Journal Experimental Botany, Vol. 64, No. 14, pp. 4559–4573, 2013 doi:10.1093/jxb/ert274  Advance Access publication 16 September, 2013 This paper is available online free all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) Research paper LEA polypeptide profiling recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance Julien Delahaie1, Michaela Hundertmark1,*, Jrme Bove1, Olivier Leprince2, Hlne Rogniaux3 and Julia Buitink4,† 1 *  Present address: Vilmorin SA, Route du Manoir, 49250 La Mnitr, France. To whom correspondence should be addressed. E-mail: julia.buitink@angers.inra.fr †  Received 29 May 2013; Revised 25 June 2013; Accepted 19 July 2013 Abstract In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering importance LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, heat-stable proteome was characterized in cotyledons legume Castanospermum australe and it was compared with that orthodox model legume Medicago truncatula. RNA sequencing identified transcripts 16 homologues out 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below critical water content 0.4  H2O g DW–1. g Characterization LEA proteome Mtabi3 seeds revealed subset LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccationsensitive genotypes, revealing new insights into mechanistic basis recalcitrant trait in seeds. Key words:  abi3, Castanospermum australe, desiccation tolerance, late embryogenesis abundant proteins, Medicago truncatula, proteomics, recalcitrant seed, RNAseq. Introduction Global agriculture and conservation plant biodiversity rely on seeds and their ability to be stored for long periods time in dedicated national and international storage facilities (Li and Pritchard, 2009; Walters et al., 2013). The terms © The Author 2013. Published by Oxford University Press on behalf Society for Experimental Biology. This is an Open Access article distributed under terms Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided original work is properly cited. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014   Universit d'Angers, UMR 1345 Institut Recherche en Horticulture et Semences, SFR 4207 QUASAV, PRES L'UNAM, 49045 Angers, France 2   Agrocampus Ouest, UMR 1345 Institut Recherche en Horticulture et Semences, SFR 4207 QUASAV, PRES L'UNAM, 49045 Angers, France 3   Institut National Recherche Agronomique, UR1268 Biopolymres, Interactions, Assemblages, Plate-forme Biopolymres-Biologie Structurale, 44316 Nantes, France 4   Institut National Recherche Agronomique, UMR 1345 Institut Recherche en Horticulture et Semences, SFR 4207 QUASAV, PRES L'UNAM, 49045 Angers, France 4560 | Delahaie et al. recalcitrant seeds is ambiguous. Work has been constrained to detect members dehydrin family and showed that they are present in range species from different habitats, while apparently being absent from others. Several studies reported presence dehydrins in recalcitrant species temperate origin, whereas these proteins could not be detected in some highly desiccation-sensitive seeds from certain tropical species (Finch-Savage et al., 1994; Farrant et al., 1996; Han et al., 1997; Hinniger et al., 2006; Panza et al., 2007; Sunderlikova et al., 2009; Ismail et al., 2010; Lee et al., 2012). In stored recalcitrant seeds Quercus robur L., dehydrin mRNA can also be induced by abscisic acid (ABA) and limited drying treatments (FinchSavage et al., 1994). Whereas presence/absence dehydrins cannot explain recalcitrant behaviour species studied to date, several other families LEA proteins exist in orthodox seeds that have not been studied in recalcitrant seeds. comparative analysis recalcitrant and orthodox seed development is an interesting alternative to identify mechanisms involved in desiccation tolerance, especially if closely related species are compared (Kermode, 1997; Oliver et al., 2011). Recently, comparison metabolomic responses drying leaves two closely related grass species (sister group contrast), one being desiccation tolerant and other desiccation sensitive, highlighted metabolic predispositions associated with desiccation tolerance (Oliver et al., 2011). In this study, recalcitrant seed species Papilionaceae subfamily was characterized to allow comparison with previous studies on orthodox seeds model legume Medicago truncatula. The phylogenetically closest recalcitrant species for which seeds can currently be obtained is Castanospermum australe A.Cunn ex Hook. (Doyle, 1995). Castanospermum australe is tropical tree native east Australia and now implanted in South Africa and Sri Lanka. In seeds this species, dehydrins were detected by western blot analysis (Han et al., 1997). The absence sequenced genome this species impedes thorough molecular comparison entire LEA proteome with orthodox seeds. Thus, high-throughput sequencing technology was used to obtain, assemble, and annotate transcriptome these recalcitrant seeds. Whereas transcripts could be detected in C. australe for most LEA genes that are present in desiccation-tolerant M. truncatula seeds, comparative analysis LEA proteome profiles revealed that abundance for number seed-specific LEA proteins was severely affected in recalcitrant seeds. In contrast, homologues several dehydrins that are expressed in seedlings or non-seed tissues M. truncatula submitted to osmotic stress accumulated to high levels in C. australe seeds. Comparison LEA proteome with desiccation-sensitive abi3 mutants M. truncatula showed comparable reduction number seed-specific LEA proteins. Materials and methods Plant material and treatments Seeds M. truncatula (A17) were produced as described in Chatelain et al. (2012). Castanospermum australe seeds were harvested during maturation and at shedding from trees growing in Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 'orthodox' and 'recalcitrant' are used to describe storage behaviour seeds. Orthodox seeds undergo maturation drying and are shed from parent plant at low moisture contents. During maturation, they acquire desiccation tolerance, allowing them to be dried to moisture contents in range 1–5% without irreversible damage. Because this ability, seeds can be stored for long periods in cold and dry vaults. Recalcitrant seeds, on other hand, do not undergo maturation drying, and are shed at relatively high moisture contents. Such seeds are highly susceptible to desiccation injury, and thus are not storable under conditions suitable for orthodox seeds (reviewed in Farnsworth, 2000; Berjak and Pammenter, 2008; Li and Pritchard, 2009). The mechanisms by which recalcitrant seeds lose viability during drying and/or storage are not well understood, which poses challenge to determine appropriate measures to better conserve these species. In orthodox seeds, isolation and analysis viviparous mutants and loss-of-function mutants impaired in embryogenesis and seed maturation resulted in identification master seed development regulator loci lec1 and abi3, regulating partial and redundant desiccation tolerance (Ooms et al., 1993; Parcy et al., 1997; To et al., 2006). third regulator, FUSCA3, appears to control seed longevity (Tiedemann et al., 2008). In Arabidopsis and maize, some target genes these activators are genes proposed to have protective role in desiccation tolerance, such as small heat shock protein genes, genes with antioxidant functions, as well as late embryogenesis abundant (LEA) genes (Kotak et al., 2007; Bies-Etheve et al., 2008; Mnke et al., 2012). LEA proteins are small hydrophilic, largely unstructured and thermostable proteins that are synthesized in orthodox seeds during mid- to late maturation and in vegetative tissues upon osmotic stress. They are thought to have range protective functions against desiccation with different efficiencies, including ion binding, antioxidant activity, hydration buffering, and membrane and protein stabilization (Tunnacliffe and Wise, 2007; Battaglia et al., 2008; Amara et al., 2012). Evidence an in vivo role for these proteins in seed desiccation comes from Arabidopsis thaliana em6-deficient mutants that show defects in maturation drying (Manfre et al., 2009). recent study in A. thaliana showed that down-regulation seed-specific dehydrins (one LEA families) reduced seed survival in dry state, although seeds did acquire desiccation tolerance (Hundertmark et al., 2011). However, precise role LEA proteins in seed desiccation tolerance remains to be ascertained for vast majority them. Genomic studies to date have identified large number LEA genes whose expression is restricted to seed tissues and/or up-regulated in response to biotic and abiotic stress in vegetative tissues (Illing et al., 2005; Hundertmark and Hincha, 2008; Amara et al., 2012; Chatelain et al., 2012). Proteomic studies demonstrate that subset polypeptides accumulate during acquisition desiccation tolerance and/or longevity in orthodox seeds (Boudet et al., 2006; Buitink et al., 2006; Chatelain et al., 2012). In desiccation-sensitive seeds Arabidopsis mutants, transcript levels several LEA genes were reduced, whereas other increased (e.g. dehydrins) (Bies-Etheve et al., 2008). The situation regarding occurrence and role LEAs in LEA proteome desiccation-sensitive seeds  |  4561 Cloning MtABI3 To obtain full-length sequence for MtABI3, genomic DNA was extracted from leaf material M. truncatula A17 using Nucleospin Food kit (Macherey Nagel). An inverse PCR was performed on 5 μg genomic DNA that was digested with EcoRI (25 U per 50 μl final volume; Promega, Madison, WI, USA), and ligated U using T4 DNA ligase (50  per 450 μl final volume; Fermentas, Vilnius, Lithuania). The full length was amplified on ligated DNA using primers indicated in Supplementary Table S1 at JXB online that were designed based on MtABI3 fragment available in expresssed sequence tag (EST) database (TC97588, DFCI Medicago truncatula Gene Index v8). The full-length genomic bp) was cloned into pJet1.2 (CloneJET kit, DNA fragment (3 458  Thermo Scientific, Bremen, Germany) and sequenced (for primers see Supplementary Table S1). RNA extraction, and sequencing and assembly For M. truncatula seeds, total RNA was extracted using nucleospin RNAplant kit (Macherey Nagel, Düren, Germany), and 10 μg total RNA from each sample were DNase treated (Turbo DNase, Ambion) and purified (RNeasy MinElute Cleanup kit, Qiagen) according to manufacturer's instructions. Total RNA was extracted with phenol from cotyledons or embryonic axes C. australe as described by Bove et al. (2005). The quantity, purity, and integrity RNA were checked using NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies) and bioanalyzer (Experion, BioRad). From 2009 RNA pool, cDNA library was prepared, normalized, and sequenced by GenXPro GmbH (Frankfurt am Main, Germany) using Illumina technology (Genome Analyser-IIx). From 2011 RNA pool, cDNA library was prepared, normalized, and sequenced by Eurofins (Ebersberg, Germany) using 454 GS FLX+ technology. Reads obtained from each sequencing were assembled novo in two steps: first with MIRA 3.4.0 (Chevreux et al., 2004) then with DNA Dragon (SequentiX, http://www.sequentix.de/software_dnadragon.php). The detailed procedure is described in Supplementary Fig. S1 at JXB online. Functional annotation and classification Contig annotation to known sequences by sequence similarity was performed using two M. truncatula nucleic databases: MT3.5 from International Medicago Genome Annotation Group (IMGAG) and MtGI11 from Dana-Farber Cancer Institute (DFCI) Medicago Gene Index. Contigs that remained unannotated after these two analyses were blasted using Blast2GO (version 2.6.0) (Gtz et al., 2008) against protein databases including all plant species: Swissprot and non-redundant protein from NCBI. Next, classification C. australe annotations in Gene Ontology (GO) was performed by Blast2GO. GO terms were retrieved from public databases and mapped to each contig, after which most specific ones were selected by an annotation rule. The detailed annotation workflow is described in Supplementary Fig. S1 at JXB online. RT–PCR 2 μg aliquot M. truncatula wild type and abi3-1 and abi3-2 RNA was reverse transcribed according to manufacturers' instructions (Thermo Scientific). The resulting cDNAs were diluted 1:3. Primer sequences and annealing temperatures are provided in Supplementary Table S1 at JXB online. PCR was performed with DreamTaq (Fermentas) according to manufacturer's instructions. Protein extraction and 2D gel electrophoresis Total soluble proteins were extracted in triplicate from 25 seeds M. truncatula (A17, R108, or Mtabi3-1 and Mtabi3-2) and 400 mg cotyledons mature C. australe from minimum three seeds for each replicate (2009 harvests) according to Boudet et al. (2006), and heat-stable proteins were recovered according to Chatelain et al. (2012). After centrifugation at 20 000 g at 4 °C, pellet was successively washed with 100 μl 80% acetone, 100% acetone, 80% ethanol, and 100% ethanol then resuspended in 300 μl rehydration buffer for 36 h according to Boudet et al. (2006). Protein concentration was assayed according to Bradford (1976). Heat-stable protein fractions M. truncatula and C. australe (150 μg), as well as 1:1 mix both protein fractions (300 μg), were rehydrated and separated on 24 cm immobilized non-linear pH 3–10 gradient strips (Bio-Rad, Hercules, CA, USA). Isoelectric focusing was performed at 20 °C, for 3  at 250  then 4  at 6  h V, h kV, followed by gradual increase to 27 kVh at 6 kV h–1 and to 40 kVh at 8 kV h–1 in BioRad Protean isoelectric focusing cell. Size separation proteins was performed on vertical polyacrylamide gels [12% (w/v) acrylamide] in Ettan Daltsix Electrophoresis system (Amersham Biosciences, Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Pietermaritzburg (Kwazulu-Natal, South Africa) in 2009 and 2011. Within 48  after collection, they were air-freighted to Angers h (France) where there were immediately processed as indicated. From 2009 harvest, embryos and cotyledons from immature and mature seeds were separated. Cotyledon tissues were used for critical water content determination or dried for 1 or 3 d over 75% relative humidity (RH) NaCl before being frozen in liquid nitrogen then stored at –80°C for RNA sequencing (Illumina) and proteomics. From 2011 harvest, cotyledons and embryos were extracted from immature (green pods), mid-mature (yellow pods), and mature (brown pods) seeds, and frozen fresh in liquid nitrogen then stored at –80 °C. The 2011 harvest was used for analysis by 454 to improve sequence assembly. Desiccation sensitivity mature C. australe cotyledons was determined on 3 × 5 × 3 mm cubes that were isolated from inner part cotyledons. Cubes were dried for indicated time intervals over saturated salt solution at 75% RH, after which they were divided into two halves. One half was used for water content determination, and other half for viability assessment following incubation for 24  in 1% (w/v) tetrazolium solution h (Sigma-Aldrich, France). Red colour was quantified by pixel intensity on image using ImageJ software (http://rsb.info.nih.gov/ ij/). Water content was determined gravimetrically by weighing seeds before and after drying in an oven for 48 h at 96 °C. Viability assays were performed on four independent drying experiments 50–100 cubes. Two M. truncatula mutants with Tnt1 insertions in ABI3 gene (NF3185, hereafter referred to as Mtabi3-1; and NF6003, Mtabi3-2) were obtained from Samuel Noble Foundation (Oklahoma, USA). Tnt1 insertions in two mutants were verified by PCR (see Supplementary Table S1 available at JXB online for primers). Mutant and wild-type lines (R108) were multiplied in growth chamber according to Chatelain et al. (2012), and lines were backcrossed once for Mtabi3-1 and twice for Mtabi32 mutants. Desiccation tolerance was determined on seeds that were harvested at different time points during development. Two to five replicates 30–50 seeds were rapidly dried to 0.09 g H2O g DW–1 over an airflow 43% RH, and rehydrated after 2 d on filter paper at 20 °C in dark. Seeds were considered desiccation tolerant when they germinated, scored by protrusion radicle through seed coat. For ABA insensitivity assays, triplicates 40–50 freshly harvested seeds just prior to pod abscission (0.8–1.0 g H2O g DW–1) were imbibed on filter paper on range ABA concentrations (mixed isomers, Sigma, St Louis, MO, USA) at 20 °C. ABA was dissolved in methanol prior to dilution in water. Control seeds were imbibed in MeOH concentration corresponding to highest ABA concentration (0.5% MeOH). Germination was scored after 14 d. For proteomic analysis, Mtabi3-1 and Mtabi3-2 seeds were harvested at point abscission, when seeds were still viable. For reverse transcription–PCR (RT–PCR) analysis, seeds were harvested at 24 days after planting (DAP). 4562 | Delahaie et al. Orsay, France) according to Boudet et al. (2006) using running buffer containing 15.6  mM TRIS (pH 8.3), 120  mM glycine, and 0.1% (w/v) SDS. Gels were stained with 0.08% (w/v) Brillant Blue G-Colloidal for 24  and destained briefly in 5% (v/v) acetic acid h, and 25% (v/v) methanol, then in 25% (v/v) methanol for 8 h. Stained gels were scanned at 63.5 × 63.5 resolution using GS 800 scanner (Bio-Rad). At least three digitalized gels from three independent experiments (extraction, focalization, and migration) were analysed using PDQuest 7.2.0 software (Bio-Rad). Spot intensities were normalized using total quantity in valid spot method. paired t-test was performed to analyse differences in intensity between C. australe and M. truncatula LEA proteins and between wild-type (R108) and Mtabi3-1/Mtabi3-2 seeds. Data submission Raw sequence data from this article can be found in Sequence Reads Archive database (NCBI) under BioProject PRJNA193308. The data on ectopic expression MtABI3 in hairy roots discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE44291 (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE44291). Results Physiological description C. australe seeds On receipt, fresh weight and water content mature, shed C. australe seeds were 45.9 ± 14.9 g per seed and 1.94 ± 0.41 g H2O g DW–1, respectively. The embryo is composed two prominent cotyledons and comparatively small axis, and is surrounded by thin brown testa (~250 μm thickness) (Fig. 1A–C). When planted, fresh seeds germinated at 100% and produced healthy seedlings. The critical water content corresponding to onset loss cell viability during rapid drying was determined on cotyledons using tetrazolium staining. During drying, loss viability as function water content showed typical pattern found in other recalcitrant seeds (Fig. 1D, E). The intensity staining remained high and constant until water content ~1.5  g H2O g DW –1 was reached, after which intensity decreased progressively with further drying (Fig. 1E). The critical water content, here defined as water content corresponding to break point for which cotyledon tissues begin to lose staining intensity, was estimated at 1.2 g H2O g DW–1. Tissues Sequencing C. australe seed transcriptome and identification LEA contigs To enable comparative analysis on molecular level between recalcitrant C. australe and its orthodox counterpart M. truncatula, sequence information on transcripts present in seeds was obtained from range tissues to capture maximum variation transcriptome at harvest: intact isolated axis, cotyledons from three developmental stages, and partially dried yet alive cotyledon tissues (Supplementary Fig. S1 at JXB online). Using Illumina and 454 technologies, sequencing normalized cDNA libraries resulted, respectively, in 7 784 004 paired reads 76  and 626 225 bp reads with an average length 376 bp (Table 1). The assembly resulted in 48 334 contigs varying between 200  and bp 14 334  long with an average length 773  (Table 1; bp bp Supplementary Fig. S1 at JXB online). total 35 050 contigs (72.5%) were annotated, which 91% were provided by IMGAG 3.5 M. truncatula database and MtGI11 version Medicago EST database. An additional 740 and 2558 remaining contigs were identified, respectively, using Swissprot and NR databases related to other plant species (Supplementary Fig. S1). Annotations were classified according to GO using Plant-GO-slim version Blast2GO (B2G). Enzyme classification (EC) numbers were retrieved with additional functionalities B2G linked to KEGG pathways. total 23 637 contigs (48.9%) were annotated with 98 615 GO terms and 8 962 EC numbers. The distribution main biological processes (BP, 45 568 annotations), molecular functions (MF, 35 110 annotations), and cellular components (CC, 17 937 annotations) is shown in Supplementary Fig. S2. Using annotated transcriptome C. australe, next step was to perform comparative analysis between LEA sequences found in both legume species. In C. australe, contigs were found for 29 LEA genes that are identified in M. truncatula genome (Supplementary Table S2 at JXB online). In mature seeds M. truncatula, proteome analysis led to detection polypeptides corresponding to 17 genes (Chatelain et al., 2012). For 16 out these 17 genes, at least one corresponding contig was detected in C. australe transcriptome (Table 2). Amino acid sequence alignment Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Mass spectrometry and protein identification Spots interest were excised from 2D gels and subjected to in-gel tryptic digestion according to Chatelain et al. (2012). Tryptic fragments were analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) spectroscopy using nanoscale HPLC (Famos-Switchos-Ultimate system, LC Packings, Dionex, San Francisco, CA, USA) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF Global, Micromass-Waters, Manchester, UK) as described in Boudet et al. (2006). Mass data were analysed with Protein Lynx Global Server software (Micromass-Waters). Protein identification was performed by comparing data with UniProt sequence databank (date release: August 2010) or with TIGR Medicago EST databank (date release: April 2010). For M. truncatula heat-stable proteome, spots linked to LEA polypeptides were identified according to reference gel published by Chatelain et al. (2012). were completely lacking red staining when dried below 0.5 g H2O g DW–1 (Fig. 1D), indicating total loss viability. This value is consistent with that reported by Han et al. (1997) on isolated axes C. australe during rapid drying using electrolyte conductivity as an indication membrane damage. In contrast, complete drying and rehydration 10  imbibed h M. truncatula cotyledons in tetrazolium solution rendered tissues red, indicating that viability was maintained (data not shown). For proteomic study, focus was on cotyledons as model for desiccation-sensitive tissues, due to their high critical water content. Cotyledons are large and surround axis, thereby possibly slowing rate dehydration latter. In M. truncatula, few differences were found between LEA abundance and composition axes and cotyledons, except for EM6 (Chatelain et al., 2012). LEA proteome desiccation-sensitive seeds  |  4563 D Drying time (h) 2 0 4 6 8 Axis Cot E 200 180 160 140 120 0.0 0.5 1.0 1.5 2.0 2.5 Water content (g H2O g 3.0 3.5 DW−1) Fig. 1.  Determination critical water content mature Castanospermum australe cotyledons. Castanospermum australe seed after shedding with (A) and without seed coat (B). (C) Embryonic axis and cotyledons mature seeds. The scale bar represents 1 cm. (D) Tetrazolium staining (red indicating living tissues) cubes (50 mm3) taken from core mature cotyledons that were first dried at 44% RH for indicated time. (E) The relationship between water content during drying and pixel intensity tetrazolium (TZ) staining. Data for four independent experiments (represented by different symbols) are shown. Table 1.  Contig features from 454 and Illumina RNAseq Castanospermum australe seeds and annotation corresponding transcriptome 454 Total number or reads after sequencing Total number contigs Average contig length N50 Number nucleotides in contigs Total number contigs annotated Contigs annotated with MT3.5 Contigs annotated with MtGI11 Contigs annotated with Swissprot Contigs annotated with NR (NCBI) Illumina 454+Illumina 626 225 36 767 869 1001 31 937 738 28 391 19 885 4 847 1 470 2 189 7 784 004 18 483 318 345 5 882 730 16 018 11 987 3 207 414 410 8 410 229 48 334 773 1020 37 365 884 35 050 25 615 6 138 739 2 558 Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Pixel intensity (TZ staining) 220 4564 | Delahaie et al. Table 2.  LEA transcripts identified in Castanospermum australe seed transcriptome Protein name Blast database M. truncatula ID C. australe contigsa E-value Alignment length Percentage identity Percentage similarity Spot on 2D gel Dehydrin DHN3 DHN-cognate BudCar5 EM6 EM1 SBP65 PM10 PM18 MP2 LEAm CAPLEA.I PM1 D113.II PM25 D-34.I D-34.II D-34.III MtGI11 Mt3.5 Mt3.5 Mt3.5 MtGI11 Mt3.5 MtGI11 MtGI11 Mt3.5 Mt3.5 MtGI11 Mt3.5 Mt3.5 MtGI11 MtGI11 MtGI11 Mt3.5 TC175037 Medtr3g117290 Medtr7g086340 Medtr4g016960 AJ498523 Medtr4g079690 TC174929 TC183861 Medtr1g061730 Medtr2g014040 TC175990 Medtr7g093170 Medtr7g093160 TC174777 Medtr1g072090 TC183570 Medtr2g076230 Ca_11990 Ca_9276 Ca_31427 Ca_14307 Ca_2036 Ca_7340 Ca_3035 Ca_330 Ca_8462 Ca_7604 Ca_8841 Ca_8304 Ca_8304 Ca_6007 ND Ca_25629 Ca_23377 2.E-09 2.E-10 5.E-06 4.E-45 9.E-40 5.E-17 4.E-43 2.E-41 3.E-31 3.E-45 1.E-31 3.E-16 5.E-17 2.E-83 198 120 119 98 100 90 274 335 226 268 141 91 91 238 47.2 53.3 62.2 81.6 78.2 58.9 59.9 56.0 57.5 48.6 66.0 64.8 65.2 75.6 52.0 65.0 77.7 90.8 85 72.2 74.8 67.2 67.1 62.4 85.8 75.8 76.1 84.4 6.E-42 3.E-06 128 24 86.7 76.0 93 87.5 3, 4 5 1 37 51 75 ND ND 9 74 18,19 ND ND 92 ND ND ND LEA_5 LEA_4 LEA_1 SMP ND, homologue not detected. Contigs were translated to calculate percentage identity and similarity. Castanospermum australe contigs that were homologues to 17 LEA gene products detected in M. truncatula seeds were identified based on MtGI11 and Mt3.5 Medicago databases (E-value 90% similarity between two species. The dehydrin family members were most divergent, with similarity that ranged only from 52% to 77% (Table 2). Other families are much more conserved between two species, such as SMP and LEA_5 family (EM1 and EM6), showing 76–86% identity and 85–91% similarity with M. truncatula, respectively. Identification heat-stable proteome C. australe Identification polypeptides corresponding to 16 LEA transcripts that were detected in C. australe transcriptome was carried out by separation heat-stable protein fraction by 2D gel electrophoresis (Fig. 2). This method has been successfully applied to characterize and quantify entire LEA proteome M. truncatula (Boudet et al., 2006; Chatelain et al., 2012). total 110 spots were sequenced using LC-ESI-MS/MS spectroscopy, out which 82 spots were identified (Supplementary Table S3 at JXB online). Polypeptides were detected for 10 16 LEA genes identified from C. australe sequence assembly (Table 2). These polypeptides include two highly abundant dehydrins [CaDHN3 (spot 3 and 4) and CaBudCar5 (spot 1)] and CaCAPLEA-1 (spot 18 and 19). Other less abundant LEA polypeptides include one more dehydrin (CaDHN-cognate, spot 5), two LEA_5 members [CaEM1 (spot 51) and CaEM6 (spot 37)], three LEA_4 members [CaSBP65 (spot 75), CaMP2 (spot 9), and CaLEAm (spot 74)], and CaPM25 (spot 92). For six LEA contigs, no polypeptides were identified in C. australe proteome, despite presence their transcripts (Table 2). In M. truncatula, four these LEA proteins are highly abundant in mature seeds and include two members LEA_5 family (CaPM10 and CaPM18) and both LEA_1 members (CaD113.I and CaPM1) (Chatelain et al., 2012). The other two LEA proteins that were not identified are members SMP family (CaD34.II and III). In addition to LEA polypeptides, other abundant polypeptides were detected in heat-stable C. australe proteome. They were identified as three pathogenesis-related proteins (spots 26, 27, and 29) (Supplementary Table S3 at JXB online), five polypeptides corresponding to small heat shock proteins (spots 2, 56, 61, 62, and 93), and four polypeptides corresponding to superoxide dismutases (38, 54, 67, and 69). Furthermore, two desiccation-related polypeptides (spots 82 and 88) were detected with homology to Lb_13-62 and PCC13-62. These genes are up-regulated in desiccation-tolerant resurrection plants Craterostigma plantagineum and Lindernia brevidens (Phillips et al., 2008) and were also recently detected in floral nectar evergreen velvet bean (Mucuna sempervirens Hemsl) (Zha et al., 2013). Comparative analysis LEA proteome between C. australe and M. truncatula The amount heat-stable proteins relative to total soluble protein fraction was lower for C. australe (20 ± 1.8%) than for M. truncatula seeds (36%; Chatelain et al., 2012). Equal amounts heat-stable protein fraction C. australe or M. truncatula were separated by 2D gel electrophoresis, and 2D profiles were compared. For most LEA polypeptides, exact position on gel differed slightly between both species (Fig. 3A, B). This made it possible to combine two extracts and separate them on same gel, allowing for an accurate comparative quantification polypeptides Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Family (PFAM) LEA proteome desiccation-sensitive seeds  |  4565 5.5 4.5 pI MW 95 76 97 10 75 9 96 85 83 5 102 82 93 79 87 4 92 91 80 110 74 72 52 57 78 25 66 1 62 54 56 50 70 67 33 39 51 40 41 46 55 27 26 28 19 18 58 31 38 108 13 24 16 23 42 22 29 37 45 32 14 17 44 35 36 Fig. 2.  Reference map heat-stable proteome mature Castanospermum australe cotyledons. 150 μg aliquot heat-stable proteins was separated by 2D SDS–PAGE using 24 cm non-linear immobilized pH gradient strips (3–10). pI and molecular mass (MW) (in kDa) are indicated. Numbers indicate polypeptides that were sequenced (see Table 2; and Supplementary Table S3 at JXB online). from both species and avoiding drawbacks associated with variations due to polypeptide migration and gel staining (Fig. 3C; Supplementary Table S4 at JXB online). In both species, dehydrin DHN3 (Fig. 3D, H, L) and LEA_4 CAPLEA (Fig. 3G, K, O) were present with high spot intensity. For six LEA polypeptides, spot intensity was much lower in C. australe compared with M. truncatula; SBP65 and MP2 (Fig. 3D, H, L), PM25 and LEAm (Fig. 3E, I, M), and EM1 and EM6 (Fig. 3F, J, N) (Supplementary Table S4). The other two dehydrins, BudCar5 and DHN-cognate, are highly abundant in C. australe (Fig. 3I, K), whereas their homologues in M. truncatula are barely detectable (Fig. 3E, G). quantitative overview comparative analysis LEA proteome, based on relative spot intensity, is presented in Fig. 4. The LEA profile is strikingly different between recalcitrant and orthodox seeds. In contrast to mature M. truncatula seeds, where dehydrins comprise 20% LEA proteome, this family represents 83% LEA proteome C. australe cotyledons. Four LEA proteins (CaEM1, CaEM6, CaMP2, and CaPM25) were 4-fold less abundant in recalcitrant seeds compared with orthodox M. truncatula, whereas CaLEAm and CaSBP65 relative abundance was reduced >20-fold. In addition, six LEA proteins were not detected in cotyledons C. australe (CaPM1, CaD113.I, two CaD34 members, CaPM10, and CaPM18). CAPLEA was present in comparable amounts in both species. Characterization LEA proteome desiccation-sensitive Mtabi3 mutant seeds and comparison with C. australe To investigate further cause–effect relationship between lack these LEA proteins and desiccation sensitivity, LEA proteome was examined in an orthodox seed that was rendered desiccation sensitive by knocking out MtABI3 gene expression. First, two independent homozygous Tnt1 insertion mutants (Mtabi3-1 and Mtabi3-2) that were backcrossed once or twice, respectively, were obtained (Fig. 5A). The Tnt1 insertions were located at 1 595 bp and 1 605 bp from start codon, respectively, just after B2 domain (Fig. 5A). RT– PCR analysis confirmed absence transcripts in two Mtabi3 mutants (Fig. 5B). The resulting freshly harvested seeds were used for physiological characterization. Like in abi3 mutants Arabidopsis (Ooms et al., 1993), mature Mtabi3 seeds retained their chlorophyll (Fig. 5C) and exhibited strongly reduced sensitivity to ABA (Fig. 5D). During seed maturation between 24 and 32 DAP, seed water content abi3 mutants remained at ~1.6 g H2O g DW–1, whereas Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 48 61 63 49 73 76 77 68 71 90 65 3 69 2 89 99 98 103 94 64 6 7 88 84 17 101 8 86 43 21.5 9.0 4566 | Delahaie et al. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Fig. 3.  Comparative analysis LEA polypeptides in heat-stable proteome cotyledons Castanospermum australe and Medicago truncatula seeds. Reference map heat-stable proteome M. truncatula (A) and C. australe seeds (B) and separation mixture equal amounts heat-stable proteins (150 μg) from both species (C). (D–O) Details different regions 2D gels proteome M. truncatula (D–G), C. australe (H–K), and both species (L-O). The indicated spots refer to Supplementary Table S4 at JXB online. in developing wild-type seeds it decreased steadily from 1.2 g H2O g DW–1 to 1.0 g H2O g DW–1 (Fig. 5E). Thereafter, in both abi3 mutants and wild type, water content decreased until 40 DAP. During latter stages drying, when pods were detached, seeds both genotypes exhibited similar rate water loss. Desiccation tolerance harvested seeds was determined as function their water content at different stages during maturation and after enforced drying (at 40 DAP) (Fig. 5F). In contrast to wild type, seed population Mtabi3 mutants started to lose their viability when water content decreased below 1.0  H2O g DW–1 and g decreased sharply below 0.5 g H2O g DW–1 (Fig. 5F). At 0.2 g H2O g DW–1, all abi3 seeds were dead. In contrast to wildtype seeds, fully mature, dried seeds did not germinate, and tetrazolium tests showed no staining, indicating that viability was completely lost (data not shown). LEA proteome desiccation-sensitive seeds  |  4567 with wild-type seeds, whereas its amount was higher in Mtabi3-1 mutant (Fig. 6; Supplementary Table S5). Normalized spot intensity (AU) 50 DHN3 DHNcognate Budcar EM1 EM6 SBP65 PM10 PM18 LEAm MP2 Caplea D113.I PM1 D34 PM25 40 30 20 10 M. truncatula C. australe Fig. 4.  Relative abundance different LEA polypeptides identified in cotyledons Castanospermum australe and Medicago truncatula seeds. Abundances were calculated based on spot intensities three replicates gels shown in Fig. 3. Next, LEA polypeptide abundance was determined using 2D gel electrophoresis on three replicates Mtabi31, Mtabi3-2, and wild-type seeds (R108 background) (Supplementary Table S5 at JXB online). To be able to compare LEA profiles among C. australe and Mtabi3 genotypes, polypeptide abundance was expressed as relative difference from M. truncatula wild type (A17 for comparison with C. australe, and R108 for comparison with Mtabi3 mutants) (Fig. 6). The intensity MtPM1 was highly variable amongst samples, irrespective M. truncatula genotypes (Supplementary Table S5). This might be due to very basic nature this protein in R108 genotype, placing it at border gel where resolution is poor. Likewise, intensity MtLEAm and D34.II could not be determined correctly (Fig. 3; Supplementary Table S5). To avoid incorrect interpretation these data, they were omitted from further analysis. Overall, abundance LEA proteome Mtabi3 mutants compared with wild-type seeds resembled that desiccation-sensitive C. australe cotyledons when compared with M. truncatula (Fig. 6). As in C. australe, abundance nine LEA polypeptides from several families was decreased in Mtabi3 mutants, namely LEA_5 (MtEM1 and MtEM6), SMP (MtPM25 and MtD34), LEA_4 (MtSBP65, MtPM18, MtPM10, and MtMP2), and LEA_1 (D113.I). The dehydrin MtDHN3 was more abundant in seeds Mtabi3 mutants than in wild-type seeds, which further underscored similarity with C. australe. The relative amount MtCAPLEA was slightly lower in seeds Mtabi3-2 mutant compared The reduction LEA polypeptides in Mtabi3 mutants raises question whether 12 reduced or absent LEA proteins in C. australe are regulated by ABI3 at gene level. In abi3 seeds Arabidopsis, transcript levels all LEA genes that are affected in desiccation-sensitive tissues (group A) were decreased (Table 3; Bies-Etheve et al., 2008). This was not case for LEA proteins that were not affected or that were more abundant in these tissues (group B). Transcript levels homologues DHN-cognate and CAPLEA1 were even increased in abi3 mutants. In addition, to investigate whether MtABI3 regulates LEA targets, advantage was taken recent transcriptome study on effect overexpressing MtABI3 in hairy roots M. truncatula (GeOmnibus GS GSE44291). The advantage this ectopic expression model is that it avoids interfering effects other B3 domain transcription factors such as FUS3 and LEC2 with ABI3 in seeds (Mnke et al., 2012). Transcript levels 9 out 11 genes coding for group LEA proteins were up-regulated by MtABI3 (Table 3). Moreover, in silico promoter analysis 8 out 10 LEA genes for which promoter sequences could be retrieved indicated that seven LEA promoters from group contain both RY (CATGCA) and ABRE (ACGTG(G/T)C) cis-regulatory elements. The RY element is known to be bound by ABI3, while ABRE motifs are implicated in binding bZIP-TFs, known to interact with ABI3 (Busk et al., 1997; Hattori et al., 2002; Guerriero et al., 2009). study on identification ABI3 regulon in Arabidopsis confirmed three LEA genes as direct targets by transient promoter activation assay or ChIP-chip analysis (Table 3). The other LEA proteins were identified as being ABI3-responsive gene products in 35S::ABI3-GR seedlings (Mnke et al., 2012). An analysis other four LEA proteins that were abundant in C. australe cotyledons (group B, Table 3) demonstrated that overexpressing MtABI3 in M. truncatula roots induced DHN3 trancripts and slightly activated BudCar5, although transcript levels were found to increase in abi3 mutants (Table 3). No effect was found on transcript levels CAPLEA-1, and DHN-cognate transcripts even decreased significantly. No RY element was retrieved in promoter analysis BudCar5 and DHN-cognate coding genes, and neither gene was part ABI3 regulon identified by Monke et al. (2012). Discussion The aim this study was to compare seed LEA proteome two legume species exhibiting orthodox and recalcitrant storage behaviour to gain further insights into panoply these protective proteins necessary for desiccation tolerance. This work shows that C. australe and M. truncatula, both from Papilionaceae subfamily Fabaceae, are phylogenetically close enough to allow for detailed sequence comparison Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 0 ABI3 regulation identified LEA proteins in relation to desiccation tolerance 4568 | Delahaie et al. LEA accumulation in relation to desiccation tolerance. Assembly normalized sequencing library identified contigs with high similarity for 16 17 M. truncatula LEA genes (Table 2) for which protein accumulation was shown in M. truncatula (Chatelain et al., 2012). This comparison was further extended to desiccation-sensitive Mtabi3 mutant M. truncatula that was obtained and characterized. It is believed that this is first report full coverage identification LEA genes and their products (the 'LEAome') in cotyledons (the predominant tissue in this species) recalcitrant seed. To date, studies have been constrained to dehydrins using an antibody against consensus sequence KIKEKLPG (Berjak and Pammenter, 2008). The comparison with M. truncatula revealed that 12 out 16 LEA proteins are less abundant or not detected in recalcitrant C. australe seed proteome (Figs 4, 6). In silico gene expression analysis M. truncatula transcriptomes demonstrated that all but one (MtMP2) these 12 genes are specifically expressed in seed tissues (Chatelain et al., 2012). Further LEA proteome analysis Mtabi3 mutants revealed that accumulation homologues these LEA proteins was affected in these desiccation-sensitive seeds. Several them (MtSBP65, MtPM25, MtEM6, MtPM18, and MtMP2) correlated with re-induction desiccation tolerance in germinated radicles M. truncatula seeds (Boudet et al., 2006). Figure 5E and F shows that seeds Mtabi3 mutants can survive drying to 0.4 g H2O g DW–1 and can be considered drought tolerant. They lose their viability after they are shed from mother plant. In C. australe, tissues did not survive drying down to 0.5  H2O g DW–1. g Collectively, these data suggest that these particular LEA proteins are needed once bulk water is removed. For most them, their role in dry state is not yet elucidated. In vitro studies EM6, PM25 (Boudet et al., 2006; Gilles et al., 2007; Boucher et al., 2010), and LEAm (Tolleter et al., 2007) demonstrated multifunctional protective capacities with different efficiencies. These include membrane (LEAm) and enzyme protection (LEAm, EM6, PM25), anti-aggregation Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Fig. 5.  Characterization abscisic acid insensitive3 (Mtabi3) mutants Medicago truncatula. (A) Gene structure, and position and B domains and Tnt1 insertions within MtABI3 gene. (B) Validation absence ABI3 transcripts in Mtabi3-1 and Mtabi3-2 mutants. Using same primer set, ABI3 was also amplified on genomic DNA. The increased size corresponds to additional introns. (C) Seed colour phenotype Mtabi3-1 and Mtabi3-2 and corresponding wild-type seeds (R108) at three stages maturation: 32 days after pollination (DAP), at pod abscission (ABS, 38 DAP), and in dry seed (DS). (D) ABA dose–response analysis during germination seeds collected at pod abscission. Germination was scored as emergence radicle. Data are average three replicates 40–50 seeds ±SE. (E) Changes in seed water content during development. Data are average three replicates three seeds ±SE. (F) Germination Mtabi3 and wild-type seeds at different stages development upon rehydration 70–80 seeds. Data are significantly different when they differ by ≥18% (χ2 test, P < 0.05). LEA proteome desiccation-sensitive seeds  |  4569 LEA protein abundance normalized to wild-type M. truncatula 3.0 EM1 EM6 D34.I PM25 SBP65 PM18 PM10 MP2 CAPLEA DHN3 D113.I 1.5 1.0 0.5 0.0 * ** * ** * ** C. australe Mtabi3-1 Mtabi3-2 against thermo-mechanical stress (EM6 and PM25), and water binding (EM6 and PM25). This work offers new model to study regulatory and mechanistic pathways implicated in desiccation tolerance through comparative analysis desiccation-sensitive cotyledon tissues from recalcitrant seeds and their orthodox counterparts. The proteome comparison with Mtabi3 seeds suggests that comparable pathways leading to LEA Table 3.   Evidence for ABI3-dependent regulation LEA homologues for which protein abundance is reduced or absent in desiccation-sensitive tissues (C. australe and Mtabi3) (group A) or unaffected or increased (group B) Protein name EM6 EM1 SBP65 PM10 LEAm MP2 PM18 PM1 D113.II PM25 D-34.I D-34.III DHN3 DHN-cognate BudCar5 CAPLEA-1 LEA group Sequence ID Nimblegen probe B B B B Medtr4g016960 AJ498523 Medtr4g079690 Medtr8g134020 Medtr2g014040 Medtr1g061730 TC183861 Medtr7g093170 Medtr7g093160 TC174777 Medtr1g072090 Medtr2g076230 TC175037 Medtr3g117290 Medtr7g086340 TC175990 Medtr_v1_022627 Medtr_v1_072582 Medtr_v1_083614 Not present on slide Medtr_v1_009629 Medtr_v1_005821 Medtr_v1_076240 Medtr_v1_045826 Medtr_v1_045826 Medtr_v1_082683 Medtr_v1_006041 Medtr_v1_012326 Medtr_v1_066754 Medtr_v1_020587 Medtr_v1_045277 Medtr_v1_085905 Cis-elementsb Medicago truncatula 35S::ABI3/ controla P-value 3.87 2.04 4.18 1.92E-06 5.53E-02 3.07E-03 2.98 3.87 –0.02 4.59 4.59 3.29 2.21 2.32 3.32 –1.47 1.57 1.06 2.34E-03 1.51E-05 8.65E-01 7,89E-07 7.89E-07 1.32E-02 4.07E-02 4.43E-02 1.10E-05 4.91E-04 3.70E-02 6.96E-02 Arabidopsis thaliana AGI 2 RY, 2 ABRE ND 2 RY, 1 ABRE ND 1 RY, 2 ABRE 0 RY, 2 ABRE ND 0 RY, 1 ABRE 2 RY, 1 ABRE ND 5 RY, 0 ABRE 0 RY, 0 ABRE ND 0 RY, 1 ABRE 0 RY, 0 ABRE ND Transcript level in abi3 seeds versus WTc ABI3 targetsd AT2G40170 AT3G51810 AT2G42560 AT5G44310 AT5G44310 AT2G36640 AT2G36640 AT5G06760 AT5G06760 AT3G22490 AT3G22490 AT3G22490 ND AT1G76180 ND AT1G52690 Down Down Down NA NA Down Down NA NA Down Down Down ND Up ND Up – T, P T, P T, P T, P T, P T, P C C T, P, T, P, T, P, ND – ND – Log ratio transcript levels (and corresponding P-values) in hairy roots overexpressing MtABI3 compared with control (empty plasmid), determined by trancriptome analysis using Nimblegen slides (GeOmnibus GSE44291). b The number RY (CATGCA) and ABRE (ACGTG(G/T)C) cis-regulatory motifs known to bind ABI3 was revealed by analysing 2 kb promoter sequence M. truncatula genes. c Relative level transcripts in mature abi3 seeds Arabidopsis compared with wild type. Data are extracted from Bies-Etheve et al. (2008). d Identification ABI3-responsive gene products in 35S::ABI3-GR seedlings by array-based transcriptome analysis (T) or qRT-PCR (P) and confirmation as direct targets by transient promoter activation assay (A) or ChIP-chip analysis (C). Data are extracted from Mnke et al. (2012). ND, not detected; NA, not analysed; WT, wild type. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Fig. 6.  The LEA protein profile from desiccation-sensitive cotyledons Castanospermum australe and seeds Mtabi3 mutants compared with desiccation-tolerant Medicago truncatula wild-type seeds. Abundance LEA proteins in C. australe and Mtabi3 (assessed as spot intensity, Supplementary Tables S4, S5 at JXB online) was normalized against their respective value obtained for wildtype M. truncatula seeds. value 1 corresponds to wild-type values (C. australe/A17 and Mtabi3/R108). Hatched bars correspond to non-seed-specific LEA proteins. Polypeptides whose abundance was not detected are indicated by asterisks. 4570 | Delahaie et al. by Wall et al. (2009) and Garg et al. (2011). More than 72% 48 334 contigs could be annotated by this approach, and 91% this annotation is provided by M. truncatulaspecific databases. This approach also enabled discovery almost all LEA transcripts for comparison with M. truncatula. However, quantitative transcriptome analysis will be needed to reveal to what extent LEA polypeptide abundance is regulated at transcriptional and/or post-transcriptional level in C. australe. Furthermore, there are many other molecular protective mechanisms that could be missing in this recalcitrant species such as antioxidant defences, nonreducing sugars, heat shock proteins, and/or induction cell wall modifications (reviewed in Berjak and Pammenter, 2008; Leprince and Buitink, 2010). The sequence assembly from normalized library will enable construction microarrays to investigate further molecular aspects desiccation sensitivity in recalcitrant seeds. striking observation was highly increased amount dehydrins in recalcitrant seeds compared with M. truncatula (Fig. 6). Two them (BudCar5 and DHN3) have also been identified in desiccation-sensitive seedlings M. truncatula submitted to osmotic stress (Boudet et al., 2006). Furthermore, in silico analysis using Medicago gene atlas shows that these dehydrins are expressed in many different organs other than seeds in stressful conditions (Benedito et al., 2008; Chatelain et al., 2012). One can speculate on functional role for such proteins in recalcitrant seeds (Berjak and Pammenter, 2008). Most recalcitrant seeds are spheroid, with large cotyledons surrounding axis. The synthesis dehydrins in cotyledons can protect axis from dehydration stress that they will undergo after shedding. Furthermore, due to their size, dehydration is likely to be slow and thus requirement for protection against only mild water deficit stress should be sufficient for maintenance seed viability as whole in seeds shed into their natural environmental habitat. Dehydrins are known to increase tolerance to osmotic stress, demonstrated by overexpression dehydrin Rab17 and Rab28 in A. thaliana plants and maize plants, respectively (Figueras et al., 2004; Amara et al., 2013). In conclusion, comparative analysis LEA proteome profiles two unrelated desiccation-sensitive tissues (cotyledons C. australe and seeds Mtabi3) with orthodox M. truncatula indicates that developmental programme leading to desiccation tolerance involves synthesis variety seed-specific LEA proteins that have been poorly characterized so far and partially involves ABI3. This developmental programme is intertwined with synthesis additional LEA proteins such as dehydrins as an apparent need to retain some tolerance against mild osmotic stress during maturation. Supplementary data Supplementary data are available at JXB online. Figure S1. Sequencing, assembly, and annotation workflow C. australe seed transcriptome. Figure S2. GO annotation sequence assembly transcriptome C. australe seeds. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 accumulation are affected in both desiccation-sensitive orthodox and recalcitrant cotyledon tissues (Fig. 6). Consistent with this observation, homologous LEA genes in Arabidopsis and M. truncatula are ABI3 responsive (Table 3). It is not known whether reduced LEA polypeptides in C. australe cotyledons are linked to reduced CaABI3 activity or defective upstream or downstream signalling pathways. Interestingly, CaABI3 contig was detected in RNA assembly, but its temporal and spatial expression, as well as its efficiency need to be assessed. In developing orthodox seeds, RY cis-elements are elements that are crucial for transactivation through ABI3/VP1-like B3-domain proteins, whereas conserved ABA-responsive elements (ABREs; PyACGTGG/ TC) mediate ABI3-related ABA signalling in conjunction with other transcription factors, such as ABI5 (Busk et al., 1997; Hattori et al., 2002; Guerrriero et al., 2009). Most LEA promoters in Medicago for which protein abundance was decreased or absent in Castanospermum were found to contain both RY and ABRE elements (Table 3). The LEA genes for which protein abundance was not affected or even increased in C. australe compared with M. truncatula do not seem to be regulated by ABI3 (Table 3). Transcript levels DHN-cognate even increased in abi3 mutants Arabidopsis and decreased in transgenic roots when overexpressing MtABI3 (Table 3). Whether this gene is negatively regulated by ABI3 is unknown. Taken together, these results strengthen idea that only LEA proteins positively regulated by ABI3 are reduced in C. australe cotyledons. However, it is likely that additional regulatory pathways intervene in accumulation these desiccation-tolerant associated proteins because in both C. australe and Mtabi3 mutants, number LEA proteins were not absent but their levels were partially reduced. Other transcription factors that regulate LEA gene expression are ABI4 and ABI5 (Bies-Etheve et al., 2008; Reeves et al., 2011). ABI3 interacts with ABI5 to regulate expression downstream genes, whereas ABI4 controls induction ABI5 (Bossi et al., 2009; Cutler et al., 2010). However, in Arabidopsis, mature seeds abi4 and abi5 mutants are desiccation tolerant. In addition, loss-offunction lec1 mutants Arabidopsis produce seeds that lose their viability during desiccation or during first few weeks after harvest (Meinke, 1992). However, an analysis direct targets LEC1 did not reveal any LEA genes (Bäumlein and Junker, 2012; Wang and Perry, 2013). Considering that homologues ABI3, LEC1, ABI5, and FUS3 were detected in C. australe RNAseq assembly, role these transcription factors in seed development warrants further investigation, particularly in relation to its recalcitrant behaviour. Sequencing C. australe transcriptome was performed by high-throughput sequencing 454 and Illumina technologies on normalized library. Library normalization improves proportion low abundant sequences and maximizes transcriptome coverage (Zhulidov et al., 2004). Both technologies have been extensively used in past few years to sequence transcriptomes non-model species without reference genome (reviewed in Schliesky et al., 2012). Table 1 confirms that hybrid novo assembly combining both sequencing technologies improves transcriptome coverage, as suggested LEA proteome desiccation-sensitive seeds  |  4571 Table S1. Primer sequences used for PCR. Table S2. Overview contigs from C. australe transcriptome matching LEA-coding genes M. truncatula. Table S3. Summary identified spots from reference gel heat-soluble protein fraction C. australe cotyledons. Table S4. Normalized intensity polypeptides heat-stable proteome M. truncatula and C. australe seeds. Table S5. Normalized intensity polypeptides heatstable proteome M. truncatula R108 (wild type), Mtabi3-1, and Mtabi3-2 seeds. Acknowledgements References Amara I, Capellades M, Ludevid MD, Pags M, Goday A. 2013. Enhanced water stress tolerance transgenic maize plants over-expressing LEA Rab28 gene. Journal Plant Physiology 170, 864–873. Boucher V, Buitink J, Lin X, Boudet J, Hoekstra FA, Hundertmark M, Renard D, Leprince O. 2010. MtPM25 is an atypical hydrophobic late embryogenesis-abundant protein that dissociates cold and desiccation-aggregated proteins. Plant, Cell and Environment 33, 418–430. Boudet J, Buitink J, Hoekstra FA, Rogniaux H, Larr C, Satour P, Leprince O. 2006. Comparative analysis heat stable proteome radicles Medicago truncatula seeds during germination identifies Late Embryogenesis Abundant proteins associated with desiccation tolerance. Plant Physiology 140, 1418–1436. Bove J, Lucas P, Godin B, Og L, Jullien M, Grappin P. 2005. Gene expression analysis by cDNA-AFLP highlights set new signaling networks and translational control during seed dormancy breaking in Nicotiana plumbaginifolia. Plant Molecular Biology 57, 593–612. Bradford MM. 1976. rapid and sensitive method for quantitation microgram quantities protein utilizing principle protein–dye binding. Analytical Biochemistry 72, 248–254. Buitink J, Leger JJ, Guisle I, et al. 2006. Transcriptome profiling uncovers metabolic and regulatory processes occurring during transition from desiccation-sensitive to desiccation-tolerant stages in Medicago truncatula seeds. The Plant Journal 47, 735–750. Busk PK, Jensen AB, Pages M. 1997. Regulatory elements in vivo in promoter abscisic acid responsive gene rab17 from maize. The Plant Journal 11, 1285–195. Chatelain E, Hundertmark M, Leprince O, Gall S, Satour P, Deligny-Penninck S, Rogniaux H, Buitink J. 2012. Temporal profiling heat stable proteome during late maturation Medicago truncatula seeds identifies restricted subset late embryogenesis abundant proteins associated with longevity. Plant, Cell and Environment 35, 1440–1455. Amara I, Odena A, Oliveira E, Moreno A, Masmoudi K, Pages M, Goday A. 2012. Insights into maize LEA proteins: from proteomics to functional approaches. Plant and Cell Physiology 53, 321–329. Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Müller WEG, Wetter T, Suhai S. 2004. Using miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Research 14, 1147–1159. Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA. 2008. The enigmatic LEA proteins and other hydrophilins. Plant Physiology 148, 6–24. Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR. 2010. Abscisic acid: emergence core signaling network. Annual Review Plant Biology 61, 651–679. Bäumlein H, Junker A. 2012. Multifunctionality LEC1 transcription factor during plant development. Plant Signaling Behavior 7, 1718–1720. Doyle JJ. 1995. DNA data in legume phylogeny: progress report. In: Crisp MD, Doyle JJ, eds. Advances in legume systematics part 7. Phylogeny . London: The Royal Botanic Gardens Kew, 11–30. Benedito VA, Torres-Jerez I, Murray JD, et al. 2008. gene expression atlas model legume Medicago truncatula. The Plant Journal 55, 504–513. Edgar R, Domrachev M, Lash AE. 2002. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research 30, 207–210. Berjak P, Pammenter NW. 2008. From Avicennia to Zizania: seed recalcitrance in perspective. Annals Botany 101, 213–228. Farrant JM, Pammenter NW, Berjak P, Farnsworth EJ, Vertucci CW. 1996. Presence dehydrin-like proteins and levels abscisic acid in recalcitrant (desiccation sensitive) seeds may be related to habitat. Seed Science Research 6, 175–182. Bies-Etheve N, Gaubier-Comella P, Debures A, Lasserre E, Jobet E, Raynal M, Cooke R, Delseny M. 2008. Inventory, evolution and expression profiling diversity LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana. Plant Molecular Biology 67, 107–124. Farnsworth E. 2000. The ecology and physiology viviparous and recalcitrant seeds. Annual Review Ecology and Systematics 31, 107–138. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 We thank our South-African colleagues (Professor P. Berjak, Professor N. Pammenter, and Professor J. Farrant) for help with collecting material. The abi3 mutants Medicago truncatula utilized in this research project, which are jointly owned by Centre National Recherche Scientifique, were obtained from The Samuel Roberts Noble Foundation, Inc. and were created through research funded, in part, by grant from National Science Foundation (NSF# 703285). No conflict interest is declared. This work was supported by grant from Rgion Pays-de-la-Loire, France (QUALISEM 2009–2013), bilateral Partenariat Hubert Curien (PHC) program France–South Africa (grant no. 25903RE to JD, OL, and JB), and post-doctoral EU FP7 Marie Curie Individual Fellowship (grant no. 252822 to MH). Bossi F, Cordoba E, Dupr P, Mendoza MS, Román CS, León P. 2009. The Arabidopsis ABA-INSENSITIVE (ABI) 4 factor acts as central transcription activator expression its own gene, and for induction ABI5 and SBE2.2 genes during sugar signaling. The Plant Journal 59, 359–374. 4572 | Delahaie et al. Figueras M, Pujal J, Saleh A, Save R, Pags M, Goday A. 2004. Maize Rabl7 overexpression in Arabidopsis plants promotes osmotic stress tolerance. Annals Applied Biology 144, 251–257. Finch-Savage WE, Pramanik SK, Bewley JD. 1994. The expression dehydrin proteins in desiccation-sensitive (recalcitrant) seeds temperate trees. Planta 193, 478–485. Garg R, Patel RK, Jhanwar S, Priya P, Bhattacharjee A, Yadav G, Bhatia S, Chattopadhyay D, Tyagi AK, Jain M. 2011. Gene discovery and tissue-specific transcriptome analysis in chickpea with massively parallel pyrosequencing and web resource development. Plant Physiology 156, 1661–1678. Gilles GJ, Hines KM, Manfre AJ, Marcotte WR. 2007. predicted N-terminal helical domain Group 1 LEA protein is required for protection enzyme activity from drying. Plant Physiology and Biochemistry 45, 389–399. Guerriero G, Martin N, Golovko A, Sundstrom JF, Rask L, Ezcurra I. 2009. The RY/Sph element mediates transcriptional repression maturation genes from late maturation to early seedling growth. New Phytologist 184, 552–565. Han B, Berjak P, Pammenter N, Farrant J, Kermode AR. 1997. The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses. Journal Experimental Botany 48, 1717–1726. Lee AK, Slovin JP, Suh JK. 2012. Dehydration intolerant seeds Ardisia species accumulate storage and stress proteins during development. Horticulture, Environment, and Biotechnology 53, 530–538. Leprince O, Buitink J. 2010. Desiccation tolerance: from genomics to field. Plant Science 179, 554–564. Li DZ, Pritchard HW. 2009. The science and economics ex situ plant conservation. Trends in Plant Science 14, 614–621. Manfre AJ, LaHatte GA, Climer CR, Marcotte WR. 2009. Seed dehydration and establishment desiccation tolerance during seed maturation is altered in Arabidopsis thaliana mutant atem6-1. Plant and Cell Physiology 50, 243–253. Meinke DW. 1992. homoeotic mutant Arabidopsis thaliana with leafy cotyledons. Science 258, 1647–1650. Mnke G, Seifert M, Keilwagen J, et al. 2012. Toward identification and regulation Arabidopsis thaliana ABI3 regulon. Nucleic Acids Research 40, 8240–8254. Oliver MJ, Guo LN, Alexander DC, Ryals JA, Wone BWM, Cushman J. 2011. sister group contrast using untargeted global metabolomic analysis delineates biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus. The Plant Cell 23, 1231–1248. Ooms J, Leon-Kloosterziel KM, Bartels D, Koornneef M, Karssen CM. 1993. Acquisition desiccation tolerance and longevity in seeds Arabidopsis thaliana. Plant Physiology 102, 1185–1191. Hattori T, Totsuka M, Hobo T, Kagaya Y, Yamamoto-Toyoda A. 2002. Experimentally determined sequence requirement ACGTcontaining abscisic acid response element. Plant and Cell Physiology 43, 136–140. Panza V, Distfano AJ, Carjuzaa P, Láinez V, Del Vas M, Maldonado S. 2007. Detection dehydrin-like proteins in embryos and endosperm mature Euterpe edulis seeds. Protoplasma 231, 1–5. Hinniger C, Caillet V, Michoux F, Ben Amor M, Tanksley S, Lin CW, McCarthy J. 2006. Isolation and characterization cDNA encoding three dehydrins expressed during Coffea canephora (Robusta) grain development. Annals Botany 97, 755–765. Parcy F, Valon C, Kohara A, Misera S, Giraudat J. 1997. The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects Arabidopsis seed development. The Plant Cell 9, 1265–1277. Hundertmark M, Buitink J, Leprince O, Hincha DK. 2011. The reduction seed-specific dehydrins reduces seed longevity in Arabidopsis thaliana. Seed Science Research 21, 165–173. Phillips JR, Fischer E, Baron M, van den Dries N, Facchinelli F, Kutzer M, Rahmanzadeh R, Remus D, Bartels D. 2008. Lindernia brevidens: novel desiccation-tolerant vascular plant, endemic to ancient tropical rainforests. The Plant Journal 54, 938–948. Hundertmark M, Hincha DK. 2008. LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9, 118. Illing N, Denby KJ, Collett H, Shen A, Farrant JM. 2005. The signature seeds in resurrection plants: molecular and physiological comparison desiccation tolerance in seeds and vegetative tissues. Integrative Comparative Biology 45, 771–787. Ismail FA, Nitsch LMC, Wolters-Arts MMC, Mariani C, Derksen JWM. 2010. Semi-viviparous embryo development and dehydrin expression in mangrove Rhizophora mucronata Lam. Sexual Plant Reproduction 23, 95–103. Kermode AR. 1997. Approaches to elucidate basis desiccation-tolerance in seeds. Seed Science Research 7, 75–96. Kotak S, Vierling E, Baumlein H, Koskull-Doring P. 2007. novel transcriptional cascade regulating expression heat stress Reeves WM, Lynch TJ, Mobin R, Finkelstein RR. 2011. Direct targets transcription factors ABA-insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors. Plant Molecular Biology 75, 347–363. Schliesky S, Gowik U, Weber APM, Bräutigam A. 2012. RNA-seq assembly—are we there yet? Frontiers in Plant Systems Biology 3, 220. Šunderlíková V, Salaj J, Kopecky D, Salaj T, Wilhem E, Matušíková I. 2009. Dehydrin genes and their expression in recalcitrant oak (Quercus robur) embryos. Plant Cell Reports 28, 1011–1021. Tiedemann J, Rutten T, Mnke G, et al. 2008. Dissection complex seed phenotype: novel insights FUSCA3 regulated developmental processes. Developmental Biology 317, 1–12. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014 Gtz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talón M, Dopazo J, Conesa A. 2008. Highthroughput functional annotation and data mining with Blast2GO suite. Nucleic Acids Research 36, 3420–3435. proteins during seed development Arabidopsis. The Plant Cell 19, 182–195. LEA proteome desiccation-sensitive seeds  |  4573 To A, Valon C, Savino G, Guilleminot J, Devic M, Giraudat J, Parcy F. 2006. network local and redundant gene regulation governs Arabidopsis seed maturation. The Plant Cell 18, 1642–1651. Walters C, Berjak P, Pammenter N, Kennedy K, Raven P. 2013. Preservation recalcitrant seeds. Science 339, 915–916. Tolleter D, Jaquinod M, Mangavel C, Passirani C, Saulnier P, Manon S, Teyssier E, Payet N, Avelange-Macherel MH, Macherel D. 2007. Structure and function mitochondrial late embryogenesis abundant protein are revealed by desiccation. The Plant Cell 19, 1580–1589. Wang F, Perry SE. 2013. Identification direct targets FUSCA3, key regulator Arabidopsis seed development. Plant Physiology 161, 1251–1264. Tunnacliffe A, Wise MJ. 2007. The continuing conundrum LEA proteins. Naturwissenschaften 94, 791–812. Zha HG, Liu T, Zhou JJ, Sun H. 2013. MS-desi, desiccationrelated protein in floral nectar evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization. Planta 238, 77–89. Wall PK, Leebens-Mack J, Chanderbali AS, et al. 2009. Comparison next generation sequencing technologies for transcriptome characterization. BMC Genomics 10, 347. Zhulidov PA, Bogdanova EA, Shcheglov AS, et al. 2004. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Research 32, e37. Downloaded from http://jxb.oxfordjournals.org/ by guest on January 15, 2014