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First published online September 8, 2006; 10.1104/pp.106.086694 Plant Physiology 142:839-854 (2006) © 2006 American Society of Plant Biologists Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches1,[W]Department of Plant Sciences, the Weizmann Institute of Science, 76100 Rehovot, Israel (A.F., R.A., H.L., G.G.); Minerva Avron Evenari Center for Photosynthesis Research, Department of Biological Chemistry, The Hebrew University, 91904 Jerusalem, Israel (I.O.); and Abteitlung Willmitzer, Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Golm-Potsdam, Germany (E.U.-W., A.R.F.)
While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
Seed maturation is characterized by a switch from maternal to filial metabolic regulation, a process that is dedicated to reserve synthesis and is coupled with the initiation of a nutrient uptake from the canopy (Weber et al., 2005  ). Maturing seeds of some plant species also gain photosynthetic capacity, which dramatically elevates the internal O2 levels leading to a partial relief of low oxygen conditions and thus ameliorating energy status of the tissue (Geigenberger, 2003; Rolletschek et al., 2005). At the metabolic level, the relatively O2-poor stage of reserve accumulation is initiated by a transient stimulation of fermentative metabolism and continues with a switch from the energy-inefficient invertase to energy-efficient Suc synthase catalyzed route of Suc degradation (Bologa et al., 2003; Rolletschek et al., 2003; Goffman et al., 2005).
Seed germination is associated with degradation and mobilization of the reserves accumulated during maturation (Bewley, 1997
Arabidopsis Seeds Maintain Comparable PSII Fluorescence through the Entire Period of Reserve Accumulation
Maturing seeds of different species possess photoheterotrophic plastids, which maintain photosynthetic activity during the period of reserve accumulation, contributing both oxygen and metabolic reassimilation of CO2 that is released by reserve biosynthesis (Rolletschek et al., 2003
Data suggests comparable photosynthesis efficiency until the desiccation period (Fig. 1B), even though the mRNA levels of the representative light-harvesting complex II and PSI genes are declining with maturity (Fig. 1C), in keeping with previous reports (see, for example, Ruuska et al., 2002 ).
We next expanded our analysis to seed primary metabolism to discern metabolic networks during seed maturation and germination. For this purpose, we utilized an established gas chromatography-mass spectrometry (GC-MS) protocol (Roessner et al., 2001
As shown in Figure 2 , the progression of seeds from 10 ± 1 DAF to 14 ± 1 and 17 ± 1 DAF was associated with major decreases in the levels of most amino acids, sugars, polyols, and organic acids, apparently reflecting their rapid utilization in the storage reserve accumulation process. The level of fatty acids that could be detected in the polar fraction of our GC-MS analysis were generally slightly increased from 10 ± 1 to 14 ± 1 DAF and then dropped from 14 ± 1 to 17 ± 1 DAF probably emphasizing the dynamic conversion of sugars into fatty acids and their subsequent incorporation into oil. This process is similarly reflected in the patterns of change in the level of Suc, which, in contrast to the general reduction of sugars, was significantly increased from 10 to 14 ± 1 DAF. The results imply that at this stage the rate of carbon import in the seeds exceeds the rate of Suc cleavage and subsequent entrance of carbon into glycolysis. As opposed to the general reduction in the levels of sugars, the level of raffinose was enhanced during this period, particularly at 14 ± 1 DAF. Metabolites of the tricarboxylic acid (TCA) cycle showed different trends of change from 10 to17 ± 1 DAF. The levels of four TCA-cycle intermediates, namely citrate, malate, succinate, and particularly 2-oxoglutarate (47-fold reduction) were progressively reduced throughout the period of reserve accumulation (Fig. 2). In contrast, the levels of fumarate and isocitrate increased from 10 ± 1 to 14 ± 1 DAF and decreased from 14 ± 1 to 17 ± 1 DAF, indicating the presence of some variations in mitochondrial metabolism/function occurring at the early (10–14 ± 1 DAF) and late (14–17 ± 1 DAF) stages of reserve accumulation (Fig. 2). It should be noted, however, that the pattern of change in the level of these two metabolites at the early to late stages of reserve accumulation varied markedly between the two seasons. In the second season, isocitrate decreased similarly to the other TCA intermediates, while fumarate increased from 10 ± 1 DAF to 17 ± 1 DAF. The levels of amino acids were also generally decreased from 10 ± 1 DAF to 17 ± 1 DAF (Fig. 2). Most noticeable though was a 184-, 81-, and 38-fold decrease in the respective levels of Gln, Asn, and Lys. These changes apparently signify the incorporation of the free amino acids into storage proteins, coupled with enhanced metabolism of Gln, Asn, and Lys (see "Discussion").
Alterations of Metabolic Profiles during the Transition from the Period of Reserve Accumulation to the Period of Seed Desiccation
The desiccation period (transition from 17 ± 1 DAF to mature seeds) was associated with a major switch in seed metabolism. As opposed to the period of reserve accumulation, which was associated with a general reduction in the levels of most metabolites (Fig. 2), the desiccation period was associated with a general increase in most amino acids, sugars (including raffinose), polyols, organic acids (with the exception of TCA intermediates), as well as in fatty acid-related compounds and slight increase in fatty acids (Fig. 3
; Supplemental Tables S2 and S4). This indicates that, as opposed to the accumulation of oil and storage proteins between 10 ± 1 and 17 ± 1 DAF, the desiccation period is associated with a major increase in levels of free metabolites. In detail, there was a dramatic accumulation of the nitrogen-rich amino acids (Asn, Lys, and Arg), the aromatic amino acids (Trp, Phe, and Tyr), Ser, Ala, and the nonproteogenic amino acid
Principal Component Analysis Suggests That Different Stages of Seed Maturation Are Associated with Distinct Metabolic Profiles
To get a global view on the metabolic changes that occur during seed development, we also analyzed the data set by a principal component analysis (PCA; Saeed et al., 2003
Semiquantitative RT-PCR Analysis of Changes in mRNA Levels of Selected Genes during the Periods of Reserve Accumulation and Desiccation We also wished to study patterns of gene expression programs of representative metabolism-associated genes during seed maturation and germination, using semiquantitative RT-PCR analysis (Fig. 5 ). The different genes were categorized into three groups based on their expression patterns during seed maturation and germination. The first group consisted of genes whose mRNA levels were relatively high during the initiation of reserve accumulation (10 ± 1 DAF), declined during the periods of reserve accumulation and desiccation (14 ± 1 DAF to dry seeds), and then increased upon the progression of germination (Fig. 5A). This group contained genes encoding photosynthesis-associated proteins (PSI), glycolytic enzymes (fructokinase, Fru bisphsophate aldolase, as well as cytosolic and plastid pyruvate kinases), two succinate dehydrogenase isozymes, and nitrogen metabolism enzymes (GABA transaminase and asparaginase). The second group contained genes whose mRNA levels increased during the periods of reserve accumulation and desiccation (10 ± 1 dry seeds) and remained at relative high levels during seed germination (Fig. 5B). This group included isocitrate lyase of peroxisomal lipid degradation, phosphoenolpyruvate (PEP) carboxykinase of gluconeogenesis, dihydrodipicolinate syntase-2 of Lys biosynthesis, and Thr synthase of Thr biosynthesis. The third group (Fig. 5C) contained genes whose mRNA levels increased during seed maturation and declined during seed germination. This group included trehalose phosphate phosphatase of trehalose metabolism, alcohol dehydrogenase, a cruciferin storage protein, enzymes of amino acid biosynthesis (Thr synthase, Trp synthase, and Asn synthase), as well as mitochondrial fumarylacetoacetate hydrolase. Fumarylacetoacetate hydrolase is the last enzyme of Tyr catabolism, producing the Kreb's cycle metabolite fumarate, which accumulates during seed desiccation (Fig. 5C).
Alterations of Metabolic Profiles Occurring during Seed Vernalization and Germination Sensu Stricto We collected and analyzed samples of imbibed Arabidopsis seeds following seed vernalization (imbibition at 4°C/dark for 72 h) and following transfer to germinative conditions (21°C/light for 24 h). As shown in Figure 6 , the vernalization period was associated with changes in the level of different metabolites. While the levels of numerous amino acids were reduced to different extents, with GABA, Gly, and Phe exhibiting major reductions over this time period, intriguingly, the levels of Asp, Thr, and Ser significantly increased. Similarly, while the levels of most sugars variously declined, the levels of Fru 6P and Glc 6P were significantly elevated. The levels of dehydroascorbate, 3-phosphoglycerate, and the TCA-cycle intermediates 2-oxoglutarate and isocitrate dramatically increased during this time period. In contrast, the levels of other acids, namely, gluconate 6P, glycerate, and threonate were significantly reduced. The transition of seeds from vernalization to germinative conditions (Fig. 7 ) was generally associated with an exacerbation of the metabolic changes observed during vernalization (with the exceptions of Arg, Asn, Gly, and Gln and fatty acids, the levels all of which were invariant). This general observation suggests that the transition between vernalization and germination sensu stricto is associated with the activation of initial important metabolic processes needed for seed germination. It is also likely that germination is associated with a follow up of additional metabolic processes, which occur slightly later during germination and therefore could not be observed by our metabolic profiling.
Changes in Gene Expression Programs Occurring during Vernalization and Germination Sensu Stricto In addition to our semiquantitative RT-PCR analysis of representative genes (Fig. 5) we addressed changes in gene expression during vernalization and germination sensu stricto using bioinformatics approach. Using publicly available microarray (AffyWatch, Nottingham Arabidopsis Stock Centre [NASC] Affymetix service; http://Arabidopsis.info/) of imbibed and germinating Arabidopsis seeds, we analyzed two independent experiments, both performed on Columbia-0 seeds by Lutz and associates (Nover, Botanisches Institut, Germany) as follows: (1) an experiment measuring the expression profile of dry seeds and seeds following 1 or 3 h of imbibition; and (2) an experiment comparing changes in mRNA levels occurring 24 h after imbibition, compared to dry seeds in wild-type plants as a part of a hormonal treatment experiment. This analysis revealed changes in the mRNA levels of genes with various functions, but we focused only on genes associated with primary metabolism. Since many specific molecular and enzymatic functions operate by gene families, in such cases we summed the GeneChip values derived from the different gene families. The function of the different genes or gene families were deduced from the AraCyc, Arabidopsis biochemical pathways (http://www.arabidopsis.org/tools/aracyc/), which contains hierarchical information of metabolic pathway including the enzymatic reaction in each pathway and the genes that encode each enzyme. In instances in which we felt that this database may be wrong, manual corrections of gene functional annotations were introduced. Table I presents the data of genes or gene families whose combined mRNA levels were significantly different (either up-regulated or down-regulated) at the 0.05% level between the dry and imbibed seeds in at least one of the experiments (see "Materials and Methods"), while the accession numbers of these genes and their grouping into gene families, whose GeneChip values were summed up, are provided in Supplemental Table S8.
As shown in Table I, imbibing seeds for 1 h caused no significant difference in the mRNA levels of any of the genes present on the GeneChip. Imbibing seeds for 3 h caused significant increase in the mRNA levels of several genes associated with various metabolic functions, including the OPPP pathway (phosphogluconate dehydrogenase), TCA cycle (succinyl-CoA synthetase), fatty acid biosynthesis (3-oxoacyl-[acyl-carrier protein] reductase), nitrogen assimilation (nitrite reductase), polyamine biosynthesis (Arg decarboxylase), ribonucleotide biosynthesis (phosphoribosylformylglycinamidine cyclo-ligase), Lys biosynthesis (dihydrodipicolinate reductase and diaminopimelate decarboxylase), and ethylene biosynthesis (1-aminocyclopropane-1-carboxylate synthase). Upon 3 h imbibition, the mRNA levels of two genes encoding enzymes of amino acid degradation as well as one enzyme of trehalose metabolism were significantly reduced, compared to dry seeds. The transition from 3 h imbibition to 24 h imbibitions was generally associated with a general enhancement in the mRNA levels of genes whose increased expression was detected already after 3 h imbibition. These generally signified major increases in mRNA levels of genes associated with biosynthetic processes and reductions in mRNA levels of genes associated with the degradation of amino acids (Gly, Leu, and Tyr). Notably, although the mRNA levels of a number of TCA-cycle enzymes were up-regulated during imbibition, those encoding the TCA-cycle enzyme oxoglutarate dehydrogenase were slightly, but significantly, down-regulated during 24 h imbibition. This may indicate an integration of the GABA shunt with the TCA cycle.
In this study we complement published observations at the transcript and protein level by performing a combined analysis of the metabolic processes occurring in Arabidopsis seeds during the periods of reserve accumulation, desiccation, vernalization, and germination sensu stricto. Although some reports on the metabolism of seed development have been published previously they have tended to focus on earlier stages of seed development and on specific biochemical pathways largely concentrating on sugar components (Baud et al., 2005 ; Baud and Graham, 2006; Gomez et al., 2006), fatty acids and oil (Cernac and Benning, 2004; Lin et al., 2004; Vigeolas et al., 2004; Gómez et al., 2005), or specific transcriptional programs (Hobbs et al., 2004; Braybrook et al., 2006). Broader transcriptional studies have also been previously conducted with an emphasis on the stages of development from 5 to 13 DAF (Ruuska et al., 2002). Here it was our intention to provide a metabolomic complement to more global studies of seed development and germination in Arabidopsis. However, discussion is focused toward the more novel aspects of our data set, i.e. the transition from seed maturation to desiccation and further to vernalization and germination sensu stricto. While we observed seasonal variation in the results obtained as well as some differences to published data, this was minor and furthermore a similar pattern of change in the metabolite levels was maintained during seed development and germination in all studies reported. This variation is, however, of interest since cardinal features of metabolic shift would be expected to be highly conserved across all studies. We highlight instances where this is indeed the case in the concluding paragraph.
Our results showed that the period of reserve accumulation (10 ± 1 to 17 ± 1 DAF) is associated with major reductions in the levels of many primary metabolites, including amino acids, sugars, polyols, and organic acids (Fig. 2). The patterns of change in the levels of the measured primary metabolites likely signify their efficient incorporation of sugars and organic acids into fatty acid and amino acid biosynthesis, on route to production of oil and storage proteins. This is specifically reflected by the characteristic changes in the levels of metabolites such as Suc and 3-phosphoglycerate (Fig. 2). These observations also indicate that Suc import between 10 ± 1 and 14 ± 1 DAF exceeds the rate of its incorporation into the glycolytic pathway, and glycolytic rate and fatty acid biosynthesis exceeds oil production.
Even though the levels of all measured amino acids were reduced during the period of reserve accumulation, the levels of Asn, Gln, and Lys were reduced much more prominently than others (Fig. 2). This apparently signifies their extensive metabolism into other amino acids. The metabolic changes occurring during the period of reserve accumulation were also associated with distinct changes in gene expression. The general reduction in sugars, polyols, and organic acids was accompanied by a progressive decline in the expression of genes encoding enzymes in glycolysis and other pathways of carbon metabolism (Fig. 5). Yet, in contrast to the reduction in the levels of amino acids, the mRNA levels of genes encoding enzymes of amino acid biosynthesis were increased during this period (Fig. 5). This signifies an increased synthetic activity associated with amino acid biosynthesis on route to their incorporation into oil and storage proteins (White et al., 2000
The major reduction in the levels of the TCA-cycle intermediates between 10 ± 1 and 17 ± 1 DAF implies that this period is characterized by a reduced flux through this pathway. This could potentially be due to a limitation in the availability of sufficient oxygen for mitochondrial respiration and production of ATP and reducing equivalents. The light reaction of photosynthesis, particularly PSII, enhances the levels of oxygen as well as ATP and redox power during seed maturation (Ruuska et al., 2004
The transition from the period of reserve accumulation to seed desiccation exhibited a continuous decrease in the levels Fru 6P and Glc 6P (Fig. 3), implying a reduced incorporation into glycolysis. Yet, the period of seed desiccation (occurring from 17 ± 1 DAF to mature seeds) was associated with a significant increase in the levels of several free amino acids as well as a number of organic acids and sugars. Prominent among these changes was that of raffinose, which has previously been shown to increase during late maturation and that was suggested as important in energy storage and membrane protection during desiccation (Baud et al., 2002
Among the amino acids the levels of the nitrogen-rich Asn, Arg, and Lys, the nonprotein amino GABA, and the aromatic amino acids Trp, Phe, and Tyr were most dramatically increased at this developmental stage (Fig. 8A). These changes were associated with high mRNA levels of genes encoding enzymes of amino acid biosynthesis, implying that amino acid biosynthesis apparently continues during the desiccation period, and that at least some amino acids significantly accumulate in their free forms rather then being incorporated into storage proteins. These lines of evidences taken in the context of current models of seed metabolism suggest that the seed desiccation process preferentially favors (1) the decrease of the C/N ratio, (2) the accumulation of free amino acids rather than storage proteins, and (3) the production of shikimate-derived secondary metabolites associated with defense (Hull et al., 2000 ; Glawischnig et al., 2004; Piotrowski et al., 2004) and indole-3-acetic acid biosynthesis (Campanella et al., 2004; Sharaf and Farrag, 2004) that are necessary triggers for seed germination (Normanly, 1997; Slavov et al., 2004). In addition to the accumulation of free amino acids and various carbon metabolites, the period of seed desiccation was also associated with small increases in the levels of various fatty acids, fatty acid-related compounds such as glycerol, as well as intermediates of fatty acid degradation (Fig. 3). These are likely derived from oil degradation, which was previously shown to occur in this period (Chia et al., 2005). The metabolic changes occurring during seed development, particularly the metabolic switch occurring in the transition between the periods of reserve accumulation and desiccation, were also illustrated using PCA, a complementary statistical tool for the global diagnosis of metabolic changes. Indeed it enables a bird view of the metabolic changes occurring along the period of reserve accumulation and the subsequent transition into desiccation. Beside the metabolic shift illustrated by the increasing distances between the different groups along these developmental periods (first and second PCA components), a further insight was shown by the third component and its effect on the distribution of the data points. This component specifically out grouped 17 ± 1 DAF data set, which signifies the transition from seed maturation to desiccation. Interestingly, the most significant loadings contributing to the discrimination of this developmental stage were mainly characterized by amino acids.
Why do maturing seeds follow a metabolic switch during seed desiccation, changing from accumulation of oil and storage protein to that of free amino acids, sugars, as well as fatty acids and also their degradation products? It has been previously hypothesized that degradation of fatty acids during seed desiccation may provide energy needed for metabolic activity in this period (Chia et al., 2005
Notably, despite the major reduction in the levels of TCA-cycle intermediates during seed maturation and their relatively low levels in mature seeds, the level of fumarate showed a very distinct pattern of change during seed maturation. Indeed it initially increased (10 ± 1 to 14 ± 1 DAF), while dropping during the second stage of reserve accumulation (14 ± 1 to 17 ± 1 DAF), eventually increasing dramatically during seed desiccation (17 ± 1 DAF to mature seeds; Figs. 2 and 3). It should be noted that in a different season, fumarate content increased throughout maturation with the most significant change during desiccation. The mechanism behind this unprecedented degree of fumarate accumulation in seeds has yet to be elucidated. It is tempting to hypothesize that it may signify a mitochondrial transition between respiratory and anaerobic activity, analogous to the bidirectional fermentative mitochondria occurring in diverse organisms (van Hellemond et al., 2003
While oil and protein reserves are long documented to be mobilized following radical protrusion (Bewley, 1997
Despite the general reduction in the levels of amino acids during vernalization, the levels of Asp and Thr were significantly increased during this period (Figs. 7 and 8B) and further boosted strongly during subsequent germination (Fig. 7). This indicates a specific activation of the Thr branch of the Asp-family pathway, which also leads to the synthesis of Met. The importance of the Asp-family pathway metabolism (through Met and S-adenosylmethionine [SAM]) to seedling growth is also supported by previous transcript profiling and proteomics studies (Ravanel et al., 2004
Our bioinformatics analysis also showed that germination-associated gene expression programs start already during seed imbibition (Table I). This generally includes the activation of genes encoding enzymes in biosynthesis pathways associated with sugar metabolism, TCA cycle, nitrogen assimilation, fatty acid production, and photosynthesis as well as the suppression of degradation pathways of some specific amino acids (see details of specific enzymes in "Results" and in Table I). Induced expression of these genes during imbibition apparently serves as a basis for a boost in biosynthetic processes commenced during subsequent germination (see next section). Notably, the mRNA levels of several genes controlling trehalose biosynthesis (Fig. 5; Table I) were down-regulated during seed imbibition, emphasizing the regulatory properties of trehalose during germination (Schluepmann et al., 2004
During postimbibition early germination we observed an increase in the levels of many metabolites, including amino acids, sugars, and organic acids, which apparently reflects the boost of biosynthetic processes. During this period, we also measured a significant increase in the level of dehydroascorbate (Fig. 6). Dehydroascorbate is derived from the antioxidant ascorbate, which is maintained at a constant steady-state level apparently because it is replenished from the hexose phosphate pool. This metabolic program may assist in scavenging of reactive oxygen species resulting from the reoxygenation of the germinating tissues and that might damage proteins and membranes (Job et al., 2005
In conclusion, in our study we show that early germination events are characterized by the efficient reactivation of metabolic pathways via the availability of key precursors as well as a coordination of energy metabolism. As summarized in the descriptive Figure 8, A and B, we show that specific sets of metabolic primers, distributed across the metabolic network, are synthesized during late maturation. These primers successively decrease during seed vernalization concomitantly to increases in specific metabolites of the central pathways but prior to major mobilization of storage reserves. Several conserved features are apparent in both harvests analyzed as well as in the literature, thus confirming a high biological relevance of these changes in the process of seedling development. The results described here also provide a strong complement to studies carried out at the transcript and protein level in the understanding of seed metabolism that is of great interest both from a fundamental and an applied perspective (Alonso-Blanco et al., 2003
Chemicals All chemicals were purchased from Sigma-Aldrich Israel with the exception of N-methyl-N-(trimethylsilyl)trifluoroacetamide (Macherey-Nagel GmbH & Co. KG).
Arabidopsis (Arabidopsis thaliana) seeds (ecotype Wassilewskija) were germinated on soil and grown for two consecutive rounds in the greenhouse during July and August, 2004 (21°C). Flowers were marked, and at given time intervals following anthesis (10 ± 1 to 18 ± 1 DAF), siliques were collected, seeds were dissected, immediately frozen in liquid nitrogen, and stored at –80°C. Ripe fruits were collected at the end of the desiccation period and stored at 4°C. Three to five thousands seeds were harvested for each extraction (30–50 mg).
Ripe seeds were germinated on moistened filter paper. Plates were placed at 4°C in the dark and following a period of 72 h transferred to standard plant growth chambers (21°C, approximately 200 µmol m–2s–1, under a 16/8-h light/dark regime) for additional 24 h. Seeds were washed twice and immediately frozen in liquid nitrogen, following excess water removal.
Chl-a fluorescence emission of PSII was measured by pulse amplitude modulated fluorometry using the PAM 101 apparatus (Heinz Walz GmbH). The intrinsic fluorescence, F0, was elicited by a modulated (1.6 Hz) 650 nm light beam (200 nmol photon m–2 s–1) and the maximal fluorescence, Fm, was elicited by a saturating white-light pulse (1 s, 5,000 µmol photon m–2 s–1). Fm – F0 = variable fluorescence, Fv, and the ratio Fv/Fm indicates the efficiency of PSII in primary photochemical reaction (Genty et al., 1989
Metabolite analysis by GC-MS was carried out by a method modified from that described previously (Roessner et al., 2001
PCA was performed on the data sets obtained from metabolite profiling with the software package TMEV (Saeed et al., 2003
Total RNA was isolated from harvested seeds at 10, 14, and 17 DAF as previously described (Ruuska and Ohlrogge, 2001
We used two publicly available data sources: (1) AraCyc, Arabidopsis Biochemical Pathways (http://www.arabidopsis.org/tools/aracyc/), which contains hierarchical information of metabolic pathways; and (2) AffyWatch, NASC Affymetrix service (http://Arabidopsis.info/), which contains expression data of more than 1,500 arrays. We used experiments from the AtGenExpress project (expression profiling of early germinating seeds and hormone treatment of seeds) that measured the expression profile of dry seeds and seeds following 3 or 24 h of imbibition. We used the expression values for each probe set following MAS5.0 analysis that is the standard procedure to measure gene expression levels using Affymetrix technology. To calculate the expression value of each enzymatic reaction we summed the expression value of all the different genes that encode the same enzyme (e.g. the expression level of the Asp kinase enzyme is the sum of the expression values of the four Asp kinase genes that are present on Affymetrix array). For each enzymatic reaction we performed a t test between values of imbibed and dry seeds. The p values were corrected using the Benjamin and Hochberg correction procedure that is the standard procedure when doing multiple tests.
Raw data were collected from the AffyWatch, NASC Affymetrix service (http://Arabidopsis.info/). Expression values for each probe set were calculated following MAS5.0 analysis, which is the standard procedure to measure gene expression levels using Affymetrix technology. To calculate the expression value of each enzymatic reaction we summed the expression values of all the different genes that encode the same enzyme (e.g. the expression level of the Asp kinase enzyme is the sum of the expression values of the four Asp kinase genes that are present on Affymetrix array). For each enzymatic reaction, a t test was performed between values of imbibed and dry seeds. The p values were corrected using the Benjamin and Hochberg correction procedure, which is the standard procedure used for multiple tests.
The t tests have been performed using the algorithm embedded into Microsoft Excel. The term significant is used in the text only when the change in question has been confirmed to be significant (P < 0.05) with the t test.
The following materials are available in the online version of this article.
We thank Ms. Tal Sadeh for the excellent care during material collection. Received July 13, 2006; accepted August 29, 2006; published September 8, 2006.
1 This work was supported by the German Federal Ministry of Education and Research within the framework of German-Israeli Project Cooperation as well as the Charles W. and Tillie K. Lubin Centre for Plant Biotechnology at the Weizmann Institute of Science. G.G. is an incumbent of the Bronfman Chair of Plant Science at the Weizmann Institute of Science. A.F. is a Postdoc fellow sponsored by the Alexander von Humboldt foundation. 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gad Galili (gad.galili{at}weizmann.ac.il).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.086694 * Corresponding author; e-mail gad.galili{at}weizmann.ac.il; fax 972–8–9344181.
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ABSTRACT
RESULTS
-ala, Beta Ala; L-AsA, ascorbate; DHA, dehydroascorbate; Fru 6P, Fru 6 phosphate; Glu 6P, Glc 6 phosphate; Myo-ino, myo-inositol; 2-OG, 2-oxoglutarate; PhoA, phosphoric acid; ShikA, shikimate; ThrA, threonate; Treh, trehalose.
-aminobutyrate (GABA; 
