Early gene expression programs accompanying trans -differentiation of epidermal cells of Vicia faba cotyledons into transfer cells

Research

Early gene expression programs accompanying trans-differentiation of epidermal cells of Vicia faba cotyledons into transfer cells Blackwell Oxford, New NPH © 1469-8137 0028-646X March 10.1111/j.1469-8137.2009.02822.x 2822 8 0 Original 877??? XX 63??? ThePhytologist Authors 2009 UK Article Publishing (2009).Ltd Journal compilation © New Phytologist (2009)

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Stephen J. Dibley, Yuchan Zhou, Felicity A. Andriunas, Mark J. Talbot, Christina E. Offler, John W. Patrick and David W. McCurdy School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales 2308, Australia

Summary Author for correspondence: David W. McCurdy Tel: +61 2 49 21 5879 Email: [email protected] Received: 17 November 2008 Accepted: 6 February 2009

New Phytologist (2009) 182: 863–877 doi: 10.1111/j.1469-8137.2009.02822.x

Key words: cDNA-AFLP, transdifferentiation, transfer cells, Vicia faba, wall ingrowths.

• Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. • Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. • A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. • Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Introduction Transfer cells (TCs) are characterized by wall ingrowths that protrude into the cytoplasm forming a complex labyrinth (Talbot et al., 2001) that acts as a scaffold for an amplified plasma membrane enriched in nutrient transporters (Offler et al., 2003). Transfer cells trans-differentiate from diverse cell types in response to developmental cues, stress or other factors (Offler et al., 2003). Despite their importance in nutrient exchange in plants and, consequently, plant development (Offler et al., 2003), little is known of the identity of genes that orchestrate their induction and building of their wall labyrinths. Several genes nominated as TC-specific have been identified in basal endosperm TCs of developing maize kernels. These

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include four defensin-like genes, BETL1-4 (Thompson et al., 2001), a novel cell wall-related protein, MEG1 (Thompson et al., 2001; Gutiérrez-Marcos et al., 2004), and a transcriptional activator, ZmMRP-1 (Gómez et al., 2002). The transcriptional activator has been shown to activate BETL and MEG1 promoters (Gutiérrez-Marcos et al., 2004), and the ZmMRP-1 promoter itself is active in regions of active transport between source and sink tissues (Barrero et al., 2009). Furthermore, a putative type-A response regulator gene, ZmTCRR-1, was shown to be specifically expressed in the basal endosperm layer of maize (Muñiz et al., 2006). While these studies provide important insights, our wider understanding of the molecular processes underlying TC development remains poor. Most TCs develop deep within complex tissues (e.g. phloem parenchyma TCs in minor veins; Haritatos et al.,

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864 Research Fig. 1 The cDNA-amplified fragment length polymorphism (AFLP) analysis of transfer cell (TC) development using adaxial epidermal peels of Vicia faba cotyledons. (a) Scanning electron microscopy (SEM) image of an isolated adaxial epidermal peel showing the epidermal cell sheet (ECS) with a tag of storage parenchyma tissue (SPT) that was removed by dissection along the dotted line indicated. (b,c) Higher magnification views of epidermal peels revealing intact cellular contents. Nuclei are clearly visible by (b) 4,6diamidino-2-phenylindole (DAPI) staining (merged bright field/fluorescence image) or visualization by SEM (c). The inner surface of the epidermal layer is shown in (a–c). Bar, 1 mm (a), 20 µm (b,c). (d) The cDNAAFLP banding patterns obtained from independently prepared and amplified cDNA; (e) cDNA-AFLP of amplified cDNA prepared from epidermal peels of cotyledons cultured for 0, 3 or 24 h. Induction (arrow head), upregulation (asterisk) and down-regulation (closed circle) of gene expression.

2000) and thus are not readily accessible for experimental analysis. However, this issue is obviated by the readily accessible epidermal cells of Vicia faba (Faba bean) cotyledons. During in planta cotyledon development, abaxial epidermal cells transdifferentiate to form TCs but adaxial epidermal cells do not (Offler et al., 1997). However, when cotyledons are cultured with their adaxial surface in contact with nutrient agar, adaxial epidermal cells form small papillate wall ingrowths within 3 h (Wardini et al., 2007b), and functional TCs with a complex, transporter-rich wall labyrinth by 48 h (Offler et al., 1997; Farley et al., 2000; Wardini et al., 2007a). Thus, the V. faba cotyledon culture system provides a large population of developing TCs that share the same induction event, and importantly, are morphologically and functionally equivalent to TCs that form in planta. To analyse transcriptional changes accompanying induction and development of TCs in adaxial epidermal cells of V. faba cotyledons, we developed an efficient and simplified cDNAamplified fragment length polymorphism (AFLP) procedure incorporating nonsaturating PCR cDNA amplification to profile transcripts derived from isolated epidermal cells. We show that large-scale changes in gene expression occur within 3 h of TC induction, with many genes being induced, upregulated or rapidly switched off specifically in the adaxial epidermal cell layer undergoing trans-differentiation. Of particular interest among genes exhibiting upregulated and selective expression in trans-differentiating adaxial epidermal cells were suites involved in auxin signalling, cell division, vesicle trafficking associated with cell wall biosynthesis, mitochondrial biogenesis and storage product hydrolysis. Blocking auxin action or vesicle trafficking inhibited wall ingrowth formation, thus confirming these

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processes as key participants in TC development. Enhanced numbers of mitotic figures present in 3-h cultured adaxial epidermal cells demonstrated that these cells underwent rapid de-differentiation on exposure to inductive conditions. Collectively, these observations provide new insights into the early gene expression events leading to the induction and formation of TCs.

Materials and Methods Plant material, cotyledon culture and tissue processing Vicia faba L. (cv. Fiord) plants were grown in controlled glasshouse and growth cabinet conditions (Talbot et al., 2001). At harvest, cotyledons of 80–120 mg FW were removed surgically from their seed coats and either fixed immediately in ice-cold ethanol and acetic acid (3 : 1, v : v) for 1 h at 4°C or cultured adaxial face down on modified Murashige and Skoog (MS) media containing 50 mm glucose and 50 mm fructose (Farley et al., 2000) for 3 h or 24 h and then fixed (see earlier). Fixed tissue was processed rapidly by rinsing briefly in distilled water before isolating sheets of adaxial epidermal cells as epidermal peels. The adhering ‘tag’ of parenchyma tissue (Fig. 1a) was surgically removed and each epidermal peel snap frozen in liquid nitrogen. Light and scanning electron microscopy (SEM) observations revealed that most epidermal cells in the peels were sheared along their anticlinal walls and their cellular contents remained mostly intact (Fig. 1b,c). Following peeling, 1-mm thick discs of storage parenchyma tissue, free of epidermal cells, were collected from 3-h cultured cotyledons using a 5-mm diameter cork borer and immediately snap frozen.

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Research

RNA extraction and cDNA amplification Epidermal peels, from a minimum of three cotyledons per treatment, were pooled and total RNA extracted using an RNeasy RNA isolation kit (Qiagen). Total RNA was extracted from corresponding storage parenchyma discs using an RNeasy RNA isolation kit following treatment with Trizol reagent (Invitrogen). Extracted RNA was reverse transcribed with Powerscript reverse transcriptase using 3′-rapid amplification of cDNA ends (RACE) CDS primer A and SMART II A oligonucleotide (Clontech; see the Supporting Information, Table S1) to generate fully transcribed first-strand cDNA tagged with short sequences complementary to the SMART II A oligonucleotide at both the 5′ and 3′ ends. This first strand cDNA was purified with phenol–chloroform and ethanol precipitated using linear polyacrylamide as a carrier (Ambion, Austin TX, USA) and then used as template for full-length cDNA amplification following the Super SMART PCR cDNA synthesis kit (Clontech) protocol. Amplification parameters were optimized empirically by electrophoretic analysis of small aliquots of PCR products from every two cycles. The resulting double-stranded amplified cDNA were purified and adjusted to equal total amounts by comparing amplification of V. faba GAPDH1 and elongation factor 1-α (VfGAPDH1-FP/RP and VfEF1α-FP/RP primer pairs, respectively; see Table S1) by semiquantitative PCR. RNA fingerprinting with cDNA-AFLP The cDNA-AFLP fingerprinting reactions were carried out using a protocol modified from Bachem et al. (1998). Briefly, equal amounts of amplified cDNA from each experimental sample were digested with MseI and ApoI (NEB, Ipswich, MA, USA). The resulting digestion fragments were ligated to enzyme-specific adaptors (Milioni et al., 2002, and Table S1) using T4 DNA ligase (MBI Fermentas, Burlington, Canada). Fragments ligated to the ApoI adaptor, biotinylated at the 5′ terminus, were collected following binding to streptavidincoated paramagnetic Dynabeads (Dynal, Oslo, Norway). A 1/10 dilution of this ligation reaction was preamplified using primers targeted to the adaptor sequences (Table S1). A 1/100 dilution of preamplification product was used for each selective PCR determined by two specified bases at the 3′ end of each primer extending into the fragment sequence. In contrast to Bachem et al. (1998), PCR primer concentration was 250 nm to allow fragment visualization by silver staining. The PCR reactions were incubated at 94°C for 10 min followed by 13 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 1 min, with the annealing temperature dropping by 0.6°C each cycle. The reactions were completed by 23 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1 min. All 256 possible PCR primer combinations were tested. Products from these reactions were run on 16 cm-long 5% polyacrylamide gels for 4.5 h at 40 mA and stained using a rapid silver staining

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procedure (Qu et al., 2005). Fragments were visualized on a Molecular Imager gel documentation XR system (Bio-Rad). Relative band intensities were determined using quantity one software (version 4.6.3; Bio-Rad), and a band was classified as differentially expressed if its intensity showed ≥ 5-fold temporal change. Transcript-derived fragment (TDF) extraction, verification and sequencing Each TDF of interest on silver-stained polyacrylamide gels was stabbed with a sterile 200 µl pipette tip and incubated in 15 µl of 10 mm Tris-HCl (pH 8.0) for 30 min at room temperature. Each fragment was reamplified using 5 µl of the fragment extract as template and subjected to the selective PCR cycle program with an additional seven cycles at an annealing temperature of 56°C. Reamplified products were separated on agarose gels, DNA bands were extracted using the Wizard gel and PCR purification kit (Promega) and cloned directly into the TA cloning vector pGEM-T Easy (Promega). Clones were sequenced using T7 primer and BDT sequencing chemistry (Invitrogen). Gene homology analysis was performed using the blast program (Altschul et al., 1997) at the NCBI website (http://www.ncbi.nlm.nih.gov/ BLAST/) and the TIGR database (http://www.jcvi.org/) with default parameters. The TDF sequences were searched against blastx and blastn of NCBI or blastx and blastn of TIGR. Promoter sequences (a maximum of 2 kb up-stream of the ATG start codon) of each Arabidopsis orthologue were screened for regulatory cis-elements by Athena (O’Conner et al., 2005). Expression of selected TDFs was validated by quantitative real-time PCR, with Platinum Taq polymerase and SYTO9 dye (Invitrogen), on unamplified cDNA produced from independently isolated RNA. Reactions were performed using a Corbett RotorGene 6000 with fluorescence acquisition through the green channel. Expression quantification utilized the ‘two standard curve’ method as described in the Corbett Rotor-Gene 6000 software package (version 1.7), using V. faba elongation factor 1-α (VfEF1-α) standard curve to normalise expression. Scanning electron microscopy of treated cotyledons Cotyledon cultures were established as described earlier except that sister cotyledons were divided between culture media with or without the specified treatment (Wardini et al., 2007b) or prepared under green light. After 15 h, adaxial epidermal peels were prepared, washed in 2% (w : v) NaOCl for 3 h and subsequently dehydrated at 4°C through a 10% step-graded ethanol–distilled H2O series, changed at 30-min intervals. Peels were critical point-dried with liquid CO2 in a critical-point drier (Balzers Union, Liechtenstein) and secured outer face down onto sticky tabs to reveal the cytoplasmic face of their outer periclinal cell walls. Samples were sputter-coated

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866 Research Table 1 Categories of verified gene expression profiles identified in adaxial epidermal cells of Vicia faba cotyledons induced to form transfer cells (TCs)

Expression profilea, b Epidermal peels Hours in culture 24

SP

Number (%) of TDFs

Epidermalspecific

3

Induced Late-Induced Early Transient-Induced Up-Regulated Rapidly Switched-Off Gradually Switched-Off

69 (15) 22 (5) 21 (4) 30 (6) 102 (22) 20 (4)

Epidermal and storage parenchyma

0

Induced Late-Induced Early Transient-Induced Up-Regulated Rapidly Switched-Off Gradually Switched-Off

116 (25) 13 (3) 19 (4) 19 (4) 26 (6) 14 (3) Total number of TDFs

471 (100)

cDNA-AFLP analysis examined expression profiles from epidermal peels of cotyledons cultured for 0, 3 or 24 h, or storage parenchyma tissue (SP) isolated from 3-h cultured cotyledons. TDFs, transcript-derived fragments. aOnly gene fragments with changes in expression levels ≥ 5-fold across the culture period are included. b Black bars are schematic representations of PCR fragments visualized on silver stained polyacrylamide gels and illustrate identified patterns of differential gene expression.

with gold to a thickness of 20 nm in a sputter-coating unit (SPI Suppliers, West Chester, PA, USA), and viewed at 15 kV with a Philips XL30 SEM. Analysis of cell division Cotyledons were cultured and fixed as described earlier. After washing briefly in phosphate-buffered saline (PBS), adaxial epidermal peels were collected and stained with 1 µg ml−1 4,6-diamidino-2-phenylindole (DAPI) for 5 min. Epidermal peels were rinsed 2 × 5 min in PBS and mounted in Mowiol (Calbiochem, San Diego, CA, USA) with 0.1% (w : v) pphenylenediamine. Tissue was viewed with a Zeiss Axiophot epifluorescence microscope equipped with a 50 W short-arc mercury lamp and a UV (365–420 nm) filter (Osram). Mitotic indices were estimated as percentages of cells containing mitotic profiles from at least 100 cells scored per replicate.

Results Isolation of RNA and amplification of cDNA from epidermal peels Recovery of total RNA obtained from either single or pooled (maximum of five) epidermal peels was not sufficient to yield reliable banding patterns using standard cDNA-AFLP protocols (data not shown). These protocols typically use up to 100 µg of total RNA for starting material (Bachem et al., 1998)

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compared with nanogram amounts retrieved from epidermal peels. We therefore incorporated a nonsaturating PCR-based cDNA amplification step based on procedures developed for cDNA microarray analysis of small tissue samples (Hertzberg et al., 2001; see the Materials and Methods section). Figure 1d shows that transcript-derived fragments (TDFs), generated by selective PCR of amplified cDNA, were consistent between technical repeats and detected temporal changes in selective gene expression (Fig. 1e). Using this modified procedure, we were able to profile gene expression in adaxial epidermal cells of freshly isolated cotyledons (no culture) or those cultured for 3 h and 24 h, and to compare these profiles with those of storage parenchyma cells from 3-h cultured cotyledons to identify changes in gene expression occurring specifically in adaxial epidermal cells and therefore likely to be related to TC induction and development (Table 1). Expression profiles deduced from our cDNA-AFLP approach (Table 1) were verified using real-time PCR on unamplified cDNA (see Fig. S1 and associated text). This analysis demonstrated that conclusions of cell-specific expression profiles could be drawn with confidence but distinction between induced and upregulated gene expression was less clear. cDNA-AFLP analysis of transcriptional regulation accompanying induction and development of TCs Analysis of the 256 primer combinations containing two basepair overhangs yielded a total of 5795 TDFs, ranging in

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Research

size from 50 bp to 500 bp, from adaxial epidermal cells and storage parenchyma tissue. From this pool of TDFs, 756 demonstrated differential expression, defined here as a ≥ 5-fold change (up or down) in band intensity detected on silver-stained gels (see the Materials and Methods section). Of these, a total of 471 fragments were verified as true cDNA-AFLP fragments by extracting each band from the polyacrylamide gel and reamplifying using the original selective primer pair. An analysis of 234 V. faba cDNAs present in GenBank revealed that 72% were cut at least once by both ApoI and MseI (data not shown). Applying this percentage to the 471 differentially expressed TDFs identified (Table 1), we estimate that TC formation may involve differential expression of c. 650 different genes. This estimate compares well with the numbers of developmentally regulated genes detected during tracheary element formation in cultured Zinnia mesophyll cells by cDNA-AFLP (562; Milioni et al., 2002) or by microarray (523; Demura et al., 2002) analyses. Furthermore, of the 471 differentially expressed TDFs, our approach identified 142 TDFs (Table 1 and hence an estimated 195 genes totally) that were induced or upregulated specifically in epidermal cells during TC formation. This number of genes displaying epidermal-specific changes in expression is within the range of preferentially expressed genes reported for epidermal cells of maize coleoptiles (130; Nakazono et al., 2003) and Arabidopsis stems at defined stages of development (180; Suh et al., 2005). These comparisons support the conclusion that our cDNAAFLP study has successfully identified the majority of genes being differentially regulated specifically in epidermal cells during TC formation. Moreover, this conclusion is supported by the finding that genes known to be expressed exclusively in epidermal layers, such as fiddlehead (Pruitt et al., 2000) and B1-type cyclin (Boudolf et al., 2004), were identified in the epidermal-specific cohort of TDFs (Table 2). Temporal patterns of expression were classified as ‘Induced’, ‘Late-Induced’, ‘Early Transient-Induced’, ‘Up-Regulated’, ‘Rapidly Switched-Off’ and ‘Gradually Switched-Off’ (Table 1). Of those genes displaying epidermal-specific changes in expression, approximately equal numbers were either induced/ upregulated or switched-off rapidly or gradually (Table 1). Responses of differential gene expression were typically rapid, with 85% of differential expression occurring within 3 h of culture (Table 1). Ontology-deduced functions of induced epidermalspecific genes relate to cell wall biosynthesis, metabolism and protein synthesis/metabolism and are potentially regulated by auxin and/or ethylene Cotyledon culture induces the formation of TCs in adaxial epidermal cells but not in cells of the underlying storage parenchyma tissue (Farley et al., 2000; Talbot et al., 2007). Consequently, attention was focused on identifying, by homology searching (see the Materials and Methods section),

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the 112 TDFs showing epidermal-specific, induced expression (Induced, Late-Induced, Early Transient-Induced; Table 1). Genes showing this expression pattern are more likely to be directly related to TC development, compared with those associated with stress responses which are expected to be expressed comparably in the adjacent storage parenchyma tissue. Functional classifications were determined by searching blast similarity matches (blast expectation values [E] of ≤ 10–3) through the Gene Ontology (http://www.geneontology.org) and KEGG BRITE (http://www.genome.jp/kegg/brite.html) databases, with confirmation by reference to the literature. This process enabled TDFs to be placed into one of nine predicted functional groups (Table 2; groupings based on the categories used by Milioni et al., 2002). Of the 112 TDFs, 44 (39%) returned no significant match to any database entry (data not shown) and a further 15 (23%) matched database entries for hypothetical or unknown proteins (Table 2; Fig. 2a). The remaining 68 TDFs showed significant alignments and were placed in functional groups. The major groups were metabolism, energy and storage (12 TDFs; 18% of 68), protein synthesis and metabolism (11 TDFs; 16%), cell wall and vesicle trafficking (9 TDFs; 13%), and transcription (6 TDFs; 9%) (Table 2; Fig. 2a). The development of TCs in tomato roots is regulated by auxin and ethylene (Schikora & Schmidt, 2001, 2002). Accordingly, promoter regions of Arabidopsis orthologues of identified V. faba TDFs (Table 2) were screened using Athena (O’Conner et al., 2005) for the presence of auxin- and ethylene-regulatory cis-element sequences. Of the 48 Arabidopsis orthologues identified, 24 (50%) contained at least one repeat of the auxin-responsive element, AuxRe (TGTCTC; Guilfoyle & Hagan, 2007) in its corresponding promoter region, while nine (19%) contained at least one ET-responsive element (GCC-box; Ohme-Takagi & Shinshi, 1995, and see Table 2). These percentages are substantially higher than the 41% and 9% for AuxRe and the GCC-box elements, respectively, found by searching all promoter regions in the Arabidopsis genome using the Data Mining application of Athena. Genes encoding hypothetical and unknown proteins are abundant in those rapidly switched off within 3 h of cotyledon culture Of the 102 TDFs whose epidermal-specific expression was rapidly switched-off (Table 1), 34 were selected for sequencing based on their size (c. 150–400 bp) and band intensity on the silver-stained gels. Of this cohort only three returned no significant hits, and of those exhibiting low E values (Table 3; Fig. 2b), a substantial proportion (14 TDFs; 45%) matched hypothetical and unknown proteins suggesting the possibility of novel functions linked with trans-differentiation of TCs. The proportion of genes distributed among the various functional groupings was generally similar to that observed for induced genes (compare Fig. 2b with 2a).

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Functional group

Clone ID

Expression profile

Size (bp)

Transcription

V39A V225E V231A V234A V36A-2 V252A V9A V43A V76A V97C-1 V134C V134F V230B V234B

I I I I ETI ETI I I I I I I I I

201 362 137 436 325 311 428 337 224 158 349 161 160 144

V40D V196B V219B V3A V92B V194C V233B V245B V113F V253C V66G V196C

ETI ETI ETI I I I I I LI LI ETI ETI

150 249 412 309 170 392 429 139 244 134 124 50

V26A V51F V146A V167A V170C

I I I I I

330 201 355 226 138

V174A V222F V224B V225F V234F V170B V243B

I I I I I LI ETI

154 108 196 577 54 243 266

Protein synthesis and metabolism

Cell wall and vesicle trafficking

© The Authors (2009) Journal compilation © New Phytologist (2009)

Metabolism, energy and storage

Gene product

Organisma

Accession numbera

E valueb

Locus IDc

Promoter motifd,e

Splicing factor Prp8 CONSTANs-like zinc finger protein KH domain RNA binding protein Mismatch repair protein GH1 protein (AUX/IAA-like) cAMP response element binding (CREB) protein Ubiquitin-protein ligase Aspartate aminotransferase Flavonoid 3-O-galactosyl transferase Polyubiquitin (UBQ12) In2-1 (glutathione-S-transferase) Serine carboxypeptidase S10 family protein Peptidase M, neutral zinc metallopeptidase Integrase, catalytic region; zinc finger, CCHC-type; peptidase aspartic Insulin-degrading enzyme Putative aminopeptidase Ribosomal protein S26 UDP-glucuronosyl/UDP-glucosyltransferase ADP-ribosylation factor (ARF1) YKT61 (similar to yeast SNARE YKT6 1) Putative clathrin coat assembly protein Glycoside hydrolase, family 17: X8 domain Pectin methylesterase inhibitor Hydroxyproline-rich glycoprotein-2 related cluster Glycoside hydrolase, family 17; X8 domain Putative ras-GTPase-activating protein, SH3-domain-binding protein Mitochondrial rpl5, rps14 and cob genes Vicilin precursor Aconitate hydratase AMP-dependent synthetase and ligase Oxidoreductase, short-chain dehydrogenase/ reductase family protein Triacylglycerol/steryl ester lipase-like protein Glyceraldehyde-3-phosphate dehydrogenase-like Malic oxidoreductase Mitochondrial NADH dehydrogenase subunit 7 (nad7) Mitochondrial cytochrome oxidase subunit I (coxI) Isoamylase isoform 1 Nitrilase/cyanide hydratase & apolipoprotein N-acyltransferase

Oryza sativa Pisum sativum Medicago truncatula Arabidopsis thaliana Glycine max Medicago truncatula Arabidopsis thaliana Medicago sativa Vigna mungo Arabidopsis thaliana Glycine max Medicago sativa Medicago truncatula Medicago truncatula

NP_001054734 AAX47173 ABE91933 AAD04176 AF016633 ABE93018 NP_201183 CAA43779 BAA36972 NP_564675 AAG34872 AAZ32845 ABE84181 ABE92232

3e-25 2e-18 1e-11 1e-56 4e-231 2e-29 2e-10 2e-48 2e-18 1e-08 2e-30 2e-04 2e-13 7e-13

At1g80070 At2g24790 At1g09660 At3g18524

Aux/Eth Aux Aux/Eth

Solanum esculentum Arabidopsis thaliana Pisum sativum Medicago truncatula Medicago sativa Arabidopsis thaliana Arabidopsis thaliana Medicago truncatula Arabidopsis thaliana Medicago sativa Medicago truncatula Trifolium pratense

CAC67408 AAN72085 AAD47346 ABE82273 DQ455181 NP_200614 AAL38763 ABE89157 NP_196116 CO513118† ABN09816 AB236858

5e-05 8e-30 2e-28 5e-22 5e-421 4e-53 3e-53 2e-102 4e-05 1e-083 9e-07 3e-041

Pisum sativum Vicia faba Oryza sativa Medicago truncatula Oryza sativa

AJ132231.1 CAA68559 ABF93861 ABE86318 ABA98146

1e-411 2e-10 1e-40 3e-25 3e-06

Medicago truncatula Medicago truncatula Medicago truncatula Beta vulgaris Pisum sativum Pisum sativum Medicago truncatula

AAR29056 ABE82032 ABN05792 ABD36076 X14409 AAZ81835 ABE91350

2e-14 5e-07 5e-12 1e-06 3e-081 1e-37 2e-38

At3g12250 At5g63780 At5g11520 At5g17030 At1g55060 At5g02790 At1g28110 At5g10540 At4g27210 At2g41790 At1g63770 At3g56340 At2g36790 At5g58060 At3g50860 At2g01630 At5g04970 At1g15825 At1g64760

Aux Aux Aux/Eth Aux Aux Aux Aux

Aux Aux

Eth Eth

At2g05710 At1g51680 At5g50590

Aux

At5g14180 At1g13440 At4g00450

Aux Aux Eth

At2g07769 At2g39930 At4g08790

Aux Aux

868 Research

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Table 2 Functional classification and highest similarity match in NCBI or TIGR databases for transcript-derived fragments (TDFs) identified by cDNA-amplified fragment length polymorphism (AFLP) to be induced specifically in adaxial epidermal cells in response to cotyledon culture

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Table 2 continued

Functional group

Clone ID

Expression profile

Size (bp)

Cell growth, division and DNA synthesis

V25C V120A V245D V24B V104A V112D V89C V165B V190B V227A V187A V178A V200B V129B V36A-1 V36B

I I I I I LI I I I I ETI I I LI ETI I

319 543 105 174 531 298 185 143 322 313 263 109 204 323 299 288

V37D V38D V42B V71D V110D V110F-1 V113E V140C V159C V202D V230C V216E V256C V165A

I I I I I I I I I I I LI LI ETI

144 111 186 109 148 137 284 107 288 273 124 118 203 298

Transport facilitation

Signal transduction

Others

Hypothetical and unknown proteins

Organisma

Accession numbera

E valueb

Locus IDc

Promoter motifd,e

AtMUS81; endonuclease/nucleic acid binding Mitotic cyclin B1-type Nucleosome/chromatin assembly factor C P-type H+-ATPase Ammonium transporter (AtAMT1;2) Vacuolar (H+)-ATPase G subunit, prokaryotic type Fiddlehead-like protein Protein kinase; Adipokinetic hormone GIL1 (gravitropic in the light) Putative nodulin protein MtN21 Protein phosphatase type 2C Epoxide hydrolase Embryonic abundant protein USP87 precursor Transferase-family protein Importin-β, N-terminal Unknown protein (clone Ps-cos16 LTR and Ogre retrotransposons) Unknown protein (clone MTH2-172C6) Unknown protein (clone MTH2-116F22) Unknown protein Conserved hypothetical protein Unknown protein (clone MTH2-39B3) Hypothetical protein T21J18_150 Hypothetical protein Unknown protein (clone MTH2-3G18) Unknown protein (clone MTH2-9B23) Unknown protein Unknown protein (clone MTH2-36J11) Unknown protein F28N24.7 Protein of unknown function DUF649 Unknown protein (clone MTH2-32M22)

Arabidopsis thaliana Glycine max Zea mays Zostera marina Arabidopsis thaliana Medicago truncatula Gossypium hirsutum Medicago truncatula Arabidopsis thaliana Oryza sativa Medicago sativa Medicago truncatula Vicia faba Arabidopsis thaliana Medicago truncatula Pisum sativum

NP_194816 BAA09467 AF384037 BAF03589 NP_176658 ABD32809 AAL67993 ABE77704 NP_851217 BAB92246 CAA72341 TA21728_3880† P21746 NP_181527 ABE90433 AY299398

6e-18 7e-41 1e-031 4e-16 1e-34 6e-25 1e-15 3e-13 2e-37 3e-14 1e-23 2e-042 7e-29 3e-12 8e-45 1e-081

At4g30870 At2g26760 At2g16780 At1g80660 At1g64780 At3g01390 At2g26250 At1g06840 At5g58960 At1g44800 At1g07160

Aux Aux Eth Aux Aux Aux Eth

At2g39980 At5g17020

Aux Aux

Medicago truncatula Medicago truncatula Arabidopsis thaliana Medicago truncatula Medicago truncatula Medicago truncatula Arabidopsis thaliana Medicago truncatula Medicago truncatula Oryza sativa Medicago truncatula Medicago truncatula Medicago truncatula Medicago truncatula

CT967315 AC174306 NP_683417 ABE89743 CT009540 TA25031_3880† AK229089 AC174350 AC139526 AAV44205 AC148482 TA9157_35883† ABE77839 AC122165

4e-101 2e-051 3e-09 0.001 2e-341 3e-082 9e-051 1e-201 2e-701 1e-21 2e-131 6e-042 2e-24 8e-131

At1g51355

Aux

Aux Eth

At1g17210

At3g52760

Expression profiles of ‘Induced’ (I), ‘Late-Induced’ (LI) and ‘Early Transient-Induced’ (ETI) are shown in Table 1. a Accession number and organism refer to the closest gene match returned from BLAST searching of NCBI database, unless noted (†) to correspond to a TIGR contig. b All values taken from BLASTx searching of NCBI, except where otherwise noted: 1, BLASTn of NCBI; 2, BLASTx of TIGR; 3, BLASTn of TIGR. cLocus ID tag provided for closest Arabidopsis match when present as a significant BLAST match (E value of < 10–3). d Auxin and ethylene response motifs identified in promoter sequences (−2000 to 0 bp upstream of ATG) in corresponding Arabidopsis genes. Aux: AuxRE (TGTCTC); Eth: GCC-Box (GCCGCC). e Promoter analysis only performed where significant Arabidopsis match (BLAST E value of < 10–3) was obtained from TDF sequence.

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Gene product

869

Functional group

Clone ID

Size (bp)

Gene product

Organism

Accession numbera

E valueb

Locus IDc

Promoter motifd,e

Transcription

V91B V140A V143A-46 V190E-61 V100B V201B V203A-73 V231D V66C-14 V118C V68A V176B

325 435 354 232 305 242 147 184 201 271 152 103

Medicago truncatula Medicago truncatula Medicago truncatula Spinacia oleracea Medicago truncatula Pisum sativum Hyacinthus orientalis Glycine max Arabidopsis thaliana Glycine max Pisum sativum Arabidopsis thaliana

TA70974_3847† ABE79807 ABE79544 S15348 ABE91897 X80854 EF468471 AAM93434 AK227260 AF516879 X06281 NM_128949

3e-083 6e-70 4e-27 7e-18 2e-14 4e-341 1e-161 1e-17 2e-05 5e-091 7e-081 3e-091

At3g24070 At1g21700 At1g27730 At5g50250 At4g13780 At2g07769

Aux/Eth Aux Eth Eth Aux

At2g17360 At3g55260

Aux/Eth Aux

At2g33980

Aux

V41C V222B V234G V40C V216C

162 173 118 173 157

Medicago truncatula Pyrgus communis Arabidopsis thaliana Arabidopsis thaliana Medicago truncatula

ABE86426 AAR25995 TA30584_3880† NP_567059.1 ABO78428

1e-15 1e-19 1e-053 9e-13 7e-13

At5g57710

Aux

At3g23750 At3g57890 At1g65320

Aux Aux

V46A V66E V91A V97A V143A-47 V163A-49 V163A-51 V184C V190E-62 V191A V203A-74 V213A V244D

425 292 315 178 274 119 193 326 90 271 270 306 185

Zinc finger, CCHC-type SWIRM Zinc finger, C2H2-type RNA-binding protein, 28K Aminoacyl-tRNA synthetase, class Ia Mitochondrial rps10, trnF and trnP 18S ribosomal RNA gene 40S ribosomal S4 protein β-N-acetylhexosaminidase-like protein Expansin (EXP1) Alcohol dehydrogenase Nucleoside diphosphate-sugar (NUDIX) hydrolase (AtNUDT22) Heat-shock protein 101 Putative senescence-associated protein Putative LRR receptor protein kinase Tubulin-specific chaperone C-related CBS (cystathionine-β-synthase)containing protein Hypothetic protein (Os12g0630700) Unknown protein (clone MTH2-34F1) Hypothetical protein Unknown protein (clone MTH2-17N24) Hypothetical protein precursor Unknown protein (clone GMW1-45c9) Hypothetical protein Hypothetical protein Unknown protein (clone MTH2-34F1) Hypothetical protein (ACLA_028940) Genomic sequence (contig VV78X023217.49) Unknown protein (clone GMW1-105h23) OSJNBa0027O01.6 protein

Oryza sativa Medicago truncatula Vitis vinifera Medicago truncatula Phillyrea latifolia Glycine max Medicago truncatula Vitis vinifera Medicago truncatula Aspergillus clavatus Vitis vinifera Glycine max Oryza sativa

NP_001067339 AC147712 CAN83392 AC138526 CAK18872 AC173960 ABO81572 CAN63098 AC147434 XP_001269594 AM430398 AC187294 TA121_57577†

2e-10 4e-04 3e-07 1e-051 1e-22 5e-201 2e-11 5e-13 2e-101 2e-07 6e-701 3e-49 1e-052

Protein synthesis and metabolism

Cell wall and vesicle trafficking Metabolism, energy and storage Signal transduction

Others

Hypothetical and unknown proteins

© The Authors (2009) Journal compilation © New Phytologist (2009)

aAccession

At5g45170 At3g16895

At2g24660 At1g41880

Aux

number and organism refer to the closest gene match returned from BLAST searching of NCBI database, unless noted (†) to correspond to a TIGR contig. All values taken from BLASTx searching of NCBI, except where otherwise noted: 1, BLASTn of NCBI; 2, BLASTx of TIGR; 3, BLASTn of TIGR. c Locus ID tag provided for closest Arabidopsis match when present as a significant BLAST match (E value of < 10–3). dAuxin and ethylene response motifs identified in promoter sequences (−2000 to 0 bp upstream of ATG) in corresponding Arabidopsis genes. Aux: AuxRE (TGTCTC); Eth: GCC-Box (GCCGCC). e Promoter analysis only performed where significant Arabidopsis match (BLAST E value of < 10–3) was obtained from TDF sequence. b

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Table 3 Functional classification of transcript-derived fragments (TDFs) identified by cDNA-amplified fragment length polymorphism (AFLP) to be rapidly and specifically switched off in adaxial epidermal cells of 3-h cultured cotyledons based on the highest match resulting from BLAST searching of NCBI and TIGR databases

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Fig. 2 Classification of predicted gene functions derived from sequenced, differentially-expressed transcript-derived fragments (TDFs). Genes showing cultureinduced, epidermal-specific (a) expression and (b) rapid down-regulation or switchingoff. Predicted functions were made on the basis of top matches obtained from BLAST searches of the NCBI and TIGR databases, using Expect (E) values ≤ 10–3 to indicate significant matches. Gene sequences returning no significant match with database entries were not included in calculating percentage values.

Similar to the cohort of induced genes, promoter analysis of the pool of Arabidopsis orthologues closely matching V. faba TDFs, which were rapidly switched off, revealed an increase in the frequency of AuxRe and GCC-box motifs within the promoter regions of these identified genes (65% and 24%, respectively; Table 3). Testing key functional pathways predicted by cDNAAFLP gene discovery – light, auxin, vesicle trafficking and cell division Predicted functions of V. faba genes deduced from ontology searches of databases, which were rapidly and specifically induced in adaxial epidermal cells, indicated possible light (e.g. Gravitropic in the light (GIL1) and Constans-like 3 (COL-3)) and auxin-mediated (e.g. GH1 and AuxRe promoter

© The Authors (2009) Journal compilation © New Phytologist (2009)

motifs) signalling pathways leading to wall ingrowth induction (Table 2). The operation of these predicted signalling pathways were tested experimentally by culturing cotyledons under green light or in the presence of the competitive auxin inhibitor, p-chlorophenoxyisobutyric acid (PCIB; Oono et al., 2003). Cotyledon culture in the absence of an early light signal had no significant effect on wall ingrowth induction (Fig. 3e). By contrast, PCIB reduced numbers of adaxial epidermal cells forming wall ingrowths by 60% (Fig. 3e). More than 10% of genes showing induced, epidermal-specific expression encoded proteins predicted to be involved in vesicle trafficking and cell wall synthesis (Fig. 2a), for example, ADP-ribosylation factor 1 (ARF1), YKT61 and a pectin methylesterase inhibitor (Table 2). To examine a requirement for vesicle trafficking in wall ingrowth formation, cotyledons were cultured in the presence of Brefeldin A, a potent inhibitor

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Fig. 3 Effect of white light, auxin and vesicle trafficking on wall ingrowth formation and measures of mitosis during transfer cell (TC) induction. (a–d) Scanning electron microscopy (SEM) images of cytoplasmic faces of outer periclinal walls of adaxial epidermal cells of Vicia faba cotyledons cultured following exposure to (a) white (control) or (b) green light, or on Murashige and Skoog (MS) media containing (c) 100 µM p-chlorophenoxyisobutyric acid (PCIB) or (d) 100 µg ml−1 Brefeldin A. Bar, 20 µm. Wall ingrowths (arrow) and starch grains (arrowhead) are labelled in (a) and (b). (e) Adaxial epidermal cell numbers containing wall ingrowths (e.g. cells marked with * in (c)) in specified treatments expressed as percentages of total cells scored (150–200 cells counted per replicate). (f) Mitotic indices of adaxial epidermal cells in control (0 h) and 3-h cultured cotyledons (100 cells scored per replicate). All values are mean ± SE of six (e) or four (f) independent replicates.

of vesicle formation (Ritzenthaler et al., 2002). Under these conditions, wall ingrowth formation was abolished (93% inhibition; Fig. 3e), demonstrating an absolute requirement for vesicle trafficking in wall ingrowth deposition. Induction of a mitotic cyclin, an endonuclease and chromatin assembly factor C specifically in epidermal cells (Table 2) suggested activation of the cell cycle upon TC induction. Comparisons of mitotic index in adaxial epidermal cells showed a dramatic rise in mitotic rates following cotyledon culture, rising from 0.5 to 7.4 in the first 3 h (Fig. 3f).

Discussion We used experimental induction of adaxial epidermal TCs in V. faba cotyledons to reveal transcriptional changes accompanying trans-differentiation of epidermal cells into functional TCs (Tables 2 and 3). Rapid (< 3 h) epidermal-specific induction of genes (Table 1) is consistent with the finding of Wardini et al. (2007b) that all biosynthetic machinery required to form wall ingrowths is transcribed within 1 h following exposure of

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cotyledons to inductive signal(s). Concurrently there is an equal number of genes rapidly switched off (122 TDFs; Table 1) upon exposure to culture, reflecting a major change in the epidermal transcriptome associated with transdifferentiation of epidermal TCs. Generic responses, including those to abiotic stress, may be distinguished from those peculiar to trans-differentiation of epidermal TCs by analysing genes specifically induced in these cells (Epidermal-specific; Table 1). This assumption is supported by the absence of gene functions associated with generic stress responses from this cohort of genes specifically induced in adaxial epidermal cells upon cotyledon culture (Table 2). The relative distribution of these genes among functional categories (Fig. 2a) matches those reported for tracheary element formation (Milioni et al., 2002) except for expression of transporter genes and those linked with cell division. Expression of transporter genes (e.g. ammonium transporter, P-type and vacuolar H+-ATPases; Table 2) is consistent with TC function (Offler et al., 2003) and further supports our conclusion that the experimental approach used here has enabled identification of gene

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expression events primarily associated with trans-differentiation into functional TCs. Signalling TC induction – role for light, auxin and ethylene? The extent of wall ingrowth formation in phloem parenchyma and companion cell TCs of Arabidopsis and pea leaves, respectively, has been shown to be dependent on incident light flux densities (Amiard et al., 2005). For the V. faba cotyledon system, exposure of their adaxial epidermal cells to white light upon cotyledon removal from seed coats may initiate a photomorphogenic signal cascade. In this context, induction of homologues of CONSTANS-like (COL-3; Datta et al., 2006) and Gravitropic in the Light (GIL1; Allen et al., 2006) and downregulation of B-EXPANSIN (Tepperman et al., 2004) is consistent with a phytochrome-driven response (Tepperman et al., 2004; Tables 2, 3). COL-3, in contrast to most COLs that function in flowering responses, has been shown to control vegetative growth patterns (Datta et al., 2006) that might include cell wall formation. However, rates of wall ingrowth initiation in adaxial epidermal TCs were found to be independent of a light signal (Fig. 3). Whether a light signal affects the extent of wall ingrowth formation in committed adaxial epidermal TCs (Amiard et al., 2005) remains to be determined. Indeed, upregulation of GIL1 (Table 2), that renders auxin transport nonpolar in the dark (Allen et al., 2006), provides a link between light and auxin signals possibly mediating induction of wall ingrowth formation. Elevated auxin levels are known to enhance formation of TCs in rhizodermal cells of a number of species, including tomato (Schikora & Schmidt, 2001). An indication that auxin levels are elevated in adaxial epidermal cells of cultured cotyledons is provided by the induced expression of a MtN21 homologue (Table 2), a signature gene for elevated levels of auxin in developing tissues (Busov et al., 2004). Observed profiles of selective gene expression in adaxial epidermal cells (Table 2) indicate that elevated auxin levels could arise from altered transport and/or enhanced biosynthesis. Inhibitory effects of flavonoids on auxin transport (Peer & Murphy, 2007) could be relieved by their enhanced metabolism through induced expression of flavonoid 3-O-galactosyltransferase (Miller et al., 2002) and glutathione-S-transferase (Smith et al., 2003). Induction of GIL1 and an aminopeptidase (Table 2) could impact on auxin transport by randomly relocalizing PIN1 proteins around plasma membranes of cotyledon cells (Murphy et al., 2005; Allen et al., 2006). These effects on auxin transport, combined with enhanced auxin biosynthesis by induced expression of a nitrilase (Table 2), catalysing hydrolysis of indole-3-acetonitrile into active indole-3-acetic acid (IAA; Vorwerk et al., 2001), could alter patterns of auxin distribution to drive wall ingrowth formation. High auxin concentrations could account for the transient induction of an early-response auxin gene, GH1 homologue (Table 2), belonging

© The Authors (2009) Journal compilation © New Phytologist (2009)

to the Aux/IAA gene family of transcriptional regulators (Guilfoyle et al., 1993). The Aux/IAA proteins interact with auxin response factors (ARFs) to confer various auxin responses alone or in combination by binding to AuxRe motifs (Guilfoyle & Hagan, 2007). These motifs are enriched (54 vs 41%) among Arabidopsis orthologues of the genes identified in our cDNA-AFLP screen (Tables 2, 3), indicating a potentially important role for auxin in orchestrating wall ingrowth formation. This conclusion is supported by finding that PCIB, an auxin analogue that inhibits auxin action by competitively binding with auxin receptors (Oono et al., 2003), reduced numbers of adaxial epidermal cells forming wall ingrowths in cultured cotyledons (Fig. 3). The proposition that ethylene may contribute to TC induction in V. faba cotyledons arises from finding a 2.7-fold enrichment of ethylene responsive cis-elements in promoter regions of Arabidopsis orthologues of differentially expressed V. faba genes (Tables 2 and 3). This proposition is supported by the finding that 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) enhanced TC formation in root epidermal cells of tomato (Schikora & Schmidt, 2002) and adaxial epidermal cells of V. faba cotyledons (F. A. Andriunas et al., unpublished). Guided by the presence of AuxRe and GCC-box motifs (Tables 2, 3), significant downstream targets of auxin and ethylene signalling pathways inducing TC development could include cellular metabolism (AuxRe), cell division (AuxRe) and vesicle trafficking/cell wall biosynthesis (GCC-box). These phenomena are discussed in the following sections. Transfer cell induction coincides with increases in cell division Induction of TC development in adaxial epidermal cells of cultured cotyledons involves reinitiation of cell division (Fig. 3f). Borisjuk et al. (1995) reported that adaxial epidermal cells of ‘Stage V’ cotyledons, equivalent to the cotyledons used in our culture experiments, have ceased division and are fully committed to cell expansion. After a 3-h culture, however, the mitotic index of adaxial epidermal cells had increased nearly 15-fold, from 0.5 to 7.4 (Fig. 3f). This rapid increase is consistent with the endopolyploid status of adaxial epidermal cells of these cotyledons (Borisjuk et al., 1995), thereby enabling entry into mitosis without a preceding interphase. This observation indicates that reinitiation of mitosis represents an important early event in trans-differentiation of epidermal TCs, most likely to set in train the substantial genomic reorganization that accompanies trans-differentiation. Epidermalspecific induction of genes involved in DNA repair (an endonuclease orthologue of AtMUS81) and chromatin remodelling (nucleosome/chromatin assembly factor), together with a mitotic B1 cyclin (Table 2) are consistent with this possibility. Mitotic cyclin B1 is the noncatalytic partner of B-type cyclin-dependent protein kinases (CDKs) belonging

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to the B1 subgroup. The B1-CDKs drive the G2/M transition in mitosis (Francis, 2007), and are expressed preferentially in epidermal cells (Boudolf et al., 2004). A B1-CDK dependent arrest at the G2/M phase accounts for the ability of these cells to rapidly (within 3 h) enter mitosis upon exposure to the inductive signal (Fig. 3f). In addition to the epidermal-specific induction of mitosis, induction of two β-1,3-glucanases (Table 2) suggests reinitiation of cytokinesis during trans-differentiation of epidermal TCs. Both induced genes are Family 17 glycoside hydrolases (Minic & Jouanin, 2006), with the Arabidopsis orthologue of V245B (Table 2) ascribed with an ancestral function in cell division/ cell wall remodelling (Doxey et al., 2007). In this instance, the β-1,3-glucanase may be a candidate for performing a specialized role during cell plate formation or, alternatively, participating in wall remodelling events required to achieve the unique morphology of reticulate wall ingrowths. Modification of energy metabolism during transfer cell development Induced genes selectively expressed in adaxial epidermal cells contributing to energy metabolism (Table 2) included components of the mitochondrial electron transport chain (nad 7, cob, cox1) and Kreb cycle (aconitase, malate dehydrogenase). Expression of mitochondrial-encoded nad 7, cob and cox1 (Table 2) are insensitive to altered oxygen tensions resulting from cotyledon excision (Rolletschek et al., 2003) but reflect expression profiles linked with mitochondrial biogenesis (Howell et al., 2007). This process is possibly orchestrated by chromatin assembly factor C (CAF-C; Table 2), which is known to influence mitochondrial numbers in yeast through the Ras/cAMP pathway (Ruggieri et al., 1989; Rigoulet et al., 2004). Consistent with this conclusion, mitochondrial matrix densities and cristae formation increase along with mitochondrial numbers in adaxial epidermal cells undergoing wall ingrowth development (Farley et al., 2000). Induced expression of NADH-dependent malic enzyme and aconitase (Table 2) is suggestive that mitochondrial activity has switched to an anaplerotic mode to meet demand for intermediates consumed in various synthetic processes underpinning wall ingrowth construction. Given that sugar demand exceeds supply during the trans-differentiation of epidermal TCs in planta (Harrington et al., 1997) and in vitro (Wardini et al., 2007a), carbon skeletons are likely to be sourced from reserves. In this context, a profile of genes potentially involved in remobilization of storage compounds were induced (Table 2), including those remobilizing lipids (triacylglycerol lipase, hydroxysteroid dehydrogenase, aconitase and malate dehydrogenase) and starch (isoamylase and glyceraldehyde-3-phosphate dehydrogenase). Oil body breakdown through triacylglyceride lipase activity would provide free fatty acids to enter glyoxysomes as described for germinating seeds (Eastmond, 2006). Within glyoxysomes,

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fatty acid molecules are oxidized to acetyl-CoA and enter the glyoxylate cycle to produce C4 precursors which can be used for energy generation or fed through gluconeogenesis into an array of biosynthetic pathways, including cell wall component biosynthesis (see previous section). An alternative energy source may be derived through starch hydrolysis catalysed by isoamylases (Hussain et al., 2003) in combination with a plastid glyceraldehyde-3-phosphate dehydrogenase (Table 2) forming part of a carbon pathway before plastid/cytosol exchange of carbon skeletons. Vesicle trafficking is essential in wall ingrowth deposition It is not surprising that genes involved in vesicle trafficking and cell wall biogenesis are induced in epidermal cells undergoing trans-differentiation into TCs. Genes induced or switched off specifically in epidermal TCs (Tables 2, 3) can be presumed to act as regulators of papillate ingrowth deposition at defined loci (Talbot et al., 2001; Fig. 3). The absence of key wall-building genes from Table 2 (listing genes expressed in epidermal cells but not in storage parenchyma) such as celluloses and sucrose synthases is explained by their generic expression in all cells undergoing expansion at this stage of cotyledon development (Borisjuk et al., 1995). Modification of vesicle trafficking upon TC induction is evident through the induction of vesicle-targeting genes ADPribosylation factor 1 (ARF1), a SNARE (YKT61) and a clathrin coat adaptor subunit (Table 2). The ARFs are considered central to orchestrating asymmetrical vesicle trafficking to effect polarity in plant cells (Xu & Scheres, 2005), a characteristic consistent with wall ingrowth deposition in adaxial epidermal cells of cotyledons. However, Class 1 ARFs (Table 2) act as intracellular regulators of trafficking, being primarily localized to the Golgi and subpopulations of post-Golgi vesicles (Matheson et al., 2008). Induction of YKT61, a SNARE located in the cis-Golgi cisternae (Chen et al., 2005), together with ARF1, is indicative of enhanced protein trafficking between Golgi and endoplasmic reticulum (ER). Upregulation of ARF1, but not SAR1, the GTPase responsible for assembly of COP11 protein coats directing vesicle budding from the ER (Memon, 2004), points to ARF1 as a key regulator of vesicle trafficking activity during wall ingrowth formation. This conclusion is supported by inhibiting this process when cotyledons were cultured in the presence of Brefeldin A (Fig. 3d,e). Interestingly, this result suggests that the contribution of cellulose synthase/sucrose synthase complexes to building papillate wall ingrowths (Talbot et al., 2007) also depends upon vesicle trafficking. Modification of trans-Golgi vesicle trafficking in TCs is indicated by induction of the clathrin coat adaptor AP-3 (Table 2). ARF1 regulates recruitment of AP-3 adaptor complex to membranes (Ooi et al., 1998) during construction of clathrin coats, and YKT61 is considered a core component of SNARE-facilitated fusion at the trans-Golgi network in

© The Authors (2009) Journal compilation © New Phytologist (2009)

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Arabidopsis (Chen et al., 2005). Since large numbers of secretory vesicles are associated with developing wall ingrowths in TCs (Wardini et al., 2007b), it is possible that, together with the early transiently upregulated putative ras-GTPase-activating protein (Table 2), ARF1/YKT61/AP-3 could constitute a specialized gene complex facilitating increased trans-Golgi vesicle delivery to the plasma membrane in a polarized manner. Genes encoding wall components and modifying enzymes are not well represented in the list of genes specifically induced or switched-off in epidermal cells (Tables 2, 3), consistent with wall ingrowths being compositionally equivalent to primary cell walls (Vaughn et al., 2007). Some exceptions are β-Nacetylhexosaminidase (Table 3), UDP-glucosyltransferase and pectin methylesterase inhibitor (Table 2). Induction of a VATPase (Table 2) in the trans-Golgi network could increase synthesis of cell wall components and trafficking to the membrane (Brüx et al., 2008). Late induction of a pectin methylesterase inhibitor (PMEI) is consistent with maintaining extensibility of developing wall ingrowths. Wall ingrowths are rich in pectins, and for V. faba cotyledon epidermal TCs these pectins are esterified (Vaughn et al., 2007 and references cited therein). Pectin methylesterases (PMEs) have a major role in pectin remodelling (Pelloux et al., 2007) through de-esterification decreasing wall extensibility (Röckel et al., 2008). Therefore, late induction of PMEI (Table 2) suggests a role in maintaining extensibility of developing wall ingrowths as they commence branching and fusing to form a fenestrated layer (Talbot et al., 2001). Conclusions Extensive, rapid and cell-specific transcriptional regulation underpins trans-differentiation of adaxial epidermal cells of V. faba cotyledons into TCs. Auxin, possibly in combination with ethylene, functions as an inductive signal to initiate wall ingrowth formation. The induction of TCs initiates re-entry into a division cycle coincidental with modification of vesicle trafficking and wall assembly machinery specifically in these cells. The rapid stepped increase in metabolic demand by the trans-differentiating epidermal cells for intermediates to support these biosynthetic activities is met by remobilization of lipid and starch stores processed through an enhanced anaplerotic pathway in newly formed mitochondria. Inhibition of pectin de-esterification in wall ingrowths could confer sufficient mechanical flexibility to form the characteristic fenestrated wall layers. The insights generated from these findings open new opportunities for further studies to expand our understanding of signalling pathways inducing, and metabolic machinery responsible for constructing, the intricate wall ingrowths of TCs.

Acknowledgements We thank Kevin Stokes for raising healthy experimental material and acknowledge funding of this project from Australian

© The Authors (2009) Journal compilation © New Phytologist (2009)

Research Council Discovery Project grants DP0556217 and DP0664626.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Transcript-derived fragment (TDF) expression patterns in unamplified cDNA using real-time PCR. Table S1 Oligonucleotide primer sequences used for cDNA synthesis and amplification, cDNA-amplified fragment length polymorphism (AFLP) and real-time PCR verification of TDF expression Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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