation step in, and rate limiting of, de novo fatty acid synthesis  <cit> .
thereafter, bax inhibitor 1-like may also increase activities of fatty acid hydroxylases and desaturases that further modify the ceramides  <cit>  before synthesis of the final sphingolipid products of glucosylceramides or glycosyl inositolphosphoceramides  <cit> .
uniform wall specific expression of an oxysterol-binding protein-related protein and a remorin  respectively point to vesicle trafficking of sterols  <cit>  and their enrichment in membrane microdomains  <cit> .
wall ingrowth deposition is linked with expression of putative sterol biosynthetic genes  such as two epoxide hydrolases  <cit>  and squalene epoxidase  <cit> .
tc transport function
expression of cell wall synthesizing and re-modelling enzymes underpins construction of the ingrowth wall that supports the amplified plasma membrane in which are embedded high densities of membrane transporters to collectively confer an enhanced transport function .
expression of cell wall biosynthesis and re-modelling genes construct the ingrowth wall
cellulose is present in the uniform wall and the inner, electron dense region, of wall ingrowths of epidermal tcs  <cit>  but cellulose biosynthesis appeared not to depend on expression of tc-specific cesas that were switched off and not replaced as the epidermal cells transited to a tc fate .
rather, cellulose deposition could be regulated post-translationally .
thus during uniform wall formation, shine  <cit>  and a gpi-anchored protein  <cit>  could regulate cesa activity with the extruded cellulose microfibrils being re-modelled by two extracellular endoglucanases  <cit> .
cellulose provides a structural scaffold to form wall ingrowths at right angles to the uniform wall  <cit> .
here, an acid endochitinase-like protein, known to associate with cellulose synthase complexes  <cit>  and an extracellular beta-glucosidase, may form part of the machinery redirecting extrusion of cellulose microfibrils .
consistent with these observations, cesa expression is down-regulated in nematode giant tcs  <cit>  while in maize and barley betcs, cesa expression is up-regulated during later stages of tc development  <cit> .
in contrast to cellulose biosynthesis, there was a substantial tc-specific induction of genes encoding enzymes responsible for synthesizing and re-modelling cell wall matrix polysaccharides  and, in particular, hemicelluloses .
to this end, a  β-d-glucan synthase, was switched off and replaced with enzymes catalyzing xylan biosynthesis  and re-modelling .
xylans in the uniform wall are consistent with their presence in flange wall ingrowths as suggested by expression of a beta-d-xylosidase and a xylosyltransferase respectively in betcs of barley  <cit>  and maize  <cit> .
xyloglucan presence in uniform walls  <cit>  is consistent with expression of xylosyltransferases  and endoxylotransglucosylase/hydrolases .
xyloglucan biosynthesis and re-modelling also dominates early developmental stages of barley betcs  <cit>  and nematode giant tcs  <cit> .
xyloglucan re-modelling coincides with tc division/expansion as indicated by co-expression of expansins .
in contrast, expansins expressed during wall ingrowth construction when cell division/expansion has ceased , must serve a yet-to-be identified function.
one possibility is to free xyloglucan chains from cellulose microfibrils rendering them available for co-operative re-modelling by alpha-xylosidases and a beta-glucosidase  <cit>  and by two extracellular isoforms of beta-galactosidase .
overall, uniform wall and wall ingrowth construction was characterized by intense xyloglucan re-modelling.
pathways leading to pectin biosynthesis and modification altered on epidermal cells transiting to a tc fate .
during uniform wall construction, synthesis of the pectin rhamnogalacturon backbone  was likely catalyzed by expression of galactofuronosyl-transferase 13-like  that significantly is essential for polarized pollen tube tip growth  <cit> .
control over the pectin gel state is conferred by co-expression of a pectin esterase inhibitor modulating catalytic activities of expressed pectin esterases  that catalyze removal of methyl ester groups from homogalacturonan backbones to allow their cross-linking with ca2+  <cit> .
this accounts for esterified pectins being the predominate type present in the ingrowth wall  <cit> .
up-regulated and wall ingrowth specific expression of two beta-galactosidases could catalyze cleavage of terminal galactosyl residues from rgi  <cit> .
overall, uniform wall formation included pectin biosynthesis and re-modelling giving way to pectin re-modelling alone for wall ingrowth deposition.
enhanced pectin biosynthesis and re-modelling also characterizes construction of barley betcs  <cit>  and nematode giant tcs  <cit> .
callose deposition is a balance between synthesis by callose synthases and degradation by β- <dig>  3-glucanases.
isoforms of both enzymes were switched off in epidermal cells on transiting to a tc fate and were not replaced during uniform wall formation .
however, udp-glucuronosyltransferase  <dig> , a callose synthase complex component essential for callose synthesis  <cit> , was expressed during wall ingrowth construction accompanied by two beta- <dig>  3-glucanase isoforms ; an expression pattern consistent with callose being confined to the outer sheath enveloping each wall ingrowth  <cit> .
this temporal profile of callose deposition corresponds with that reported for nematode giant tcs  <cit>  but contrasts with callose synthesis/turnover being restricted to the cell division phase of barley betc development  <cit> .
transcripts encoding enzymes of the phenylpropanoid pathway occurred across ingrowth wall formation .
expressed abc transporters  could efflux the monolignol precursors to the cell wall where class iii peroxidases  catalyze their polymerization to lignin  <cit> .
significantly, the full complement of phenylpropanoid pathway enzymes is expressed in nematode giant tcs  <cit> , and a detailed histochemical study of maize betcs  <cit> , provides strong evidence for their lignification.
extensins are expressed in tcs forming flange  <cit>  and reticulate  <cit>  wall ingrowths.
a large number  of extensin family members of hydroxyproline-rich glycoproteins were expressed in developing epidermal tcs , implying a key role played by extensins in shaping the ingrowth wall and, in particular, wall ingrowths.
a key enzyme in the post-translational modification pathway generating functional extensins, prolyl 4-hydroxylase  <cit> , was expressed throughout ingrowth wall formation .
upon being exocytosed into the cell wall space, extensin monomers are envisaged to cross-link to form networks, mediated by redox activity of type-iii apoplasmic peroxidases .
expression of a proline-rich-extensin-like receptor kinase , regulating extensin/pectin interactions  <cit> , may generate supramolecular structures  <cit>  to function as templates for ingrowth wall formation.
significantly, coincident with wall ingrowth construction, was the expression of two nodule-specific extensins that support polarized tip growth in elongating infection threads  <cit>  and a dz-hrgp homolog that contributes to polarized tip growth of pollen tubes  <cit> .
a large array of membrane transporters is specifically expressed in tcs
the large number of transporter genes switched off upon transiting to an epidermal tc fate  suggests a substantial change in transport function.
a striking feature of the  <dig> tc-specific membrane transporters, whose expression did not change during trans-differentiation, nine transport inorganic and organic nitrogen compounds .
five transport amino nitrogen compounds including two aquaglyceroporins .
two nucleoside proton symporters, adenine/guanine permease azg <dig>  <cit>  and equilibrative nucleoside transporter  <dig>  <cit> , likely function to retrieve apoplasmic nucleosides released from degradation of endosperm and inner cell layers of seed coats crushed by expanding cotyledons.
collectively, expression of these transporters reflects a demand for nitrogenous precursors to support protein biosynthesis.
expression of a putative plasma membrane potassium transporter and tonoplast potassium proton antiporter  ensure potassium homeostasis.
investment into nitrogen transporters was enhanced during uniform wall formation .
this included a chloride channel  that may function as a nitrate sensor to regulate development by modulating auxin transport  <cit> .
two additional transporters were recruited for potassium homeostasis; a skor channel  and a potassium antiporter .
most pronounced was the large number  of expressed abc transporter genes  linked with expression of flavonoid biosynthetic and defense genes .
however, along with effluxing xenobiotics, members of the abcb transporter subfamily facilitate plasma membrane transport of iaa  <cit>  contributing to the altered iaa homeostasis of epidermal tcs .
with the onset of wall ingrowth deposition, appearance of two sugar transporter transcripts  reflects a growing dependence on an extracellular carbon supply  <cit> .
further investments were made in amino nitrogen and potassium transporters .
an ongoing expression of abc transporters  corresponded with expression of flavonoid biosynthesis and defense genes .
CONCLUSIONS
in epidermal cells undergoing trans-differentiation to a tc-morphology, only a small proportion of their transcriptome  was found to be tc-specific.
within the cohort of tc-specific transcripts, those encoding pathways regulating signalling and intracellular organization were fine-tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a tc fate.
each phase of ingrowth wall construction was linked with an unique sub-cohort of expressed genes.
comparison of transcriptomes of cells forming flange or reticulate wall ingrowths detected subtle differences in their transcript profiles, with the major difference being the absence of a two-component signalling system from the latter.
availability of supporting data
the cdna sequence datasets of raw reads and assembled reference transcriptome library supporting the results of this article are available in the repository of the european nucleotide archive  with the ena accession number: prjeb <dig>
additional files
additional file 1:
figure s <dig>  table s <dig> and accompanying text.
temporal formation of the uniform wall and wall ingrowths.
additional file 2: figure s <dig>  quality assessment of total rna extracted from adaxial epidermal and storage parenchyma cells of v. faba cotyledons.
additional file 3: table s <dig>  quality assessment of total rna extracted from, and cdna libraries of v. faba cotyledons used for illumina sequencing.
additional file 4: table s <dig>  annotation and expression levels of all genes specifically expressed in the epidermal cells of cultured v. faba cotyledons trans-differentiating to epidermal transfer cells.
additional file 5: table s <dig>  annotation and expression levels  of selected unigenes expressed specifically in trans-differentiating adaxial epidermal cells of v. faba cotyledons  <cit> .
additional file 6: table s <dig>  primer sequences used for qrt-pcr determination of expression levels of specified genes to validate rnaseq data sets.
additional file 7: figure s <dig>  species distribution of genes with highest sequence similarity to unigenes in the reference transcriptome library of trans-differentiating adaxial epidermal cells of cultured v. faba cotyledons.
additional file 8: table s <dig>  reads generated from illumina sequencing used to identify gene cohorts linked with uniform wall and wall ingrowth deposition in trans-differentiating epidermal cells of v. faba cotyledons.
additional file 9: table s <dig>  temporal profile of unigene numbers expressed in adaxial epidermal or storage parenchyma cells of cultured v. faba cotyledons.
additional file 10:
figure s <dig> and associated text.
cell division and expansion of adaxial epidermal cells of cultured v. faba cotyledons.
additional file 11: table s <dig>  genes encoding proteins generating or transducing developmental signals switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology.
additional file 12: table s <dig>  genes encoding proteins involved in regulation of the cytoskeleton and vesicle trafficking and sphingolipid/sterol synthesis and transport that are switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology.
additional file 13: table s <dig>  genes encoding cell wall biosynthetic and remodelling enzymes and structural proteins switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology.
additional file 14: table s <dig>  genes encoding membrane transporters switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology.
additional file 15: figure s <dig>  validation of expression patterns of unigenes obtained from illumina sequencing using quantitative rt-pcr.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jwp and ceo initiated the research and designed the experimental approach.
hmz, xx and sw performed the experiments.
hmz processed the data and hmz, jwp, ceo, rr and hw interpreted the results and prepared the manuscript.
all authors have read and approved the final manuscript.

 transfer cells trans-differentiate from a range of existing cell types belonging to the major tissue systems of dermal, ground and vascular. once tc development is completed, a 10- to 20-fold amplification of their plasma membrane surface area, containing high densities of solute transporters, confer these cells with an extraordinarily high capacity for nutrient exchange between apo- and symplasmic compartments located at bottlenecks for long-distance transport of nutrients throughout the plant body. an intricate invaginated complex of cell wall ingrowths provides structural scaffolding on which the amplified plasma membrane is arrayed to enhance nutrient flows. the tight coupling of this structure/function relationship is graphically illustrated by compromised seed filling of mutants in which tcs, located at the maternal/filial interfaces of both eudicots and monocots, exhibit an attenuated construction of their cell wall ingrowth complex. since a number of major crop species, including cereals and grain legumes, contain tcs positioned at the maternal/filial interfaces of their developing seeds, the shrivelled seed phenotype exhibited by tc mutants underscores the important role these cells play in determining crop yields. the cell wall ingrowth complex is organized into one of two architectural types – flange or reticulate. flange ingrowths form as ribs or bands of wall material while the more commonly occurring reticulate wall ingrowths arise as numerous wall papillae that develop at right angles to the original wall. the extent of their reticulation varies from cylindrical papillae alone to ones in which papillae branch and fuse to generate fenestrated wall layers. immediately preceding construction of reticulate, but not flange wall ingrowths, a structurally distinctive wall-layer, the so-called uniform wall, is rapidly laid down over the pre-existing primary wall of the trans-differentiating cell to a thickness that can extend up to 50% of that of the pre-existing primary wall. collectively, the uniform wall and reticulate wall ingrowths form the ingrowth wall. despite the central role the ingrowth wall, and particularly its wall ingrowth component, plays in underpinning transport function of tcs, little is known about the mechanisms responsible for inducing and then orchestrating its construction. transcriptional analyses of cells committed to a developmental pathway leading to forming a tc-morphology have identified up-regulated expression of genes encoding components of signalling pathways for abscisic acid, auxin, ethylene, reactive oxygen species and ca2+ in developing seeds and for auxin and ethylene in giant cells induced by nematode infection. events downstream from the inductive signals largely have been identified by transcriptome analyses of developing cereal seeds and, in particular, for flange wall ingrowths formed in basal endosperm tcs of maize and barley. a more limited analysis has been reported for the formation of reticulate wall ingrowths in eudicots. while there are undoubtedly some shared features, given the disparate architectures of flange and reticulate wall ingrowths, we hypothesize that signalling pathways, cell wall biosynthesis and delivery of polysaccharides to the wall matrix will have features that are peculiar to each architectural type of ingrowth wall. this characteristic likely extends to distinctive elements responsible for constructing the uniform wall and wall ingrowth papillae. to this end, we used vicia faba cotyledons, in which on transfer to culture, their adaxial epidermal cells spontaneously undergo trans-differentiation to a tc-morphology. this experimental system provided the opportunity to undertake a transcriptome analysis of tc-specific gene networks. in addition, because of the temporal sequence for the deposition of the uniform wall followed by wall ingrowth papillae, gene cohorts associated with these two developmental events could be distinguished. hereafter, wall ingrowth papillae, which represent the initial stage of wall ingrowth construction, are referred to as wall ingrowths. methods plant growth conditions, cotyledon culture and collection of tissue samples for sequencing v. faba l. plants were raised under controlled environmental conditions. cotyledons of harvested pods were surgically excised and cultured aseptically on liquid ms medium for specified times before being fixed in 75% ethanol and 25% acetic acid for 1 h at 4°c. peels of the adaxial epidermis and blocks of storage parenchyma cells were surgically removed from each fixed cotyledon. collected tissues were immediately snap-frozen in liquid nitrogen and stored at -80°c until used for rna extraction. selection of times to obtain tissue samples from cultured cotyledons was based on the temporal pattern of uniform wall deposition preceding that of wall ingrowths. to this end, for the reference library, a representative balance of genes induced to regulate uniform wall and wall ingrowth construction was obtained by collecting 6 mg of epidermal peels from each of freshly harvested cotyledons and cotyledons cultured for and 3 h, and 9 h. to identify expression of tc-specific genes regulating uniform wall and wall ingrowth construction, epidermal peels and storage parenchyma tissue were sampled from replicate batches of freshly harvested cotyledons and cotyledons cultured for 3 h and 12 h.figure tissue sampling times informed by the sequential temporal deposition of the uniform wall followed by wall ingrowth papillae. note that across the period sampled, wall ingrowth papillae continue to be deposited and have not commenced to form the first fenestrated layer. ec, epidermal cell. to verify that each sampled population of cultured cotyledons was trans-differentiating, percentages of epidermal cells forming wall ingrowths of a sub-set of cotyledons at 12 h of culture were scored. rna isolation, cdna-library construction and illumina sequencing total rna was extracted using qiagen rneasy plant mini kits. contaminating genomic dna was removed using dnase i. for preparation of an epidermal-specific tc transcriptome reference library, 1 μg of total rna was aliquoted from the total rna extracts of each specified harvested time point. the rna aliquots were combined to generate a temporally mixed rna library. to identify genes responsible for uniform wall and wall ingrowth formation, 2 μg aliquots of total rna were sampled from each biological replicate of epidermal and storage parenchyma rna extracts. total rna quality was verified by determining the integrity of the 25s and 18s rna with an agilent bioanalyzer. the cdna libraries were prepared from poly-a mrna isolated from 1 μg of total rna using a truseq® rna v sample prep kit according to manufacturer’s instructions. cdna quality was evaluated by determining size and purity using an agilent bioanalyzer. cdna fragments, ranging from – 700 bp, were selected by agarose gel purification. for the reference library, selected cdna fragments were 100 bp pair-end sequenced in a single lane on an illumina hiseq platform. to characterize gene cohorts linked with uniform wall and wall ingrowth deposition, only biological replicates with the anticipated 70% of their epidermal cells at 12 h of cotyledon culture containing detectable wall ingrowths were processed for illumina sequencing. in three compliant biological replicates for each specified harvest time, cdna libraries were prepared from total rna extracts of epidermal peels and storage parenchyma tissues as indicated above. cdna fragments were purified separately from each sample and indexed with unique nucleic acid identifiers. the indexed cdna libraries were diluted to an average concentration of nm and pooled in equal volumes to generate the final mixed cdna pool for sequencing. the pool was then 100 bp pair-end sequenced in two lanes on an illumina hiseq platform. using illumina casava pipeline version. raw reads were trimmed with adaptor filtering and a read length cut-off of 50%. thereafter, filtered reads with over 20% of their nucleotides having a q score <  or their sequences having a n reading over 5% were removed. de novo assembly, annotation and go classification of transcriptome library filtered reads from the cdna library were assembled de novo into contigs by beijing genomics institution using trinity software release- with a k-mer of and a minimum k-mer threshold abundance of. the reads were then mapped back to contigs to assemble unigenes using trinity butterfly that filtered out transcriptional artifacts, misassembled transcripts and poorly supported transcripts. potential redundant sequences were grouped, using the tgi clustering tool set with a minimum 40% sequence overlap and over 80% sequence identity. assembled unigene sequences were annotated by alignment to the following publically available databases: ncbi nr; swissprot; kegg; cog, using blastx with an e-value threshold of 1e- unigenes with no hit in blastx were predicted using estscan. mapping reads to the reference library, determining differentially expressed genes and go-enrichment analysis reads sequenced by the illumina hiseq platform from mrna extracts of cotyledon epidermal peels and storage parenchyma tissues were analyzed by bgi. raw reads were filtered as described for the reference transcriptome library. clean reads were aligned to the reference sequences using the soapallgner/soap pipeline. no more than mismatches per read were allowed to ensure high quality alignment. sequence coverage of unigenes in each sample was calculated. during alignment, soapallgner/soap reported the number of mapped reads per kilo base per million reads as a measure of transcript abundance of each unigene. transcripts with rpkms <  were considered not to be expressed and were removed from the data sets. differentially expressed genes were determined using a computational algorithm based on digital gene expression profiles to perform pairwise differential expression analysis. multiple testing of the comparisons was corrected using the fdr method. sequences with a corrected fdr p value of no more than were selected. after this correction, genes exhibiting a differential expression of two-fold or more were identified as degs. subcellular localization of selected proteins encoded by unigenes was predicted using the wolf psort algorithm. quantitative rt-pcr validation a collection of unigenes with different expression patterns was selected. cdna was converted from the same rna samples sent for illumina sequencing using quantitect reverse transcription kit. primers were designed using primer plus and synthesized by sigma-aldrich australia. for each qrt-pcr reaction a 15 μl system containing  μl sybr green master mix,  μl of forward and reverse primers,  μl of nuclease free h2o and 5 μl cdna was set up. the following pcr cycle was used: 95°c for 5 min, 95°c for 15 s, 60°c for 20s, 72°c for 30 s; steps to were repeated times. high-resolution melting curves following the final pcr cycle checked the specificity of the pcr products. for each cdna sample, technical duplicates in each of three biological replicates were tested. four housekeeping gene candidates were assessed using genorm. relative expression levels of each unigene were determined using the two standard curves method.  de novo assembled transcriptome library for ingrowth wall deposition sequencing of reference library cdna fragments on an illumina hiseq platform generated, pair-end reads. after filtering raw reads,, high-quality reads, in which 97% of nucleotides have a phred quality score of ≥ q level were de novo assembled into contigs with a n value of 980 bp and an average length of 423 bp. clustering yielded unigenes with consensus sequences grouped into distinct clusters and singletons. this unigene population had a n value of 1723 bp and an average length of 1076 bp. high integrity and accuracy of the assembly was indicated by % of filtered reads mapping to the assembled transcriptome with % of reads uniquely mapping to the transcriptome. the uniquely mapped reads had an average coverage depth of.table raw, filtered and mapped reads used for the de novo assembly of the reference transcriptome library for adaxial epidermal cells of freshly harvested and cultured v. faba cotyledons undergoing trans -differentiation to a tc morphology category number percentage blastx searches of publically available protein databases, using the putative unigenes as query sequences, yielded matches for % of the unigenes. blastx-derived alignments predicted sequence orientation for of the unigenes and identified open reading frames. taxonomic distribution of the annotated unigenes by blast2go demonstrated that over 85% unigenes had their best hits with their homologs in other legume species. the raw reads and assembled unigene sequences are available at european nucleotide archive, accession number: prjeb global gene expression patterns during early phases of trans-differentiation to a tc-morphology total rna was extracted from epidermal peels and storage tissue blocks at and 12 h of cotyledon culture and processed for rnaseq on an illumina platform. between – million clean reads were generated for each biological replicate. an average of 85% of the clean reads mapped to expressed unigenes. of these reads, 60% mapped with a perfect match and 40% with a mismatch less than 5 bp. total numbers of unigenes, detected within epidermal and storage parenchyma cells across cotyledon culture were temporally stable. some 70% of the unigenes exhibited expression levels >  rpkm within a cell type and these were dominated by unigenes with expression levels ranging from to rpkm. an estimated unigenes were expressed in adaxial epidermal cells of developing v. faba cotyledons. of these epidermal cell unigenes, were shared with the underlying storage parenchyma cells leaving unigenes that were epidermal cell specific. upon transiting to a tc fate, expression of unigenes in the precursor epidermal cells was switched off. the combined transcriptomes of trans-differentiating tcs at and 12 h of cotyledon culture totalling unigenes approximated those detected by rnaseq analyses of differentiating betcs in barley grains and of nematode giant tcs in rice roots. seventy-three % of the epidermal tc transcriptome was represented by ongoing expression of genes by their precursor epidermal cells whilst 27% were induced. the percentage of induced genes was comparable to the proportion of up-regulated genes in nematode giant tcs. of the induced genes, 66% were tc specific and the remainder was shared with the underlying storage parenchyma cells. ten % of the epidermal cell genes were silenced in transiting to an epidermal tc identity and these were replaced 3-fold by induced genes. tc specific genes were inspected manually to remove duplicates and those with < 70% coverage to yield a total of unigenes.figure numbers of unigenes expressed preferentially in, or shared between, cell types and uniform wall and wall ingrowth formation occurring within adaxial epidermal cells of cultured v. faba cotyledons. only unigenes with a rpkm value >  in all three biological replicates were defined as ‘expressed’ in one or more cell types/phases of ingrowth wall deposition. for, only unigenes with a coverage of > 70% were listed for further analysis. ec- adaxial epidermal cell; tc – epidermal tc trans-differentiated from adaxial epidermal cell; sp- storage parenchyma cell; uw - uniform wall; wi- wall ingrowth. transcriptome networks specifically expressed during uniform wall or wall ingrowth formation the temporal sequence of uniform wall followed by wall ingrowth deposition provided the opportunity to separate the tc-specific cohort of expressed genes into groups linked with the two phases of ingrowth wall construction and those shared between these phases as follows. the latter were identified by manual inspection of the 3- and 12-h gene expression profiles. the remaining expressed genes were then specifically linked with either uniform wall or wall ingrowth formation and these were identified on the following grounds. at 12 h of cotyledon culture, uniform wall construction had ceased and tc-specific gene expression is solely committed to deposition of wall ingrowths. thus the expression profile at 12 h contained transcripts specifically related to constructing wall ingrowths alone. in contrast, at 3 h of cotyledon culture, uniform wall construction is proceeding rapidly concurrent with a smaller population of cells starting to deposit wall ingrowths. hence at 3 h of culture, genes involved in uniform wall and wall ingrowth construction would be co-expressed. however, those exclusively directing deposition of wall ingrowths would be expressed at relatively lower levels. thus subtracting the 12-h from the 3-h gene expression profile identified those genes specifically expressed during uniform wall construction. the above exercise demonstrated that of the tc-specific expressed genes, 66% were selectively linked with uniform wall or wall ingrowth formation with the remainder being expressed throughout ingrowth wall formation. this latter cohort of genes was categorised according to their relative expression levels in the two phases of ingrowth wall formation, that is, no change and up-regulated during uniform wall or wall ingrowth deposition. genes whose expression levels did not change accounted for 60% of the common cohort.table numbers of annotated expressed genes ascribed to specified functional categories for genes switched off in epidermal cells transiting to a tc fate and for those specifically expressed in epidermal cells undergoing trans -differentiation to a tc morphology functional category epidermal switched-off genes transfer cell specific expressed genes uw/wi no change uw up-regulated uw specific wi up-regulated wi specific total number of expressed genes total number of annotated genes dna synthesis and modelling transcription/translation signalling ros/ca2+ intracellular organization cell wall enzymes and structural proteins membrane transporters defense flavonoid synthesis and compartmentation tc specific genes separated into those genes expressed throughout uniform wall and wall ingrowth formation and those that are specific to each of these wall-building phases. genes expressed throughout ingrowth wall formation are separated into groups depending on their differential expression patterns of no change, up-regulated during uw or wi formation. blastx searches, at a stringency of < e- established that 41% of the tc-specific genes and 34% of epidermal cell genes switched off shared homology with known genes listed in public databases. the 59/ % of un-known transcripts equates with a similar value reported for the transcriptome of developing barley betcs. table  provides a summary of the changes in expression of selected functional categories of tc-specific genes that are known to be central to tc development and function. for all categories, numbers of epidermal cell genes switched off in transiting to a tc fate were replaced by increased numbers of tc-specific transcripts between 3- to 12-fold. amongst these, expression of transcription/translation, receptor kinase/kinases, cytoskeleton/vesicle trafficking, matrix polysaccharides and defense genes contributed to the two-fold greater number of genes expressed during uniform wall compared to wall ingrowth construction. in addition, a portion of uniform wall associated expressed genes were linked with a co-occurring burst in cell division/expansion. genes encoding the entire flavonol pathway were switched on during tc development. a more detailed analysis was undertaken of the temporal expression of tc-specific genes contained in functional groups that are at the heart of regulating development of the tc’s structural specialization of a polarized ingrowth wall that realises their functional capacity to support high rates of nutrient transport across their plasma membranes. these categories included hormonal and ros/ca2+ signal generation and their signalling pathways, cytoskeleton/vesicle trafficking and lipid biosynthetic enzymes related to microdomain formation, cell wall biosynthetic and remodelling enzymes along with cell wall structural proteins and membrane transporters. these data are supplemented with estimates of their annotation and relative expression levels at specified stages of ingrowth wall construction along with noting whether their homologues have been detected in transcriptomes of betcs and nematode giant tcs. temporal patterns of expression of selected genes from the targeted functional categories determined by qrt-pcr were consistent with those derived from illumina sequencing, suggesting that the illumina data are relatively reliable. discussion the ability to obtain peels of cotyledon epidermal cells permitted cell-specific transcriptomes of these cells to be identified as they trans-differentiated to a tc-morphology. in addition, developmental stage-specific transcriptomes were isolated by taking advantage of the temporal disjunction between uniform wall and wall ingrowth construction. we interrogated these tc-specific transcriptomes to discover genes encoding proteins of functional categories considered to contribute to developing a transport capable tc. transcriptional cascades are initiated by auxin and ethylene along with ros and ca2+ serving as secondary messengers and positional signals. the substantial numbers of expressed receptor kinases/kinases and transcription factors, point to these forming a significant component of the signalling network. re-organization of the cytoskeleton and vesicle trafficking together with membrane microdomains, under control of the inductive signalling network, would be expected to direct targeted delivery of cargos containing cell wall biosynthetic enzymes and cell wall components to construct the polarized uniform wall and localized wall ingrowths. the latter provide a scaffold to support an amplified plasma membrane surface area to which membrane transporters are targeted through a polarized endomembrane secretory system to confer enhanced tc transport function. in addition, targeted positioning of transporters is responsible for generating the polarized extracellular ros and cytosolic ca2+ signals and possibly altering positioning of auxin transporters that may contribute to generating the auxin maximum. the following discussion focuses on expressed genes belonging to these three functional categories namely, signals and signalling pathways, intracellular organization and transport function. the discussion starts with seeking out candidates responsible for generating the developmental signals and downstream signalling pathways. in all cases, statements regarding gene identity and function should be considered as putative.figure regulatory interrelationships between the functional categories of expressed genes that contribute to developing a functional transfer cell. signals and signalling pathways hormonal signals, acting in series with ros and ca2+, likely activate expression of receptor kinases/kinases and transcription factors to induce tc trans-differentiation. auxin, ethylene and abscisic acid form a complex of primary signals putatively regulating tc development consistent with previous findings for epidermal tcs and those reported for nematode giant tcs, induction of trans-differentiation was accompanied by changes in auxin activity increasing five-fold between uniform wall and wall ingrowth formation driven by an auxin maxima generated by enhanced auxin biosynthesis and disrupted polar transport. for instance, auxin biosynthesis could be elevated by expression of indole-3-acetate o-methyltransferase iaa, that catalyzes synthesis of a biologically more active iaa, methyl-indole-3-acetic acid and indole-3-glycerol phosphate synthase that starts the tryptophan independent iaa biosynthetic pathway. polar auxin transport was likely disrupted by de-localized distribution of expressed auxin efflux and influx carriers by co-expression of kan and hd-zipiii. as a point of distinction between mechanisms regulating flange and reticulate wall ingrowths, in contrast to the latter, differentially expressed auxin-related genes were not detected in developing betcs. initiation of epidermal tc development is linked with a substantive burst in ethylene biosynthesis, regulated by an auxin-induced expression of acc synthases, driving expression of ethylene response factors and particularly so for uniform wall formation. for example, erf and shine expression could invoke cell wall biosynthesis through respectively up-regulating callose and cellulose biosynthesis. strong expression of ethylene signalling genes during uniform wall formation corresponded with that reported for early phases of wall ingrowth construction in barley betcs and nematode giant tcs. a switch to a specific aba signalling pathway throughout ingrowth wall formation was suggested by expression of pyl an aba receptor, lec tightly connected with aba signalling regulating ab and ab and erf rap that acts through an ab14-mediated signalling pathway. aba also acts during wall ingrowth formation. aba action throughout ingrowth wall formation in epidermal tcs contrasts with that for barley betcs where aba induces a maturation program once wall ingrowth deposition ceases. three gibberellin biosynthetic genes with the biological activities of their products being modulated by two gibberellin 2-oxidases and short internode related during uniform wall formation are expressed. these gibberellins likely regulate cell division and expansion. an absence of gibberellin activity during wall ingrowth construction was suggested by co-expression of short internode related and gibberellin 2-beta-dioxygenase 8-like. residual cytokinin levels in the developing epidermal tcs would be depleted through degradation by the expressed cytokinin dehydrogenase and by conjugation to an inactive form catalyzed by the five cytokinin-o-glucosyltransferases. no components of the salicylic acid or brassinosteroid signalling pathways were detected in the tc-specific transcriptome indicating that these pathways likely did not play a role in trans-differentiation to a tc state. reactive oxygen species and ca2+ serve as secondary messengers directing ingrowth wall construction extracellular ros, produced by ethylene-induced respiratory burst oxidases, regulate biosynthesis and polarized deposition of the uniform wall in epidermal tcs. similarly, rboh expression co-occurs with wall ingrowth formation in barley betcs. in contrast, no differentially expressed rbohs were detected in nematode giant tcs. interestingly, ros-related gene expression was dominated by those involved in extra- and intracellular ros homeostasis suggesting that ingrowth wall formation depends upon a spatio/temporal fine-tuning of ros. for instance, antioxidant capacity could be down regulated by ascorbic acid oxidation catalyzed by an extracellular l-ascorbate oxidase whilst concurrent ros degradation by peroxidation could be mediated by carboxymethylenebutenolidase-like protein, secreted to the cell wall. in contrast to epidermal tcs, differentially expressed peroxidases were not detected in barley betcs and were strongly down-regulated in nematode giant tcs. loci at which wall ingrowths are deposited are defined by a cytosolic ca2+ signal spatially organized into narrow plumes shaped by co-operative activities of plasma membrane ca2+-permeable channels organized into clusters surrounded by ca2+-atpases. however, unlike the unknown dhp receptor ca2+-permeable channels responsible for generating the ca2+ signal, ion conductances of the expressed skor, annexin and cyclic nucleotide gated channels are not inhibited by verapamil or nifedipine. this, together with channel expression coinciding with uniform wall construction, points to an additional role for ca2+ signalling in transiting precursor epidermal cells to a tc fate. a conclusion that accommodates ca2+ signalling genes being expressed in both flange and reticulate forms of developing tcs. ca2+ signalling would appear to be mediated through ca2+-dependent protein kinases during uniform wall formation while a calmodulin signalling route could operate when wall ingrowths are being deposited as suggested by expression of three iq-domain containing proteins that bind calmodulins to facilitate their function. receptor kinases/kinases represent a significant component of the signalling network tc-specific receptor kinases/kinases represented a major component of the signalling networks expressed within the epidermal tcs. while their functions are unknown, similar to nematode giant tcs, members of a two component signalling system present in barley betcs, was not detected in the epidermal tc cohort of receptor kinases/kinases. thus, the presence/absence of a two component signalling system could underpin the distinction between flange and reticulate tc wall architectures. intracellular organization polarized accumulation of extracellular ros regulating uniform wall synthesis and cytosolic ca2+-plumes determining loci for wall ingrowth formation respectively depend upon a finely-tuned intracellular distribution of rbohs and ca2+-permeable channels. this requirement is undoubtedly met through modifying cytoskeleton and vesicle trafficking combined with re-configuring microdomains in the plasma membrane abutting sites of ingrowth wall deposition. tc-specific expression of cytoskeleton and vesicle trafficking confers tc polarity microtubule and actin-associated genes switched off on transiting to a tc fate were predominantly linked to cell division that declined rapidly from three to six hours. consistent with polarized cell wall deposition, tc- specific genes indicative of remodelling cytoskeletal elements were expressed either across ingrowth wall deposition or only during one phase of the process. uniform wall specific gene expression indicated substantial polymerization/reorganization of actin filaments and polarization of transport vesicles and organelles consistent with ros-regulated polarization of the uniform wall. wall ingrowth specific expression of 65-kda microtubule-associated protein 3-like and villin- respectively suggests establishing a stable microtubule and actin network to orchestrate deposition of wall ingrowths at loci. in root hair growth of arabidopsis, atvln regulated actin organization is ca2+-dependent pointing to an interaction between actin and ca2+plumes that define wall ingrowth loci. rapid polarized/localized cell wall deposition during formation of the ingrowth wall is dependent upon exo/endocytotic activity. regulation of cycling of er-derived proteins through assembly and directed transport of coat protein complex ii and arfs across ingrowth wall deposition is consistent with asymmetrical vesicle trafficking to effect polarity. expression of uniform wall- and wall ingrowth-specific exocyst complex components and v- and t-snares, to direct targeted vesicle delivery to the plasma membrane, confirms the specialized nature of each phase of ingrowth wall deposition. two of four genes encoding proteins involved in membrane trafficking are potential regulators of localized endocytosis supporting wall ingrowth deposition. these two dynamin genes represent two subfamilies of drps that interact and assemble with clathrin at discrete foci in the plasma membrane of arabidopsis cells to regulate endocytosis and cellulose deposition. this profile of cytoskeleton and vesicle trafficking genes is comparable to that reported for betcs and nematode giant tcs. formation of membrane microdomains may sub-compartmentalize the plasma membrane polar domain sphingolipids and sterols form major components of membrane microdomains. indeed, genes encoding enzymes in their biosynthetic pathways are expressed during development of flange wall ingrowths in barley betcs. sphingolipids consist of a polar head group linked to an amino alcohol long-chain base with the amine group acylated with a fatty acid with c of lcb being or not being hydroxylated. bax inhibitor 1-like post-translationally activates the c hydrolase thus promoting flux through this route for acylation by very long-chain fatty acids. significantly, synthesis of vlcfas may be enhanced by expression of four 3-ketoacyl-coa synthase isoforms, a elongation of fatty acid protein and a long-chain-fatty-acid-coa ligase that form part of the elongase complex. an increased precursor flow into the vlcfa pathway is supported by up-regulated expression of biotin carboxyl carrier protein of acetyl-coa carboxylase, a sub-component of acetyl-coa carboxylase catalyzing the first carboxylation step in, and rate limiting of, de novo fatty acid synthesis. thereafter, bax inhibitor 1-like may also increase activities of fatty acid hydroxylases and desaturases that further modify the ceramides before synthesis of the final sphingolipid products of glucosylceramides or glycosyl inositolphosphoceramides. uniform wall specific expression of an oxysterol-binding protein-related protein and a remorin respectively point to vesicle trafficking of sterols and their enrichment in membrane microdomains. wall ingrowth deposition is linked with expression of putative sterol biosynthetic genes such as two epoxide hydrolases and squalene epoxidase. tc transport function expression of cell wall synthesizing and re-modelling enzymes underpins construction of the ingrowth wall that supports the amplified plasma membrane in which are embedded high densities of membrane transporters to collectively confer an enhanced transport function. expression of cell wall biosynthesis and re-modelling genes construct the ingrowth wall cellulose is present in the uniform wall and the inner, electron dense region, of wall ingrowths of epidermal tcs but cellulose biosynthesis appeared not to depend on expression of tc-specific cesas that were switched off and not replaced as the epidermal cells transited to a tc fate. rather, cellulose deposition could be regulated post-translationally. thus during uniform wall formation, shine and a gpi-anchored protein could regulate cesa activity with the extruded cellulose microfibrils being re-modelled by two extracellular endoglucanases. cellulose provides a structural scaffold to form wall ingrowths at right angles to the uniform wall. here, an acid endochitinase-like protein, known to associate with cellulose synthase complexes and an extracellular beta-glucosidase, may form part of the machinery redirecting extrusion of cellulose microfibrils. consistent with these observations, cesa expression is down-regulated in nematode giant tcs while in maize and barley betcs, cesa expression is up-regulated during later stages of tc development. in contrast to cellulose biosynthesis, there was a substantial tc-specific induction of genes encoding enzymes responsible for synthesizing and re-modelling cell wall matrix polysaccharides and, in particular, hemicelluloses. to this end, a β-d-glucan synthase, was switched off and replaced with enzymes catalyzing xylan biosynthesis and re-modelling. xylans in the uniform wall are consistent with their presence in flange wall ingrowths as suggested by expression of a beta-d-xylosidase and a xylosyltransferase respectively in betcs of barley and maize. xyloglucan presence in uniform walls is consistent with expression of xylosyltransferases and endoxylotransglucosylase/hydrolases. xyloglucan biosynthesis and re-modelling also dominates early developmental stages of barley betcs and nematode giant tcs. xyloglucan re-modelling coincides with tc division/expansion as indicated by co-expression of expansins. in contrast, expansins expressed during wall ingrowth construction when cell division/expansion has ceased, must serve a yet-to-be identified function. one possibility is to free xyloglucan chains from cellulose microfibrils rendering them available for co-operative re-modelling by alpha-xylosidases and a beta-glucosidase and by two extracellular isoforms of beta-galactosidase. overall, uniform wall and wall ingrowth construction was characterized by intense xyloglucan re-modelling. pathways leading to pectin biosynthesis and modification altered on epidermal cells transiting to a tc fate. during uniform wall construction, synthesis of the pectin rhamnogalacturon backbone was likely catalyzed by expression of galactofuronosyl-transferase 13-like that significantly is essential for polarized pollen tube tip growth. control over the pectin gel state is conferred by co-expression of a pectin esterase inhibitor modulating catalytic activities of expressed pectin esterases that catalyze removal of methyl ester groups from homogalacturonan backbones to allow their cross-linking with ca2+. this accounts for esterified pectins being the predominate type present in the ingrowth wall. up-regulated and wall ingrowth specific expression of two beta-galactosidases could catalyze cleavage of terminal galactosyl residues from rgi. overall, uniform wall formation included pectin biosynthesis and re-modelling giving way to pectin re-modelling alone for wall ingrowth deposition. enhanced pectin biosynthesis and re-modelling also characterizes construction of barley betcs and nematode giant tcs. callose deposition is a balance between synthesis by callose synthases and degradation by β- 3-glucanases. isoforms of both enzymes were switched off in epidermal cells on transiting to a tc fate and were not replaced during uniform wall formation. however, udp-glucuronosyltransferase, a callose synthase complex component essential for callose synthesis, was expressed during wall ingrowth construction accompanied by two beta- 3-glucanase isoforms; an expression pattern consistent with callose being confined to the outer sheath enveloping each wall ingrowth. this temporal profile of callose deposition corresponds with that reported for nematode giant tcs but contrasts with callose synthesis/turnover being restricted to the cell division phase of barley betc development. transcripts encoding enzymes of the phenylpropanoid pathway occurred across ingrowth wall formation. expressed abc transporters could efflux the monolignol precursors to the cell wall where class iii peroxidases catalyze their polymerization to lignin. significantly, the full complement of phenylpropanoid pathway enzymes is expressed in nematode giant tcs, and a detailed histochemical study of maize betcs, provides strong evidence for their lignification. extensins are expressed in tcs forming flange and reticulate wall ingrowths. a large number of extensin family members of hydroxyproline-rich glycoproteins were expressed in developing epidermal tcs, implying a key role played by extensins in shaping the ingrowth wall and, in particular, wall ingrowths. a key enzyme in the post-translational modification pathway generating functional extensins, prolyl 4-hydroxylase, was expressed throughout ingrowth wall formation. upon being exocytosed into the cell wall space, extensin monomers are envisaged to cross-link to form networks, mediated by redox activity of type-iii apoplasmic peroxidases. expression of a proline-rich-extensin-like receptor kinase, regulating extensin/pectin interactions, may generate supramolecular structures to function as templates for ingrowth wall formation. significantly, coincident with wall ingrowth construction, was the expression of two nodule-specific extensins that support polarized tip growth in elongating infection threads and a dz-hrgp homolog that contributes to polarized tip growth of pollen tubes. a large array of membrane transporters is specifically expressed in tcs the large number of transporter genes switched off upon transiting to an epidermal tc fate suggests a substantial change in transport function. a striking feature of the tc-specific membrane transporters, whose expression did not change during trans-differentiation, nine transport inorganic and organic nitrogen compounds. five transport amino nitrogen compounds including two aquaglyceroporins. two nucleoside proton symporters, adenine/guanine permease azg and equilibrative nucleoside transporter, likely function to retrieve apoplasmic nucleosides released from degradation of endosperm and inner cell layers of seed coats crushed by expanding cotyledons. collectively, expression of these transporters reflects a demand for nitrogenous precursors to support protein biosynthesis. expression of a putative plasma membrane potassium transporter and tonoplast potassium proton antiporter ensure potassium homeostasis. investment into nitrogen transporters was enhanced during uniform wall formation. this included a chloride channel that may function as a nitrate sensor to regulate development by modulating auxin transport. two additional transporters were recruited for potassium homeostasis; a skor channel and a potassium antiporter. most pronounced was the large number of expressed abc transporter genes linked with expression of flavonoid biosynthetic and defense genes. however, along with effluxing xenobiotics, members of the abcb transporter subfamily facilitate plasma membrane transport of iaa contributing to the altered iaa homeostasis of epidermal tcs. with the onset of wall ingrowth deposition, appearance of two sugar transporter transcripts reflects a growing dependence on an extracellular carbon supply. further investments were made in amino nitrogen and potassium transporters. an ongoing expression of abc transporters corresponded with expression of flavonoid biosynthesis and defense genes.  in epidermal cells undergoing trans-differentiation to a tc-morphology, only a small proportion of their transcriptome was found to be tc-specific. within the cohort of tc-specific transcripts, those encoding pathways regulating signalling and intracellular organization were fine-tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a tc fate. each phase of ingrowth wall construction was linked with an unique sub-cohort of expressed genes. comparison of transcriptomes of cells forming flange or reticulate wall ingrowths detected subtle differences in their transcript profiles, with the major difference being the absence of a two-component signalling system from the latter. availability of supporting data the cdna sequence datasets of raw reads and assembled reference transcriptome library supporting the results of this article are available in the repository of the european nucleotide archive with the ena accession number: prjeb additional files additional file 1: figure s table s and accompanying text. temporal formation of the uniform wall and wall ingrowths. additional file 2: figure s quality assessment of total rna extracted from adaxial epidermal and storage parenchyma cells of v. faba cotyledons. additional file 3: table s quality assessment of total rna extracted from, and cdna libraries of v. faba cotyledons used for illumina sequencing. additional file 4: table s annotation and expression levels of all genes specifically expressed in the epidermal cells of cultured v. faba cotyledons trans-differentiating to epidermal transfer cells. additional file 5: table s annotation and expression levels of selected unigenes expressed specifically in trans-differentiating adaxial epidermal cells of v. faba cotyledons. additional file 6: table s primer sequences used for qrt-pcr determination of expression levels of specified genes to validate rnaseq data sets. additional file 7: figure s species distribution of genes with highest sequence similarity to unigenes in the reference transcriptome library of trans-differentiating adaxial epidermal cells of cultured v. faba cotyledons. additional file 8: table s reads generated from illumina sequencing used to identify gene cohorts linked with uniform wall and wall ingrowth deposition in trans-differentiating epidermal cells of v. faba cotyledons. additional file 9: table s temporal profile of unigene numbers expressed in adaxial epidermal or storage parenchyma cells of cultured v. faba cotyledons. additional file 10: figure s and associated text. cell division and expansion of adaxial epidermal cells of cultured v. faba cotyledons. additional file 11: table s genes encoding proteins generating or transducing developmental signals switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology. additional file 12: table s genes encoding proteins involved in regulation of the cytoskeleton and vesicle trafficking and sphingolipid/sterol synthesis and transport that are switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology. additional file 13: table s genes encoding cell wall biosynthetic and remodelling enzymes and structural proteins switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology. additional file 14: table s genes encoding membrane transporters switched off in epidermal cells transiting to a tc fate and those specifically expressed in epidermal cells undergoing trans-differentiation to a tc morphology. additional file 15: figure s validation of expression patterns of unigenes obtained from illumina sequencing using quantitative rt-pcr. competing interests the authors declare that they have no competing interests. authors’ contributions jwp and ceo initiated the research and designed the experimental approach. hmz, xx and sw performed the experiments. hmz processed the data and hmz, jwp, ceo, rr and hw interpreted the results and prepared the manuscript. all authors have read and approved the final manuscript. 
auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and aba genes were evenly expressed across ingrowth wall construction. key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. each phase of ingrowth wall construction was linked with unique cohorts of expressed genes. transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. an rnaseq study of an inducible epidermal transfer cell system in cultured vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. genes encoding ethylene and ca2+ signal generation and transduction pathways were enriched during uniform wall construction. uniform wall and wall ingrowth specific expression of exocyst complex components and snares suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. transfer celltranscriptomecell wall constructionuniform wallwall ingrowthtransportersseed developmentvicia fabaissue-copyright-statement© the author 2015 transfer cell specific expression of cellulose synthases was absent. pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. all functional groups of genes examined were expressed before and following transition to a transfer cell fate. what changed were the isoform profiles of expressed genes within functional groups. extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. the ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of abc transporters. however, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood.
 transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. the ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. however, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood.  an rnaseq study of an inducible epidermal transfer cell system in cultured vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. all functional groups of genes examined were expressed before and following transition to a transfer cell fate. what changed were the isoform profiles of expressed genes within functional groups. genes encoding ethylene and ca2+ signal generation and transduction pathways were enriched during uniform wall construction. auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and aba genes were evenly expressed across ingrowth wall construction. expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. uniform wall and wall ingrowth specific expression of exocyst complex components and snares suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. transfer cell specific expression of cellulose synthases was absent. rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. more striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of abc transporters. sugar transporters were less represented.  pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. each phase of ingrowth wall construction was linked with unique cohorts of expressed genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transfer celltranscriptomecell wall constructionuniform wallwall ingrowthtransportersseed developmentvicia fabaissue-copyright-statement© the author 2015 