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First published online December 16, 2005; 10.1104/pp.105.071167 Plant Physiology 140:263-278 (2006) © 2006 American Society of Plant Biologists Repressing the Expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE Gene in Pea Embryo Causes Pleiotropic Defects of Maturation Similar to an Abscisic Acid-Insensitive Phenotype1,[W]Institut für Pflanzengenetik und Kulturpflanzenforschung, D–06466 Gatersleben, Germany
The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic activity of plastids, and regulated proteolysis.
Maturing seeds differentiate from meristem-like tissues into highly specialized storage organs. In legumes, this occurs sequentially, involving a decrease in mitotic activity, Suc uptake, cell expansion, and accumulation of storage products (Borisjuk et al., 1995  ). Maturation is evident by morphology, changes in gene expression, and metabolite profiles (Weber et al., 2005). In Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare) seeds, fundamental transcriptional reprogramming accompanies maturation, evident by up-regulated genes related to storage metabolism (Ruuska et al., 2002; Sreenivasulu et al., 2004). On the metabolite level, specific sugars correspond to development in fava bean (Vicia faba) embryos. Glc in nondifferentiated premature regions correlates with mitotic activity (Borisjuk et al., 1998), whereas high Suc is present in maturing, actively elongating, and starch-accumulating cells (Borisjuk et al., 2002). Local ATP concentrations (energy state) depend on differentiation grade. Low ATP levels are present before maturation, whereas the highest values are found in differentiated, storage-active regions (Borisjuk et al., 2003). Thus, maturing embryos adapt to specific environmental and nutrient conditions, which leads to adjustment of metabolic fluxes and energy state (Rolletschek et al., 2002; Borisjuk et al., 2003).
Controlling the switch from cell division to maturation during the transition phase is particularly interesting for crop seeds because of the initiation of storage metabolism. Work on legume and Arabidopsis seeds suggests regulation mediated by sugars and hormones and their interaction (Wobus and Weber, 1999
The hormone abscisic acid (ABA) regulates a wide range of developmental events and mediates responses to stress. In seeds, ABA is necessary to proceed through maturation, acquire desiccation tolerance, and prevent precocious germination (Phillips et al., 1997
SUCROSE NONFERMENTING-1 (Snf1)-RELATED PROTEIN KINASES (SnRK1s) are metabolic regulators in yeast, animals, and plants. In yeast, Snf1 is expressed in response to nutrient depletion and mediates usage of alternative substrates. It works in concert with histone acetylation to regulate transcription (Lo et al., 2005
Plants contain several isoforms of the three components common to the yeast and mammalian Snf1/AMPK heterotrimeric complex: the kinase
Reduction of SnRK1 in transgenic plants mainly affects cleavage and use of Suc in potato (Purcell et al., 1998 To understand the role of SnRK1 for the switch from cell proliferation to differentiation during transition stages, SnRK1 was repressed by an antisense approach in transgenic pea (Pisum sativum) seeds. This caused pleiotropic defects of maturation similar to an ABA-insensitive phenotype in Arabidopsis. Array-based gene expression analysis of transgenic embryos indicates a developmentally younger status and an inability to cope with stress and to adjust metabolism to environmental conditions. It is concluded that SnRK1s are regulators of embryo maturation in legumes and interact with ABA-dependent and -independent pathways.
Characterization of SnRK1 in Fava Bean and Pea Seeds
A 200-bp SnRK1 cDNA fragment was cloned from a cDNA library of fava bean cotyledons using reverse transcription-PCR. A library screen revealed 12 clones representing a single isoform, designated VfSnRK1 (
DNA gel-blot hybridization of fava bean at high stringency conditions after BamHI digestion yielded three bands, larger than expected from cDNA restriction, suggesting that VfSnRK1 contains introns. HindIII digestion revealed a single band. Because the cDNA does not have HindIII restrictions sites, additional sites may exist within introns (Fig. 2 ). The restriction pattern indicates that VfSnRK1 is present as a single- or low-copy gene. Tissue-specific RNA gel-blot analysis revealed expression in all tissues with decreasing transcript abundance in roots, gynoecia, embryos, pods, and sink and source leaves (Fig. 3A ). In growing embryos, transcript levels were highest between 19 and 22 d after fertilization (DAF), corresponding to the transition stage. Levels continuously decreased from 23 to 30 DAF (Fig. 3B).
VfSnRK1 kinase activity was measured in protein extracts from developing embryos using a SAMS peptide (HMRSAMSGLHLVKRR) kinase assay. Because the concentration of storage proteins strongly increased at later stages, kinase activity was calculated as nanomoles of phosphate incorporated per minute per milligram of plant material and not on a protein basis. The temporal profile of SAMS peptide kinase activity was similar to that of VfSnRK1 transcripts, but the peak of activity occurred 2 d later (Fig. 3C).
VfSnRK1 was fused in antisense orientation to the seed-specific vicilin and unknown seed protein (USP) promoters and cloned into the binary vector pGPTV-bar. Vic-SnRK1 antisense (pVicilin-SnRK1-as) and USP-SnRK1 antisense (pUSP-SnRK1-as; Fig. 4 ) were used for pea transformation. Nine independent transformants containing Vic-SnRK1-as and three transformants with USP-SnRK1-as were regenerated. To obtain stable lines, the plants were allowed to self-pollinate and PCR-positive plants were further propagated.
Approximately 30% of the antisense plants were partially or completely sterile. Because promoters of storage protein genes are active in pollen (Zakharov et al., 2004 ), strong inhibition of SnRK1 activity in pollen may cause abortion and subsequent sterility. Cytological analysis revealed high percentages of smaller and wrinkled pollen in these plants (data not shown). Similar effects of pollen abortion have been described in transgenic barley plants expressing the SnRK1 antisense construct under the control of the wheat (Triticum aestivum) HMG subunit promoter (Zhang et al., 2001). Nonfertile pea plants showed extensive branching, probably caused by an altered sink-source relationship due to the failure of seed development. Because we aimed to study the role of SnRK1 for seed development, transgenic lines were chosen without obvious reduction in fertility. Seeds from three independent transgenic lines (Vic-34 and Vic-14 transformed with Vic-SnRK1-as, and U-5 transformed with USP-SnRK1-as) of the F4 to F6 generation were further analyzed. DNA gel-blot analysis revealed one band indicating single inserts in each line. The 5-kb hybridizing band corresponds to the endogenous PsSnRK1 (Fig. 5 ). The plants have no obvious growth phenotype. RNA gel-blot analysis revealed that PsSnRK1 mRNA levels were decreased in seeds during the transition stage of lines Vic 14, Vic-34, and U-5 (Fig. 6 ). SnRK1 activity was estimated using a SAMS peptide assay in protein extracts of embryos of lines Vic-34, U-5, and the wild type at 16, 18, 21, and 24 DAF. Activity was significantly reduced by approximately 50% to 70% at 16, 18, and 21 DAF, the period that corresponds to the transition stage. At later stages (24 DAF), activities were not significantly different (Fig. 7 ).
Taken together, the antisense expression of VfSnRK1 causes a decrease of 50% to 70% of both PsSnRK1 mRNA and SAMS peptide activity in embryos during the transition stage.
Detailed analysis of growing seeds was done for line Vic-34. Compared to the wild type (Fig. 8, A and C ), the Vic-34 seeds (Fig. 8, B and D–F) are growth retarded and could be classified into two categories. A smaller fraction of seeds was more strongly growth inhibited, remained smaller, and frequently aborted before having filled the seed coat (Fig. 8, E and F). Abortion occurred at various stages from the transition stage onward. The fraction of aborted seeds was approximately 10%, but changed slightly from one generation to the other, although we grew the plants under constant conditions. Probably small differences of environment might play a role. The majority of seeds were fully developed and able to germinate (Fig. 8D). However, they were somewhat delayed in growth as shown by lower fresh-weight accumulation rates (Fig. 9 ). Transgenic seeds frequently showed alterations in cotyledon shape, surface, and symmetry (Figs. 8, D–F, and 10A). Dry, mature seeds of all three lines have a remarkable greenish phenotype instead of the yellow color of wild-type seeds (Fig. 10B ). Occasionally, seeds germinate precociously, as shown, for example, in the U-5 line (Fig. 10B). After desiccation, the viviparous seeds are not able to germinate.
Suc and starch were analyzed in cotyledons of lines Vic-34, Vic-14, U-5, and the wild type during the early storage phase (21–24 DAF). Suc levels at that stage were clearly higher in all transgenic cotyledons (Fig. 11A ), whereas starch was not different (Fig. 11B). The higher Suc-to-starch levels correspond to the observed growth retardation and indicate that conversion of Suc into storage compounds is affected. Seed storage compounds were then measured in mature, dry seeds. Starch levels were not different (data not shown). However, the globulin content was significantly lower by approximately 10% to 20% in seeds of all transgenic lines (Fig. 11C), whereas albumins were less affected and significantly lower only in Vic-14 seeds (Fig. 11D). Correspondingly lower total nitrogen was measured for all lines (Fig. 11E), whereas total carbon content was slightly, but significantly, increased (Fig. 11G). Thus, the SnRK1 antisense seeds have an increased carbon-to-nitrogen ratio (Fig. 11G). Individual seed weight was decreased by 10% to 20% in all three lines (Fig. 11H).
These results indicate that SnRK1 antisense seeds have general defects in maturation indicated by growth retardation, impaired globulin accumulation, lower final dry weight, and occasional vivipary.
Differentially expressed sequences were arranged in two clusters. Cluster A gene transcripts are highly abundant in wild-type seeds during the prestorage phase (13–15 DAF) with a continuous decrease toward maturation. In Vic-34 embryos, this group showed higher mRNA levels between DAF 17 to 19 (Fig. 12A ) and was designated as delayed down-regulated. Cluster A contains 67 genes with annotated and 36 with unknown function. Cluster B gene transcripts are low abundant in wild-type embryos at prestorage, with a continuous increase toward maturation (Fig. 12B). In Vic-34 embryos, this group showed lower values at 13 to 19 DAF and was designated as delayed up-regulated. Cluster B contains 59 genes with annotated and 22 genes with unknown function.
Delayed Down-Regulated Functions Functional classification was based on homology (BLAST2) and literature searches. Therefore, all statements on gene identity and function have to be considered as putative. Transcript abundance does not necessarily reflect transcriptional activity, protein content, or enzyme activity. However, for simplicity reasons, higher or lower transcript levels were referred to as down- or up-regulated gene expression.
Delayed down-regulated ESTs from cluster A were divided into seven functional groups (Table I; Supplemental Fig. 1). The largest group (21 genes) is related to cell cycle and mitotic activity. Fourteen sequences encode histones H1, H1.41, H2A, H2B, H4, and H3C. mRNAs of core histones are synthesized in a cell cycle-dependent pattern at the beginning of the S-phase to allow nucleosome formation on duplicated DNA (Koning et al., 1991
The second group of 12 ESTs is related to transcriptional and translational activity with 10 ribosomal proteins of different classes, one poly(A)-binding, and another RNA-binding protein.
The third group represents 10 sequences related to primary and secondary metabolism. Four have possible roles in hormone synthesis: two ABA-2-like short-chain dehydrogenases involved in steroid hormone synthesis, which are Suc and GA inducible in watermelon (Citrullis vulgaris) seeds (Kim et al., 2003
The fourth group contains eight ESTs with different regulatory and signaling functions. Four are putatively stress induced. Three are associated with Ca2+-signaling pathways: calreticulin, calmodulin, and acyl-CoA-binding protein. The latter has Ca2+-binding and second messenger activity. Others encode polygalacturonase inhibitor, a cell wall protein belonging to the Leu-rich repeat family with functions in development and defense (Di Matteo et al., 2003
The fifth group of six sequences is related to mitochondrial functions. Three are associated with the mitochondrial respiration chain: succinate dehydrogenase (complex I), cytochrome c oxidase (complex IV), and ATP synthase
The sixth group contains six transport-related ESTs encoding a putative sugar transporter of unknown function and an aquaporin-like protein, which are GA and stress up-regulated (Jang et al., 2004
The seventh group of four sequences is associated with photosynthesis with plastocyanin, chlorophyll a/b-binding protein, transketolase, and Leu zipper fatty acid desaturase (FAD); the latter is light up-regulated in tobacco (Nicotiana tabacum) and required for chlorophyll synthesis (Liu et al., 2004
Delayed up-regulated genes due to SnRK1 repression are divided into eight functional groups (Table II; Supplemental Fig. 2). The largest group is related to storage activity and contains 19 ESTs. Seven encode 11S storage proteins, six vicilins, and one legumin. Another six encode seed storage or late embryogenesis-related proteins, four USPs, three lectins, one pea albumin, a Pro-rich, cold-inducible lipid transfer protein, and a secreted embryo-specific lipoprotein. One EST represents a biotin carboxyl carrier protein subunit involved in storage lipid synthesis. From the 19 genes, at least 15 are known to be ABA inducible and at least seven are related to drought and desiccation tolerance.
The second group consists of 11 sequences related to transcriptional and/or translational activity. Four encode 40S and 60S ribosomal proteins: two methionyl and lysyl tRNA synthetases, one a translationally controlled tumor protein involved in protein elongation, and one a small nuclear ribonucleoprotein involved in rRNA modification, ribosomal function, polyploidy, and chromosome rearrangement (Brown et al., 2003 ). One sequence encodes a pea DEAD-box RNA helicase, a multifunctional protein involved in protein synthesis (Pham and Tuteja, 2000), and histone H3A, which in yeast is the target of Snf1 phosphorylation, which enhances transcription of genes activated in response to cell-signaling events by changes in chromatin structure (Nowak and Corces, 2004).
The third group consists of eight ESTs related to stress tolerance. Three encode alcohol dehyrogenase-1, which in Arabidopsis is ABA inducible and related to drought tolerance (DeBruxelles et al., 1996
The fourth group of six sequences is related to regulatory and signaling functions. Four are directly involved in ABA signaling: Abi-3 transcription factor, which is a key regulator of seed maturation, confers ABA sensitivity and activates vicilin promoters (Zeng et al., 2003
The fifth group encodes five ESTs related to primary and secondary metabolism: ceramide glucosyltransferase possibly involved in glycolipid synthesis, cytosolic NAD-malate dehydrogenase possibly involved in the production of malate from oxalacetate for amino acid production (Scheibe, 2004
A sixth group contains three ESTs involved in cell wall synthesis and modification, cellulose synthase,
The seventh group contains three sequences related to protein degradation, the 20S proteasome
The eighth group contains three ESTs involved in storage activity of plastids: an oxalacetate/malate translocator, with a possible role in import of carbon skeleton into plastids for amino acid synthesis; an ADP/ATP translocator, which is rate limiting for the import of ATP into plastids for biosynthesis; and 5-enolpyruvylshikimate-3-P synthase 2 (EPSP2 synthase), an enzyme of the shikimate pathway involved in defense-related aromatic biosynthesis (Forlani et al., 1994
In plants, abiotic stress and/or environmental conditions regulate gene expression as well as development. Accordingly, internal signals in seed development are nutrient status, drought/osmotic conditions, oxygen, and/or energy. During maturation, the developmental program has to be adjusted to the changing physiological and metabolic status. Snf1-like kinases are ubiquitously present in eukaryotes. Their classic role is to adapt metabolism to changing environmental conditions, such as nutrition, energy, and stress (Hardie, 1999 ). We show here that in legume seeds SnRK1 kinase interacts with ABA signal transduction and is a key regulator controlling developmental programming associated with the switch from prestorage to maturation.
VfSnRK1 is expressed in various tissues, preferentially sink organs (Fig. 3). In growing embryos, highest expression as well as kinase activity occurred during the transition stage. To analyze SnRK1 functions in seed development, we used antisense expression in pea seeds, which reduced both SnRK1 mRNA and SAMS peptide kinase activity by 50% to 70%. Phenotypic analysis reveals seed abortion of 10% to 20% at various stages of early maturation. Obviously, these seeds cannot develop enough sink strength to grow sufficiently and therefore do not reach maturity. The majority of seeds, although growth retarded, reach maturity. Such a phenotype can arise when SnRK1 repression is near the threshold level of the minimal activity required to proceed through maturation. The fact that a 50% to 70% reduction of SnRK1 transcripts and activities caused partial seed abortion indicates that the residual SnRK1 activity is around a critical level, which represents a kind of threshold for initiation of seed maturation. A similar pleiotropic and heterogeneous phenotype has also been observed for seeds of the lh-2 mutant (ent-kaurene oxidase), which, although possibly different from the SnRK1 action, also affects seed sink strength (Swain et al., 1995
Viable seeds clearly show maturation defects. Lower rates of fresh-weight accumulation and higher Suc levels indicate that utilization and/or conversion of Suc into storage products is affected. Seed composition analysis shows that storage defects concern globulin synthesis rather than starch. Lower mature seed weight, greenish appearance of seeds, and occasional precocious germination indicate that the seeds have problems reaching final maturation and full desiccation tolerance. Such a phenotype is reminiscent of Arabidopsis mutants with defects in ABA synthesis or sensitivity (Finkelstein et al., 2002
Comparison of gene expression between wild-type and Vic-34 embryos reveals 183 differentially expressed genes in two clusters (Fig. 11). Cluster A genes are highly abundant during prestorage, decrease toward maturation, and are delayed down-regulated in Vic-34 embryos (Table I; Supplemental Fig. 1). Cluster B genes are stage specifically induced in wild-type embryos at maturation and are delayed up-regulated in Vic-34 embryos (Table II; Supplemental Fig. 2).
Gene expression related to GA and/or BR synthesis is increased. GAs work additively with BRs and are antagonistic to ABA. In young Arabidopsis seeds, the GA-to-ABA ratio can regulate developmental fate (Gazzarrini et al., 2004
A switch from mitotic to cell expansion growth, during which sugar signals are involved, is characteristic for transition-stage embryos. Hexoses are correlated to mitotic activity, whereas Suc feeding disrupts the meristematic state and induces cell expansion (Weber et al., 1996
Vacuolar H+ transport (four genes) is up-regulated, which is an ATP-consuming process essential for vacuole enlargement, cell growth, and expansion (Shiratake et al., 1997
From 57 annotated and delayed up-regulated genes, 17 are directly related to protein storage and 10 to mRNA translation. This is consistent with decreased storage protein synthesis (Fig. 11) and indicates that its gene expression requires SnRK1. The majority of these storage protein genes are also up-regulated by ABA. Remarkably, expression of Abi-3 is strongly repressed in Vic-34. Abi-3 is a major regulator of seed maturation and Arabidopsis abi-3 mutants display an ABA-insensitive phenotype with severe maturation defects like failure to synthesize storage proteins, Suc accumulation, and loss of desiccation tolerance (Finkelstein et al., 2002
A larger number of repressed genes are related to stress tolerance, involving drought/desiccation (vicilins, USP, alcohol dehydrogenase), genotoxic stress (mismatched repair protein, NIMA-related kinase), and biotic stress (snakin-1). Other stress tolerance genes are UDP-Glc:flavonol 3-O-glucosyltransferase, a key enzyme of anthocyanin synthesis (Winkel-Shirley, 2002
From the histones especially, isoform H3A is repressed in Vic-34 embryos. Histone H3 is the target of Snf1 phosphorylation in yeast, which results in gene activation probably through chromatin modification (Lo et al., 2005 Cell wall synthesis and modification, a cellular function related to maturation, is repressed in Vic-34 embryos. Massive cell enlargement during legume seed maturation requires de novo synthesis and/or reconstruction of cell wall constituents. Another repressed function is related to the biosynthetic activity of plastids. Together with the effect on mitochondrial function, this shows that during embryo maturation a phase-dependent switch occurs from mitochondrial function to biosynthetic activities of plastids. In Vic-34 embryos, this process is affected, suggesting that SnRK1 could play a role.
Other repressed genes encode enzymes directly or indirectly involved in storage activity like amino acid biosynthesis (malate dehydrogenase, acetohydroxy acid isomeroreductase), glycolipid synthesis (ceramide glucosyltransferase), and aromatic biosynthesis (riboflavin synthase, EPSP2). Two genes have regulatory and signaling functions related to development: a transcription factor of the class III homeodomain Leu zipper family, which in Arabidopsis is an early marker of procambial cells and may promote vascular differentiation (Baima et al., 2001
Three repressed genes are associated with regulated proteolysis (20S and 26S proteasomal subunits and subtilisin protease). Phase transitions in plant development involve altered patterns of protein expression. Regulated proteolysis by the proteasome complex is a key regulatory component for this process. In Arabidopsis, ubiquitin-specific proteolysis is essential for early embryo development (Doelling et al., 2001
Plant Material Pea (Pisum sativum cv Erbi) plants were grown in 2-L pots in growth chambers under a light-to-dark regime of 16 h light (20°C) and 8 h dark (18°C). Plants were fertilized once a week with nitrate and ammonium in order to keep nonlimiting nitrogen conditions. For the isolation of embryos, pods were tagged according to DAFs, collected in the middle of the light phase, and processed further. For biochemical analysis, seeds were harvested, and embryos were immediately isolated and snap frozen in liquid nitrogen.
The fava bean (Vicia faba) SnRK1 full-length cDNA was cloned under the control of the vicilin promoter or the USP promoter (Fiedler et al., 1993
Embryo material (100 mg) was ground in liquid nitrogen with 0.009 g Polyclar AT and suspended in 500 µL extraction buffer (0.25 M mannitol, 50 mM HEPES, 50 µM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM dithiothreitol [DTT], 0.1 mM phenylmethylsulfonyl fluoride [PMSF], pH 8.2). After centrifugation, ammonium sulfate was slowly added to the supernatant to 40% saturation while stirring for 20 min at 4°C. Precipitated protein was suspended in 50 µL fractionation buffer (50 mM Tris-HCl, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM DTT, 0.1 mM PMSF, 0.02% [v/v] Brij-35, 10% [v/v] glycerol, pH 8.2) and concentration was determined according to the Bradford method. SAMS peptide kinase activity was performed as described by Davies et al. (1989)
To obtain full-length clones of VfSnRK, the PCR-amplified DNA bands of approximately 200 bp were used to screen a
Two cDNA libraries were constructed from growing pea embryos and seed coats from four different stages covering transition to midmaturation stages using the pBluescript II XR cDNA library construction kit (Stratagene). Equal amounts of RNA from different stages were mixed and used for library construction. Average insert size of the libraries was estimated between 1,000 and 1,600 bp; 5,538 cDNA clones from embryo (PSC, Pisum sativum cotyledon) and 7,680 cDNAs from seed coat (PSS, Pisum sativum seed coat) libraries were sequenced from 3' ends with an average length of 620 nucleotides. A total of 8,414 ESTs (4,958 from embryo and 3,756 ESTs from seed coat) were used for clustering using StackPack software (www.egenetics.com), resulting in 1,082 clusters (1,465 contigs) of 6,061 sequences and 2,353 singletons. Chimeric clones were removed manually. Contigs with a high number encode vicilins (136 ESTs), histone H3 (132 ESTs), vicilin precursors (119 ESTs), embryonic-abundant protein (101 ESTs), and chlorophyll a/b-binding proteins (97). The EST set was annotated using BLASTX2 (www.ncbi.nlm.nih.gov). The set of 8,414 ESTs was annotated with reference to gene function using BLASTX2 comparisons with the NRPEP protein database (ftp://ftp.ncbi.nih.gov/blast/db/FASTA/nr.gz). EST sequence information is available at http://pgrc.ipk-gatersleben.de/est/index.php. A unigene set of 4,548 clones was PCR amplified and spotted on nylon filters. Macroarrays were used to investigate mRNA abundances of wild-type and Vic-34 embryos at 11, 13, 15, 17, 19, and 21 DAF and 13, 15, 17, and 19 DAF, respectively, from the end of the prestorage to the midmaturation phase covering the transition stage.
Array hybridization signals were detected by phosphor imager (Fuji BAS 2000; Fuji Photo Film) and analyzed using ArrayVision (Imaging Research), J-EXPRESS (Dysvik and Jonassen, 2001
To allow comparison of signal intensities between the two experiments, the median of the logarithmically scaled (log2) intensity distribution for each experiment was set to zero (median centering of arrays; Eisen et al., 1998 Because median centering does not yield information about signal intensity, we used the data after the first round of median centering of arrays to calculate nonlogarithmic, normalized signal intensities. To exclude fragments with signals close to background, the normalized nonlogarithmic signal intensities have to exceed three arbitrary units for at least one experiment.
The complete dataset was reduced to cDNA fragments with differential expression. To identify significant regulated genes, the signals showing 3-fold and higher differences in temporal expression profiles were selected. To group together genes with similar properties, cluster analysis was done using the program J-EXPRESS (Dysvik and Jonassen, 2001
After ethanol extraction, the starch-containing insoluble material was solubilized in 1 N KOH for 1 h at 95°C and neutralized with 5 N HCl. Starch was hydrolyzed with amyloglucosidase and determined enzymatically. To determine albumin and globulin fractions of extractable proteins, powdered samples were extracted in acetate buffer (50 mM acetate, 1 mM KCl, 10% [v/v] DMSO, 0.5% [v/v] butanol, pH 4.5) and, subsequently, in phosphate buffer (100 mM KH2PO4, 100 mM Na2HPO4, 500 mM KCl, pH 7). Protein was measured with bovine serum albumin as standard. Relative content of total carbon and nitrogen in dried, powdered samples of cotyledons was measured using an elemental analyzer (Vario EL; Elementaranalysensysteme). Statistical analysis was done using Student's t test with Sigma Stat software (Jandel Scientific). Sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers AJ971809 (VfSnRK1) and AJ971810 (PsSnRK1).
We are grateful to Katrin Blaschek, Elsa Fessel, and Angela Schwarz for excellent technical assistance. We thank Isolde Saalbach for help with pea transformation and Ulrich Wobus for discussions and continuous support. We also thank Uwe Scholz, Thomas Rutkowski, and Matthias Lange for support with bioinformatics; Lothar Altschmied for help in EST annotation, clustering analysis, and array preparation; and Ursula Tiemann and Karin Lipfert for help with preparation of the artwork. Received September 8, 2005; returned for revision November 1, 2005; accepted November 1, 2005.
1 This work was supported by the Deutsche Forschungsgemeinschaft and by Institut für Pflanzengenetik und Kulturpflanzenforschung (cDNA sequencing).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with journal policy described in the Instructions for Authors (http://www.plantphysiol.org) is: Hans Weber (weber{at}ipk-gatersleben.de).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.071167. * Corresponding author; e-mail weber{at}ipk-gatersleben.de; fax 49–39482–5500.
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ABSTRACT
RESULTS
- or Snf1 subunit, the regulatory subunit 
or Snf4, and the 
- or GAL83/SIP subunit with adapter function (Buitink et al., 2003
ZAPII (Stratagene) cDNA library from fava bean cotyledons (Heim et al., 1993
