Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism

Authors


*(fax +039482 500; e-mail weber@ipk-gatersleben.de).

Summary

The application of nitrogen to legumes regulates seed metabolism and composition. We recently showed that the seed-specific overexpression of amino acid permease VfAAP1 increases amino acid supply, and the levels of N and protein in the seeds. Two consecutive field trials using Pisum sativum AAP1 lines confirmed increases in the levels of N and globulin in seed; however, compensatory changes of sucrose/starch and individual seed weight were also observed. We present a comprehensive analysis of AAP1 seeds using combinatorial transcript and metabolite profiling to monitor the effects of nitrogen supply on seed metabolism. AAP1 seeds have increased amino acids and stimulated gene expression associated with storage protein synthesis, maturation, deposition and vesicle trafficking. Transcript/metabolite changes reveal the channelling of surplus N into the transient storage pools asparagine and arginine, indicating that asparagine synthase is transcriptionally activated by high N levels and/or C limitation. Increased C-acceptor demand for amino acid synthesis, resulting from elevated levels of N in seeds, initiates sucrose mobilization and sucrose-dependent pathways via sucrose synthase, glycolysis and the TCA cycle. The AAP1 seeds display a limitation in C, which leads to the catabolism of arginine, glutamic acid and methionine to putrescine, β-alanine and succinate. Mitochondria are involved in the coordination of C/N metabolism, with branched-chain amino acid catabolism and a γ-amino-butyric acid shunt. AAP1 seeds contain higher levels of ABA, which is possibly involved in storage-associated gene expression and the N-dependent stimulation of sucrose mobilization, indicating that a signalling network of C, N and ABA is operating during seed maturation. These results demonstrate that legume seeds have a high capacity to regulate N:C ratios, and highlight the importance of mitochondria in the control of N–C balance and amino acid homeostasis.

Introduction

Legume seeds represent an important plant-derived protein source, and improving the protein content and seed composition is desirable. However, as many failed metabolic engineering strategies have demonstrated, any successful improvement requires a profound understanding of seed metabolism and its regulation. Legume embryos occur in a two-phase development. During the initial pre-storage phase, organogenesis and morphogenesis occur together with high mitotic activity. The maturation phase is characterized by cell expansion and storage activity (Weber et al., 1997a, 1998, 2005). Maturation and storage activities are initiated during the phase transition. At this stage, active sucrose and amino acid uptake also begin (Miranda et al., 2001; Weber et al., 1997b), leading to an increased nutrient status of the seed.

Seed metabolism is connected to maturation, which is initiated by a signalling network involving sugars, ABA and sucrose-non-fermenting-kinase 1 (SnRK1), (Brocard-Gifford et al., 2003; Gibson, 2004; Radchuk et al., 2006). Sucrose has a dual function in nutrition and in the signalling of storage-associated processes (Borisjuk et al., 2002), a process that interacts with the ABA signalling pathway (Finkelstein et al., 2002). Sucrose possibly increases the ABA sensitivity or ABA levels (Smeekens, 2000), or modulates the response to sugar signals. Several ABA-synthesis and ABA-insensitive (ABI) mutants are also sugar-sensing mutants, indicating that sugar signalling requires the ABA transduction chain (Rook et al., 2001). ABA action in seeds may involve SnRK1 kinases (Radchuk et al., 2006; Rolland et al., 2006). Decreased levels in ABA affects the proper metabolism of sucrose in the SnRK1-repressed pea (Radchuk et al., 2006; R. Radchuk, R.J.N. Emery, H. Weber, unpublished data). SnRK1 may exert its effect on ABA via the ABI3 homologue, which is downregulated in these seeds (Radchuk et al., 2006).

In seed development, nitrogen represents another important metabolic regulator. Amides are taken up via the H+ co-transport of membrane-integrated amino acid permeases (AAPs; Tegeder et al., 2000; Miranda et al., 2001). Cotyledons exhibit the full capacity for amino acid biosynthesis (Macnicol, 1977). Thereby, imported amides are deaminated, and the released ammonia N is incorporated into other amino acids. To fuel adequate amino acid synthesis, additional C has to be diverted to the key carbon acceptors, oxalacetate and α-ketoglutarate, precursors for amino acids of the aspartate and glutamate family members (Golombek et al., 2001; Miflin and Lea, 1977).

The AAPs are encoded by multigene families displaying organ-specific expression and low transport selectivity (Okumoto et al., 2002). AtAAP1 is expressed seed-specifically and may control storage protein synthesis (Hirner et al., 1998). Two AAP isoforms in pea, PsAAP1 and PsAAP2, are expressed in seed coats, epidermal transfer cells and storage parenchyma cells, as well as in vegetative organs (Tegeder et al., 2000). VfAAP1, which is orthologous to PsAAP2, is expressed in embryonic storage parenchyma cells at early maturation, shortly before maturation starts (Miranda et al., 2001).

Seed storage biosynthesis is generally dependent on the N state in the seeds of pea, soybean, maize and barley (Balconi et al., 1991; Hernández-Sebastiàet al., 2005;Müller and Knudsen, 1993; Salon et al., 2001). Pea and Vicia embryos strongly accumulate free amino acids at stages when storage protein formation is still low (Radchuk et al., 2007). Similarly, the 11S and 7S globulin null mutant of soybean accumulates high levels of amino acids (Takahashi et al., 2003), suggesting an important role for transporters in the accumulation of amino N, regardless of storage protein synthesis supporting the notion that seed N status is an important metabolic signal.

Despite the wealth of information on amino acid permeases, a rate-limiting role must be verified experimentally using transgenesis. It is important to study the situation in crop plants, which, in contrast to Arabidopsis, are selected for high yield, and display high metabolic activity and fluxes in seeds. It is also advantageous to test such plants in the field. In addition to identifying the possible strategies for crop improvement, studying seed metabolic phenotypes can improve our understanding of pathway regulation and interaction.

We have previously shown that the overexpression of an amino acid permease, VfAAP1, increases amino acid supply, total seed N and protein content (Rolletschek et al., 2005). Here, we present an in-depth analysis of pea lines overexpressing VfAAP1 using a combination of transcript and metabolite profiling, which offers a powerful strategy to monitor the complex phenotype. The aims of this study were twofold: first, to evaluate whether the increase of seed protein is stable under field-grown conditions and, second, to analyse the effect of increased amino acid supply on seed metabolism. We could confirm, in two consecutive field trials, that increases in seed N and globulin content occur. However, these are associated with compensatory changes of sucrose/starch and individual seed weight, and have profound consequences on metabolism. The imbalance in the N:C ratio initiates compensating mechanisms, including pronounced amino acid catabolism and γ-amino-butyric acid (GABA) shunt activity. The results clearly reveal that legume seeds have a high capacity to regulate and adjust N:C ratios, and highlight the importance of mitochondria in controlling the N–C balance and amino acid homeostasis.

Results

Generation of pea lines overexpressing VfAAP1 in the seed

AAP1 was fused to the LeB4 promoter (Bäumlein et al., 1992), inserted into PZP 200 and was introduced into pea. Co-transformation enabled the selection of transgenic lines without resistance genes (bar) (Rolletschek et al., 2005). Four independent, PCR-positive lines (lines 10, 14, 18 and 20) were checked by Southern gel blot analysis for copy number (data not shown). Northern gel blot analysis performed on mid-maturation embryos revealed that all four lines exhibited strong transgene expression (Figure 1). In mature seeds of the T2 generation, total N levels were elevated by 20% (Figure 1). Line 14 is free of the bar marker gene and contains a single insert, and homozygous plants of this line were chosen for all subsequent analyses (Figure 1).

Figure 1.

 Characterization of VfAAP1-overexpressing pea lines. Independent lines 10, 14, 18 and 20 were analysed for transgene integration, copy number, total seed N content and VfAAP1 mRNA levels. Values of total N are means (n = 5–10) ± standard deviations; statistically significant differences, P ≤ 0.05.

AAP1 seeds have a higher N content under field conditions

AAP1 seeds grown under growth-chamber conditions revealed significant increases in nitrogen and protein content (Figure 1). As such growth conditions are artificial, pea plants were tested in field trials, as well as in a soil bed under glass from March to June, without additional light or temperature regulation, in order to create field-like conditions. In two field experiments, the seed yield per plant was unchanged (data not shown), although field-grown plants produced twofold more seed mass compared with the growth-chamber plants. Mature field-grown AAP1 seeds have higher levels of total nitrogen and seed globulin, whereas the albumin content was invariant or slightly lower, with significant differences only in the 2006 field trial (Table 1). However, AAP1 seeds had a lower carbon state, as revealed by the decreased total carbon content (significant in field trial 2006), starch (significant in field trials 2005 and 2006) and sucrose levels (significant in field trials 2006 and in the greenhouse 2007). In addition, there was a clear reduction of individual seed size (significant in field trial 2006 and in the greenhouse 2007).

Table 1.   Seed compositional analysis of mature AAP1 seeds grown in field trials, and in the greenhouse under field-like conditions
 Field trail 2005Field trail 2006Greenhouse 2007
AAP14WildtypeAAP14WildtypeAAP14Wildtype
  1. Values are means (n = 5–10) ± standard deviations.

  2. a ≤ 0.05, b ≤ 0.01, c ≤ 0.001.

Total nitrogen (%)4.30 ± 0.31a3.96 ± 0.19 4.33 ± 0.28c4.02 ± 0.274.49 ± 0.11c3.53 ± 0.17
Total carbon (%)42.27 ± 0.6542.33 ± 0.68 41.57 ± 0.30b41.98 ± 0.6943.09 ± 0.0943.19 ± 0.41
Albumins (mg g−1)18.57 ± 1.9420.26 ± 2.8321.11 ± 1.43c23.00 ± 2.2720.74 ± 0.9821.10 ± 0.62
Globulins (mg g−1])93.01 ± 7.17c79.33 ± 7.69 115.15 ± 10.56c100.77 ± 0.5587.08 ± 3.55c68.52 ± 6.91
Starch (mg g−1)367.82 ± 17.21a384.79 ± 17.29 383.03 ± 35.38a404.14 ± 30.64380.56 ± 16.76385.56 ± 15.88
Sucrose (mg g−1)65.54 ± 14.0466.66 ± 7.63 57.37 ± 5.80b63.20 ± 7.1171.59 ± 4.4776.46 ± 7.67
Seed weight (mg)320 ± 10.46325.5 ± 6.36 270.51 ± 33.01a286.76 ± 55.96286.53 ± 8.30c311.57 ± 10.74

AAP1 overexpression alters the N:C ratios

Embryos from field trial 2006 were harvested at four stages [18, 22, 26 and 30 days after pollination (DAP)]. The fresh weight was lower (Figure 2A) and the total N content was higher at mid maturation (22 and 26 DAP; Figure 2B), whereas the total C content was unchanged (Figure 2C). Starch levels were lower (22 and 30 DAP; Figure 2D) and sucrose levels remained unchanged (Figure 2E). Free amino acids were higher at 18 DAP, and were partially raised at 22 DAP, but not at 30 DAP (Figure 2). ABA levels at mid maturation were higher in the transgenic seeds (Figure 2G).

Figure 2.

 Compositional analysis of maturing AAP1 embryos (field-grown), (line 14).
(A) Embryo fresh weight.
(B) The percentage of total N in embryos.
(C) The percentage of total C in embryos.
(D) The starch levels in embryos.
(E) The sucrose levels in embryos.
(F) The amino acid levels (sum from Figure 3) in embryos.
(G) Levels of abscisic acid in embryos.
(H) Sucrose synthase activity in embryos. The data points are the means of five replicates ± the standard error, with significant differences according to a Student’s t-test: aP < 0.05; bP < 0.01; cP < 0.001.

Individual amino acids, GABA and citrulline were analysed by HPLC from greenhouse-grown AAP1 embryos (15, 18, 22, 26, 30 and 35 DAP). In the wild-type embryos, levels were highest at earlier maturation (15 DAP), and were lower at later maturation, with Ala, Gln, Arg, Thr, Glu and Asn being the most abundant. AAP1 embryos contain higher levels of most of the amino acids: especially at early maturation (18 DAP). Ile, Met, His, Gln, Glu, Leu, Ala, Val, Gly, Ser and Phe were significantly higher only at that stage. Asn and Thr were also increased at later stages, and Pro was higher between 22 and 30 DAP (Figure 3). Lys and Tyr were invariant. Significantly lowered levels in AAP1 embryos could only be detected for Asp, and only at 26 DAP, whereas at 22 DAP the levels were higher. Citrulline, an intermediate in the Arg pathway, was decreased at 22 and 26 DAP. In contrast, levels of GABA were higher at mid maturation (18–22 DAP).

Figure 3.

 Levels of free amino acids in AAP1 embryos and wild-type embryos during maturation (for greenhouse grown plants). Data points are means from four replicates ± the standard error, with significantly higher levels, according to a Student’s t-test: aP < 0.05; bP < 0.01; cP < 0.001. Significantly lower levels, according to the Student’s t-test: xP < 0.05; yP < 0.01; zP < 0.001.

Transcript profiling of AAP1-overexpressing embryos

We analysed differential gene expression in AAP1 and wild-type embryos, field-grown in 2006, using microarrays based on seed-expressed genes (Radchuk et al., 2006). The field trial from 2006 was chosen because weather conditions were better during this season, which also resulted in less plant disease. Four stages were analysed (18, 22, 26 and 30 DAP), and genes upregulated by at least a factor of 1.7 were regarded as differentially expressed. Transcript abundances do not necessarily reflect transcriptional activity, protein content or enzyme activity; therefore, all the following statements on gene identity and function have to be considered as ‘putative’. For reasons of simplicity, higher or lower transcript levels were subsequently referred to as up- and down regulated. A total of 30, 148, 243 and 117 genes were upregulated (0.5, 2.4, 4 and 1.9% from 5246) at 18, 22, 26 and 30 DAP, respectively. From these, 16, 61, 96 and 38 genes (corresponding to 53, 41, 41 and 32% of the upregulated genes in the respective stage) show no homology to annotated sequences. A total of 44, 97, 72 and 35 genes were downregulated (0.7, 1.6, 1.2 and 0.5% of the total) at 18, 22, 26 and 30 DAP, respectively. From these, 16, 55, 34 and 11 genes (corresponding to 36, 57, 57 and 31% of the downregulated genes in the respective stage) show no homology to annotated sequences. The number of differentially expressed genes increases from early to mid maturation. Except at 18 DAP, 2–3-fold more genes are upregulated than downregulated. Genes with known annotation have been assembled into six functional groups: metabolism, stress and pathogen response, transcription and translation, maturation, growth and development, transport and protein processing and signal transduction (Figure 4, Tables S1–S4).

Figure 4.

 Numbers of genes up- or downregulated (numbers at left, top and bottom, respectively) in AAP1 embryos (field-grown) at 18, 22, 26 and 30 days after pollination (DAP). The figure summarizes the genes that are allocated to the functional gene group, which are presented in detail in Tables S1–S4.

Genes encoding ribosomal proteins, histones, and initiation and elongation factors are predominantly upregulated, together with genes involved in protein transport and processing (Tables S1–S4), indicating stimulatory effects on gene transcription and protein biosynthesis. Seven upregulated sequences represent chaperones and chaperonines involved in protein processing and translocation across membranes. Another seven genes are related to vacuolar transport, inorganic pyrophosphatase, H+ transporting ATPase and α-tonoplast intrinsic protein (α-TIP), whereas others are involved in protein vesicle transport. A hexose transporter (22 DAP), a Zn/Fe-transporter (26 DAP) and a plastidial outer-envelope acid-selective-channel OEP16 (26 and 30 DAP; Pohlmeyer et al., 1997) are upregulated, whereas two genes encoding amino acid permeases are downregulated (30 DAP).

AAP1 overexpression affects genes involved in amino acid and sugar metabolism

In AAP1 seeds, 14 genes are upregulated that are potentially involved in amino acid turnover (Table 2). Seven are involved in amino acid synthesis: aspartate aminotransferase (18 DAP), Asn synthetase, ASN1 (22, 26 and 30 DAP), plastidial threonine synthase (22 DAP), mitochondrial Ser/Gly hydroxymethyltransferase (26 and 30 DAP), imidazoleglycerol-phosphate dehydratase (26 DAP), Met synthase (30 DAP) and Gln synthetase, GS1 (26 and 30 DAP). Three genes are downregulated: threonine aldolase, involved in Thr/Gly interconversion (18 and 22 DAP), Gln synthetase GS1 (22 DAP) and plastidial acetohydroxy acid synthase small subunit (AHAS; 26 DAP). AHAS is inhibited by all branched chain amino acids, with a particularly strong effect for Leu and Val (Binder et al., 2007). Four upregulated genes are involved in amino acid catabolism: Arg decarboxylase (22 DAP), a pyridoxal-dependent decarboxylase with homology to Arg decarboxylase (26 DAP), and mitochondrial aminomethyltransferase (22 DAP), involved in Gly degradation and isovaleryl-CoA-dehydrogenase (IVD, 26 DAP). The latter is upregulated by more than sixfold, and catalyses the conversion of isovaleryl-CoA and/or isobutyryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the Leu/Val catabolic pathway. Its gene expression is inducible by sugar starvation (Däschner et al., 2001).

Table 2.   Up- and downregulated genes in AAP1 seeds involved in amino acid and sugar metabolism
Seq. IDAnnotationDAPaP-valueFactorFunction
  1. aDAP, days after pollination.

Amino acid synthesis, upregulated
03092Aspartate aminotransferase180.041.7Asp metabolism
04409Asparagine synthase, ASN122, 26, 300.04, 0.00, 0.001.9, 3.5, 2.1Asn synthesis
05021Threonine synthase, plast.220.00,2.3Thr synthesis
04128Ser/Gly hydroxymethyltransferase, mitochondrial26, 300.00, 0.0032.9, 2.9Ser/Gly interconversion
01308Imidazoleglycerol-phosphate dehydratase260.042.1His synthesis
04228Methionine synthase300.0142.1Methionine synthesis
00518Glutamine synthetase GS1260, 0.0051.9, 1.8Glu synthesis
Amino acid synthesis, downregulated
02292Threonine aldolase18, 220.01, 0.041.9, 2.3Thr–Gly interconversion
02864Glutamine synthetase GS1, cytosolic220.002.6Gln synthesis
01320Acetohydroxy-acid synthase small subunit, plast.260.002.1Val biosynthesis
Amino acid catabolism, upregulated
04625Arginine decarboxylase; P.s.220.012.1Arg catabolism
04364Aminomethyltransferase, mitochondrial220.022Gly catabolism
03136Isovaleryl-CoA Dehydrogenase260.006.2Leu, Val catabolism
02433Pyridoxal-dependent decarboxylase260.001.8Amino acid decarboxylation
Sugar metabolism, upregulated
01278, 04390, 03580Sucrose synthase 118, 220.00, 0.01, 0.002.7, 2.5, 2.4Sucrose cleavage
04443Sucrose synthase 3180.001.8Sucrose cleavage
03580Sucrose synthase220.003.2Sucrose cleavage
04847Phosphoglycerate kinase, cytosolic220.00,1.9Glycolysis
04285, 02459GAPDH, cytosolic220.00, 0.003, 2.1Glycolysis
04156, 04213Enolase220.00, 0.001.8, 1.7Glycolysis
04424, 01749Triosephosphate isomerase220.00, 0.001.7, 1.7Glycolysis
05180Neutral invertase260.002Sucrose cleavage
02303Sucrose-phosphate synthase260.001.9Sucrose synthesis
01342Phosphoglycerate mutase300.0216.4Glycolysis
Sugar metabolism, downregulated
05180Neutral invertase220.023Sucrose cleavage
04847Phosphoglycerate kinase, cytosolic260.001.9Glycolysis
01309Phosphoglucomutase, cytosolic260.002.8Glycolysis

In total, 15 sequences of the sucrose cleavage and glycolytic pathway are upregulated (18 and 22 DAP); five encode sucrose synthase, the major sucrose cleavage enzyme in legume seeds (Weber et al., 1996). This is in accordance with increased sucrose synthase activity (Figure 2H). A neutral invertase is downregulated at 22 DAP, but is upregulated at 26 DAP. Seven cDNAs, upregulated at early maturation (22 DAP), encode enzymes from the upper glycolysis: cytosolic phosphoglycerate kinase (one isoform), cytosolic GAPDH, enolase and triosephosphate isomerise (each with two isoforms). A sucrose-P-synthase gene was upregulated at 26 DAP. At a later stage (26 DAP), the cytosolic isoforms of phosphoglycerate kinase and phosphoglucomutase were downregulated (Table 2). Increased mRNA levels of enzymes involved in UDP-Gal (UDPG-4-epimerase) and UDP-Glc metabolism (UDP-Glc 6-dehydrogenase and UDP-glucuronate decarboxylase; Figure 5), can play a role in cell-wall biosynthesis, along with upregulated caffeoyl-CoA O-methyltransferase, UDP-glucosyltransferase, polygalacturonase, α-l-arabinofuranosidase and cinnamyl-alcohol dehydrogenase (Table S3). UDP-Gal together with myo-inositol reacts to produce galactinol, which is increased in the AAP1 seeds (Figure 5). The possible increased demand for C in AAP1 seeds is in accordance with the upregulation of hexose transporter (22 DAP), plastidial starch phosphorylase (22 DAP) and glucan-water-dikinase (26 DAP; Edner et al., 2007), which plays a role in starch degradation.

Figure 5.

 mRNA levels as increased in AAP1-seeds of enzymes involved in sucrose mobilization, and in uridine 5′-diphosphate (UDP)-sugar metabolism (in green); also see Tables S1–S4. The numbers in parentheses indicate the number of days after pollination.

AAP1 overexpression stimulates mitochondrial gene expression

The 16 genes associated with mitochondrial metabolism are upregulated (Table 3). Two are involved in Gly and Leu/Val catabolism (aminomethyltransferase and IVD), and one is involved in Ser/Gly interconversion (Ser/Gly hydroxymethyltransferase; 26 and 30 DAP). Four are involved in the tricarbonic acid cycle (TCA): pyruvate dehydrogenase E1 and E3, succinyl-CoA ligase and succinate dehydrogenase (26 DAP). Three others are part of the electron transfer chain: one electron transfer flavoprotein:ubiquinone oxidoreductase, complex 1 (ETFQO) and two ubiquinol-cytochrome-c reductase complex 3 (UQCytC). They are associated with the inner mitochondrial membrane. ETFQO accepts proteins from IVD via electron transfer flavoprotein (ETF), and passes them via ubiquinone (UQ) to UQCytC. IVD and ETFQO are induced under sugar starvation conditions (Ishizaki et al., 2005, 2006), and the pathway is involved in the catabolism of branched-chain amino acids (Sweetlove et al., 2007). Two genes encode γ-aminobutyrate transaminase (GABA-T) and succinic semialdehyde dehydrogenase gabD (SSDH) involved in the GABA shunt (Fait et al., 2008). This pathway bypasses the TCA cycle and channels carbon from Glu into succinate, thereby coordinating N metabolism with C metabolism. Two genes are involved in mitochondrial protein transport, TIM17/22 and TIM50 inner membrane translocases, and two encode mitochondrial processing, peptidase-β and dnaK-type molecular chaperone PHSP1.

Table 3.   Upregulated genes in AAP1 seeds involved in mitochondrial metabolism and function
Seq. IDAnnotationDAPaP-valueFactorFunction
  1. aDAP, days after pollination.

Related to mitochondrial metabolism (upregulated)
04364Aminomethyltransferase220.022Gly catabolism
03136Isovaleryl-CoA dehydrogenase260.006.2Leu, Val catabolism
04128Ser/Gly hydroxymethyltransferase 26, 300.00, 0.002.9, 2.9Ser/Gly interconversion
04151Flavoprot:ubiquinone oxidoreduct. (complex 1)260.001.7Respiration
05077, 03247Ubiquinol-cytochrome-c reductase (complex 3)260.00, 0.001.7, 1.8Respiration
02739Pyruvate dehydrogenase, E1260.001.8Glycolysis/TCA cycle
03519Pyruvate dehydrogenase, E3260.001.8Glycolysis/TCA cycle
00209Succinyl-CoA ligase260.001.7TCA cycle
03915Succinate dehydrogenase260.002TCA cycle
00802Gamma-aminobutyrate transaminase260.001.8GABA-shunt
01944Succinic semialdehyde dehydrogenase gabD260.001.8GABA-shunt
01338Mitochondrial processing peptidase beta260.002.1Protein processing
00166dnaK-type molecular chaperone PHSP1260.001.9Chaperone
01176TIM17/22 Mitoch. inner membrane translocase260.001.7Protein transport
01371TIM50 Mitoch. inner membrane translocase300.004.7Protein transport

AAP overexpression stimulates maturation-associated gene expression

In maturing seeds ABA is the major hormone that is linked to nutrient responses (Wobus and Weber, 1999). AAP1 overexpression increased amino acids (Figure 3) and ABA levels (Figure 2G), in accordance with upregulated ABA-2, encoding a short-chain dehydrogenase/reductase, and 9-cis-epoxycarotenoid dioxygenase1 (NCED9) (Table 5). Both encode key enzymes for ABA synthesis, of which overexpression in Arabidopsis and tobacco led to elevated ABA levels (Lin et al., 2007; Qin and Zeevaart, 2002). Increased ABA levels and transcripts of ABA key biosynthesis enzymes correspond to the upregulation of 19 genes associated with seed storage and maturation (Table 4). Eight encode storage proteins, six encode vicilins and two encode legumins. One encodes a late-embryogenesis (LEA) protein, and four others encode ferritins: all are potentially ABA inducible (Lobréaux et al., 1993). Others encode dehydration-responsive, ripening related and dormancy-associated proteins and Cys proteinase inhibitor.

Table 5.   Upregulated genes in AAP1 seeds involved in hormonal metabolism
Seq. IDAnnotationDAPaP-valueFactorFunction
  1. aDAP, days after pollination.

Related to hormonal metabolism (upregulated)
01719Auxin and ethylene responsive GH3-like protein22, 300.04, 0.022.1, 1.7Hormone signaling
02168Auxin-resistance protein AXR1300.002.2Signal transduction
04459GAST-like protein, GA-regulated, Snakin-1220.001.7Broad function
04341, 03779ABA-2260.00, 0.002.7, 2.3ABA synthesis
041239-cis-epoxycarotenoid dioxygenase1260.002ABA synthesis
Table 4.   Upregulated genes in AAP1 seeds involved in seed maturation
Seq. IDAnnotationDAPaP-valueFactorFunction
  1. aDAP, days after pollination.

Maturation related, upregulated
01245, 03272Ferritin180.02, 0.002.5, 4Fe storage
01245, 03272Ferritin260.00, 0.003.3, 4Fe storage
00263Dehydration-responsive protein, ankyrin-domain220.002Maturation
05042Ripening related protein220.011.9Maturation
04249, 05000, 04987Dormancy-associated protein22, 260.00, 0.00, 0.001.9, 2, 1.7Maturation
05134LEA protein, ABA-induced260.003.3Maturation
04876Cys proteinase inhibitor260.002Maturation
04615Vicilin220.001.8Storage protein
04635, 00633Vicilin, 47K300.00, 0.014.9, 2.3Storage Protein
04038, 02231Vicilin A300.01, 0.003.2, 2.2Storage Protein
04616Vicilin300.002.8Storage Protein
02001, 01284Legumin B300.04, 0.002.4, 2.2Storage Protein

AAP overexpression stimulates the gene expression of specific transcription factors

AAP1 seeds represent models with altered metabolic pathways and specific changes in the nutrient content, and as such can be helpful to identify putative transcriptional regulators potentially involved in metabolic regulation and/or nitrogen signalling. Table 6 shows seven transcription factors that are found to be specifically upregulated on the transcriptional level in AAP1 seeds. Two transcription factors (seq. ID 04321 and 04774; 22 and 26 DAP, respectively) were initially described in Vicia faba seeds, one represents AP2/EREBP transcription factor ERF-2 (22 and 26 DAP), two bZIP transcription factors (26 and 30 DAP), one HD Zip homeotic protein (26 DAP) and a BTF3 transcription factor (26 DAP).

Table 6.   Upregulated genes in AAP1 seeds encoding transcription factors
Seq. IDAnnotationDAPaP-valueFactorFunction
  1. aDAP, days after pollination.

Transcription factors (upregulated)
04321Transcription factor, V. faba220.002Transcriptional regulation
04336AP2/EREBP transcription factor ERF-222, 260.02, 0.001.7, 1.8Transcriptional regulation
04407bZIP transcription factor26, 300.00, 0.031.9, 1.8Transcriptional regulation
04538bZIP transcription factor ATB2300.042.3Transcriptional regulation
03119HD Zip homeotic protein260.012.8Transcriptional regulation
04756BTF3 transcription factor260.001.7Transcriptional regulation
04774Transcription factor, V. faba260.001.7Transcriptional regulation

Metabolic profiling of AAP1 embryos

To further evaluate the metabolic basis of AAP1 overexpression, metabolic profiling was applied by GC-MS on growth-chamber-grown AAP1 embryos at five stages (18, 22, 26, 30 and 35 DAP). As already shown by the HPLC analysis (Figure 3), most of the changes occurred in early development. At 22 DAP the levels of ornithine, Asn, Thr, Arg, Gln, Homo-Ser, Ile, Ser and Met were increased; however, at later stages levels were unchanged (Figure 6). Pro and Trp were slightly increased at 30 DAP, whereas Lys, Glu, Ala, Phe and Tyr remained the same. The level of putrescine, but not of spermidine, was increased in early development. Putrescine is synthesized by the decarboxylation of Arg or ornithine (Groppa and Benavides, 2008): the levels of both were 8–10-fold higher in AAP1 embryos (Figure 6). Expression of Arg decarboxylase was stimulated at 22 DAP (Table 2). β-Alanine is increased at 18 and 22 DAP, conceivably because of the degradation of polyamines (Cona et al., 2006). The findings suggest a new catabolic pathway of Arg in seeds via putrescine and β-Ala. Stimulation of mitochondrial gene expression is in keeping with changes in the pool sizes of metabolites of the TCA cycle, revealing significantly higher levels of citrate, iso-citrate and aconitate at 22 DAP. In contrast, succinate, fumarate, malate and 2-oxo-glutarate (2-OG) were unchanged, the latter even being slightly decreased at 30 DAP. Such an asymmetrical distribution of TCA cycle intermediates may indicate an increased usage of 2-OG for amination reactions.

Figure 6.

 Relative changes of metabolites in AAP1 embryos compared with wild type, as determined by GS-MS. Key: dark-grey, increased at the significance level P < 0.05; light-grey bars, increased at the significance level P < 0.1; black bars, decreased at the significance level P < 0.05.

Discussion

Seed uptake of reduced amino nitrogen via membrane-localized H+ co-transporters exhibits considerable metabolic control. Pea seeds overexpressing the amino acid permease VfAAP1, represent models with an altered metabolic state, and are valuable tools to study nutrient (nitrogen) sensing, and to unravel the specificity of cotyledonary N metabolism and pathway interactions. This study had two aims: evaluating whether the increase of seed protein achieved by AAP1 overexpression is stable under field-growth conditions, and analysing the effect of increased amino acid supply on seed metabolism using a combination of transcriptome and metabolite profiling. Because pea is a crop plant, and is important because of its high seed protein content, the outcome of this study holds high potential for agronomic application.

Amino acid supply stimulates cotyledonary protein synthesis and increases the N:C ratio

In two consecutive field trials, and under greenhouse conditions, the increase in total seed N content could be confirmed in mature AAP1 seeds. Apparently, amino acid supply specifically stimulates globulin synthesis. AAP1 seeds have higher seed N content from mid to late seed maturation (Figure 2B). However, the levels of most free amino acids are increased only at early, but not at late, maturation, indicating that the amino acids are used up for storage protein synthesis during maturation. This coincides with results from transcriptional profiling demonstrating an induction of genes involved in transcription and translation, and in different aspects of protein synthesis, transport, processing, maturation, deposition and vesicle trafficking (Tables S1–S4), and of eight genes encoding storage proteins (Table 4). This clearly demonstrates and confirms a general stimulatory effect of N supply on protein synthesis.

Pea seeds accumulate starch and proteins, and as such, a tight control of N:C partitioning and metabolism is required. Remarkably, mature AAP1 seeds have a lower carbon status, as seen at the levels of starch (Figure 2D), total seed carbon and sucrose (Table 1; field trial 2006). This can explain the lower seed (Table 1) and embryo fresh weight (Figure 2A). An alternative explanation is the well-known negative correlation that exists between the carbon costs that have to be spent per gram and seed yield (Sinclair, 1998). Given that a protein storing seed must invest more carbon for the synthesis of a gram seed than a starch-storing seed, it follows that seed protein production is energetically more expensive than that of starch (Vertregt and de Vries, 1987). However, seed size alone is an imprecise criterion. Other research with transgenics has shown that there is often a great deal of compensation among seed mass, seed number and seed growth rate, as well as compensation by altered seed fill duration and overall plant growth. This also strongly depends on the growth conditions. For example, Vicia narbonensis and pea seeds expressing an amino acid transporter have larger seeds when grown in the phytochamber (Götz et al., 2007; Rolletschek et al., 2005).

AAP1 seeds display altered amino acid metabolism

Legume seeds import mainly amides, and have the full capacity for amino acid synthesis. The fact that most of this biosynthesis occurs in the plastids is consistent with the upregulation of a plastidial amino acid selective outer-envelope channel, OEP16, at 26 and 30 DAP (Tables S3 and S4). The outer envelope of plastids contains abundant solute channels, the substrate specificity and function of which have not been fully defined (Pohlmeyer et al., 1997). OEP16 belongs to a distantly related family of protein and amino acid transporters, called PRAT (Rassow et al., 1999). It seems reasonable to speculate that OEP16 is induced by high levels of amino acids, and plays a role in the import of precursors, thereby triggering the de novo synthesis of amino acids within the plastid. Two other genes encoding amino acid permeases are downregulated at 30 DAP (Table S4), indicating a negative feedback mechanism on the endogenous uptake system of the seeds. Miranda et al. (2001) showed a repressing effect of exogenous amino acids on amino acid permease gene expression of V. faba seeds. However, in the AAP1 seeds, amino acid levels are predominantly increased at earlier stages, but not at 30 DAP (Figure 3).

The major changes from the combined transcriptome and metabolic profiling of AAP1 seeds in comparison with wild type are summarized in Figure 7. Asn biosynthesis is clearly increased (18, 26 and 30 DAP; Figure 3), together with transcript levels of Gln-dependent Asn synthase (22, 26 and 30 DAP; Table 2). Asn is an important metabolite, used for both transport and transient storage. It frequently accumulates under carbohydrate limitation and/or starvation (Baldet et al., 2002; Devaux et al., 2003), and upon stresses such as senescence. Asn synthase is transcriptionally activated under conditions of high N:C ratio and/or N solute addition and sugar starvation (Brouquisse et al., 1992; Chevalier et al., 1996), thereby redirecting N under N-surplus conditions into transient storage pools. Bearing these facts in mind, we conclude that in AAP1 seeds Asn synthase responds to the increased N status and/or C-limiting conditions. However, signals and mechanisms for this activation remain as yet unclear. It would, however, appear unlikely that low levels of 2-OG and/or oxalacetate is a signal, given that Asn synthetase is also transcriptionally increased in V. narbonensis seeds overexpressing phosphoenolpyruvate (PEP)-carboxylase, along with higher levels of organic acids, whereas levels of sucrose and starch were lowered, and levels of storage proteins and the N:C ratio were raised (Radchuk et al., 2007; Rolletschek et al., 2004). Furthermore, pea seeds with repressed ADP-Glc pyrophosphorylase have lower starch content, but have increased levels of soluble sugars, and do not show induction of Asn synthase, but rather display an upregulation of asparaginase transcripts (K. Weigelt, I. Saalbach and H. Weber, unpublished data). Thus, the most probable regulator of Asn synthetase in seeds is the lowered sugar content.

Figure 7.

 Summary of the changed transcript and metabolite levels in AAP1 embryos. Data are derived from Figures S1–S4 (transcripts) and Figures 3 and 6 (metabolites). Colour coding: red, downregulated; green, upregulated in AAP1 embryos with respect to the wild type.

Asn biosynthesis via Asn synthase competes with several of the Asp-derived amino acids for the common substrate Asp. In the AAP1 seeds, only Thr is considerably increased, Leu, Val, Ile and Met are increased to an intermediate level, and no change was observed in the levels of Lys (Figure 3). Apparently, Lys does not accumulate because of possible feedback inhibition of dihydrodipicolinate synthase by Lys, reported in other systems (Azevedo et al., 1997, 2006). These results show that in AAP1 seeds, amino acid supply and/or the increased N:C ratio does not strongly stimulate the biosynthesis of Asp-derived amino acids. In particular, the important compound Lys is not altered. Instead, such conditions stimulate Asn synthase, and thereby the flux into the transient storage product Asn. Another transient N storage pool is that of Arg, which normally represents up to 40% of the total N in seed storage proteins, and is the most abundant free amino acid in pea seeds (Slocum, 2005). In the AAP1 seeds, Arg is increased at both 20 and 30 DAP (Figure 3). In summary, it can be concluded that some of the surplus N resulting from AAP1 overexpression is channelled into the transient storage pools Asn and Arg, rather than into Asp-derived amino acids (Figure 7). The levels of Glu and Asp are either unchanged (Glu) or slightly increased at 18 DAP, and are decreased at 26 DAP (Asp), which indicates rapid metabolism of both amino acids.

AAP1 seeds display features of amino acid catabolism

Two genes upregulated in AAP1 seeds are involved in mitochondrial Gly and Leu/Val degradation (aminomethyltransferase and IVD). In addition, extensive metabolism of Met may occur, which is only increased slightly at 18 DAP, together with transcripts of Met synthase (22 DAP), followed by SAM synthase (30 DAP) and SAM-decarboxylase (26 DAP). This pattern points to the conversion of Met to SAM and polyamines. The strong accumulation of β-Ala in the AAP1 seeds (18, 20 and 30 DAP; Figure 6), which in maize shoots is formed upon degradation of polyamines (Cona et al., 2006; Terano and Suzuki, 1978), supports this hypothesis. It has been demonstrated in yeast and Medicago truncatula that β-Ala is converted to co-enzyme A (Broeckling et al., 2005; White et al., 2001); interestingly, panthotenate kinase, which is transcriptionally upregulated at 26 DAP, is involved in this pathway.

There is also evidence for Arg catabolism by Arg-decarboxylase (upregulated at 22 DAP). Putrescine, a downstream product, is strongly increased (18 and 20 DAP; Figure 6), and can be further metabolized via spermine/spermidine to β-Ala (increased at 18, 20 and 30 DAP). Alternatively, putrescine can be converted by diamine oxidase to GABA (increased at 18 and 22 DAP). Polyamines may exert their functions through the cytosolic formation of GABA (Xing et al., 2007). Further metabolism of GABA occurs in the mitochondria, with the GABA shunt providing a considerable augmentary route of succinate production to that supplied via the operation of the TCA cycle (Studart-Guimaraes et al., 2007). Three genes within this alternative route of electron provision to the mitochondrial electron transport chain are upregulated: those encoding GABA-T, SSDH and succinate-dehydrogenase. However, the increased GABA levels in AAP1 seeds could also be derived from the direct decarboxylation of Glu. A reaction-scheme model is presented in Figure 8 that takes into consideration changed transcript and metabolite levels, and which shows the degradative pathways of Arg, Glu and Met that are initiated in the AAP1 seeds.

Figure 8.

 Reaction scheme, taking into consideration the changed transcript and metabolite levels, showing the possible degradation pathways of Arg, Glu and Met into putrescine, β-Ala and succinate in AAP1 embryos. Colour coding: red, downregulated; green, upregulated in AAP1 embryos with respect to wild type.

Metabolic changes in AAP1 seeds involve mitochondrial activity

A large portion of the metabolic changes in AAP1 seeds involve a response in mitochondria. First, the provision of amino acids appears to result in a shift in the capacity to interconvert Ser and Gly from Ser/Gly hydroxymethyltransferase (Li et al., 2003). Second, the mitochondria are involved in the degradation of Gly, Leu and Val via the concerted action of aminomethyltransferase (Li et al., 2003) and IVD; (Däschner et al., 2001). IVD works together with the mitochondrial electron transfer chain; genes encoding ETFQO and UQCytC are upregulated at 26 DAP. Together, this may lead to the coordinated dehydrogenation of branched-chain amino acids Leu/Val, and its oxidation via the ETF (Sweetlove et al., 2007). A detailed demonstration of the operation of this pathway has been confined to dark-induced senescence in leaves (Ishizaki et al., 2005, 2006). However, the data presented here suggest a role in heterotrophic tissues under conditions of fluctuating nutrition. Third, mitochondria are involved in the catabolism of Glu via the GABA shunt (Bouché and Fromm, 2004; Fait et al., 2008). Within this bypass, Glu is decarboxylated to GABA (increased at 18 and 26 DAP; Figure 3), and is subsequently, via the action of GABA-T and SSDH (both of which are upregulated at 26 DAP), further metabolized to succinate, which is then converted via succinate dehydrogenase (upregulated at 26 DAP) to fumarate. Thus, in seeds, GABA is involved in general nitrogen metabolism, and possibly in the storage/transport and/or reallocation of nitrogen (Fait et al., 2008). Finally, the AAP1 embryos have an enhanced capacity for degradation of carbohydrates, via pyruvate dehydrogenase E1 and E3, towards the production of citrate and isocitrate (both of which are increased at 22 DAP; Figure 6). The reaction scheme presented in Figure 9 takes into consideration the transcript and metabolite levels associated with Glu/Gln and Asp/Asn metabolism that appear to be initiated upon AAP1 overexpression. This cross-compartmental pathway may operate under the surplus N conditions apparent within the AAP1 seeds. Metabolic routes that are likely to be upregulated in the AAP1 seeds include: (i) production of Asn from Asp/Gln as a transient N storage pool; (ii) decarboxylation of Glu and metabolization via the GABA shunt; (iii) transamination of Asp and conversion of its C backbone to Pyr via the plastidial malate-dehydrogenase and NADP-malic enzyme (both of which are upregulated at 26 DAP), with subsequent entry in the TCA cycle catalysed by Pyr-DH; (vi) dehydrogenation of Leu/Val via IVD, with electrons entering the mitochondrial ETF via ETFQO and UQcytC. The results indicate that seeds have a high capacity to tightly regulate N:C ratios, and that any deviation, such as that observed within the AAP1 seeds, gives rise to a compensatory response. Seed mitochondria play a vital role in readjusting N:C ratios and amino acid metabolism.

Figure 9.

 Reaction scheme taking into consideration the changed transcript and metabolite levels in AAP1 embryos, and explaining the metabolism of Glu/Gln and Asp/Asn that is initiated upon AAP1 overexpression. See text for details.

AAP1 seeds are carbohydrate limited, and reveal stimulated sugar metabolism

As many as 15 upregulated genes are associated with sucrose metabolism and the glycolysis/TCA cycle. Five encode sucrose synthase, which in legume seeds is very important for carbohydrate mobilization (Barratt et al., 2001; Heim et al., 1993; Weber et al., 1996). AAP1 seeds also have a higher sucrose synthase activity, which coincides with activated gene expression associated with cell wall synthesis (Figure 5; Table S3). Sucrose synthase yields UDP-Glc, which is the central precursor for the synthesis of cell wall polysaccharides (Brett, 2000). In wheat stems, the enzyme is important for gene expression involved in diverting UDP-Glc to cell wall synthesis (Xue et al., 2008). C mobilization is also in accordance with the upregulation of two genes involved in starch degradation and one hexose transporter. Seeds normally require C-acceptors for amination reactions, and AAP1 seeds with increased levels of amino acids need even more, which potentially causes carbon limitation and subsequent sucrose mobilization. It is apparent that increased levels of amino acid, and/or an increase in the N:C ratio, signals carbon mobilization via a sucrose synthase-dependent pathway, and further metabolism through glycolysis and the TCA cycle. This is in accordance with the reduced sucrose and starch levels observed in mature AAP1 pea seeds. Three upregulated genes involved in amino acid metabolism and mitochondrial electron transport can be activated by sugar starvation: Asn-synthase (Brouquisse et al., 1992;Chevalier et al., 1996; Devaux et al., 2003), ETFQO (Ishizaki et al., 2005) and IVD (Däschner et al., 2001).

In conclusion, the higher N:C ratio of AAP1 seeds increased the demand of C-acceptors, which may lead to C limitation and the initiation of sucrose mobilization, as well as to a readjustment of C:N ratios, via mitochondrial amino acid degradation and GABA shunt, and the channelling of N into the transient storage pools of Asn and Arg.

AAP1 seeds displays a signalling network of C, N and ABA signals

Transcriptional profiling performed on sucrose-starved Arabidopsis suspension cultures identified protein phosphatases and protein kinases, as well as a range of transcription factors, as possible components of the starvation response (Contento et al., 2004). The activation of genes of branch-chain amino acid degradation in Arabidopsis also involves Ser/Thr protein kinases, and is prevented by kinase inhibitors (Fujiki et al., 2002). In the AAP1 seeds, several genes encoding Ser/Thr kinases are upregulated at 22 and 26 DAP (Tables S2 and S3); however, direct evidence is missing for their involvement in sugar starvation signalling. The same is true for seven upregulated genes encoding transcription factors (Table 6). In contrast with other sugar-starvation models (Contento et al., 2004; Yu, 1999), in the AAP1 seeds protein biosynthesis and storage are activated. It is likely that such stimulating effects can derive from N signalling, as has been shown before for Medicago embryos (Sreedhar and Bewley, 1998). Accordingly, transcriptional profiling of Arabidopsis exposed to N also shows the induction of gene expression involved in organic acid generation and protein synthesis (Scheible et al., 2004). In maize kernels, N supply increases sucrose synthase, aldolase, phosphoglucomutase, Ala- and Asp-aminotransferases, and acetolactate synthase activities and zein-N contents, indicating N signalling upon C mobilization (Singletary et al., 1990). There may also be a role for ABA, of which levels are also increased (Figure 2G). ABA in seeds is the major maturation hormone, and its action is linked to that of nutrients (Weber et al., 2005; Wobus and Weber, 1999). In the AAP1 seeds, increased ABA synthesis is accompanied with upregulated gene expression of ABA-2 and NCED9 (Table 5), which are the key enzymes for ABA synthesis (Lin et al., 2007; Qin and Zeevaart, 2002). This coincides with the induction of storage and maturation associated gene expression (Table 3). ABA could be involved in the stimulation of sucrose metabolization and breakdown, a pathway that probably involves SnRK1 kinase. Pea embryos that are deficient of SnRK1 kinase have decreased ABA levels, lower sink strength and maturation defects. Accordingly, sucrose levels are increased and the conversion of sucrose into storage products is impaired (Radchuk et al., 2006).

Although open questions remain concerning the hierarchical nature of the control of seed metabolism, the results reveal that maturing seeds exhibit a complex C/N-responsive network, which is responsive to aspects of ABA metabolism. In addition, it is demonstrated that it is the balance of C and N, rather than the levels of C or N per se, that is important for normal seed maturation, and that mitochondrial metabolism plays a crucial role in C/N homeostasis in seeds.

Experimental procedures

Plant material and transformation

Pea plants (Pisum sativum L., cv. Eiffel) from the first four generations were grown in 2-l pots in growth chambers under a light/dark regime of 16-h light (20°C)/8-h dark (18°C). For metabolite measurements and enzyme assays of embryos, pods were tagged according to the number of DAP, and were collected in the middle of the light phase, with the isolated embryos snap-frozen in liquid nitrogen. Pea transformation was described in Rolletschek et al. (2005).

Field trials

Field trials were performed with the homozygous line 14 (generations T5/T6) in spring and early summer 2005 and 2006. A 100-m² field was grown with 100 transgenic and 100 wild-type control plants. Field trails were performed according to German law, with permission from the state of Saxony-Anhalt (Az.6786-01-0158). At maturity, whole plants were harvested by cutting the stem and bagging the plants. Mature seeds were collected and processed in the laboratory.

RNA isolation and hybridization techniques

Nucleic acids were isolated and northern hybridization was performed as described in Heim et al. (1993). The VfAAP1 cDNA fragment was used as a probe after labelling with [32P]dCTP as described in Miranda et al. (2001).

Determination of starch, globulins/albumins, and total carbon and nitrogen levels, and enzyme assay

Extraction, and the determination of starch and globulins/albumins, was described in Rolletschek et al. (2002). Relative contents of total carbon and nitrogen in dried, powdered samples were measured using an elemental analyzer (Vario EL; Elementar Analysensysteme GmbH, http://www.elementar.de). Sucrose synthase activity was performed as described in Heim et al. (1993). Statistical analysis was performed using a Student’s t-test using sigma stat software (SPSS, http://www.systat.de).

Ps6kOLI1 microarrays, microarray hybridizations and data evaluation

An array definition file for the Ps6kOLI1 microarrays has been deposited in the ArrayExpress database (http://www.ebi.ac.uk/microarray/) under accession number A-MEXP-142. This microarray is based on 5246 70-mer oligonucleotide probes (P. sativum Genome Oligo Set Version 1.0; Operon Biotechnologies, https://www.operon.com) representing expressed sequence tag (EST) clusters primarily derived from pea seed coat and cotyledon cDNAs. Each probe is represented by three replicate spots per microarray.

Total RNA (20 μg) was used to synthesize the Cy3- and Cy5-labelled cDNA targets described by Küster et al. (2004). Ps6kOLI1 microarray hybridizations were performed using three independent biological replicates. Hybridization of targets, image acquisition and analysis of image data were described previously (Hohnjec et al., 2005), using a HS4800 hybridization station and an LS Reloaded microarray scanner (Tecan, http://www.tecan.com) according to the manufacturer’s instructions. Image processing data were evaluated using the emma 2.0 software (Dondrup et al., 2003). A Lowess normalization was performed for each microarray hybridization using a floor value of 20, and a Student’s t-test was subsequently applied to identify differentially expressed genes (P < 0.05). The complete transcriptome profile datasets can be viewed at http://www.ebi.ac.uk/arrayexpress under ArrayExpress accession: E-MEXP-1557; experiment name, VfAAP1ox_field 2006_pea seed.

Amino acid determination by HPLC

Ethanol extracts (100 μl) were dried, resuspended in 250 μl H2O and then centrifuged for 5 min, at 10 000 g and 4°C, and the supernatants were stored at −20°C. Derivation was performed using the AccQ•Tag method (Waters, http://www.waters.com) according to the manufacturer’s instructions. The separation was performed by a AccQ•Tag 3.9 × 150-mm column installed in a Waters HPLC system (600 controller, 717plus autosampler) at 37°C. The solutions used were: (A) 140 mm sodium acetate (pH 5.8), 7 mm triethanolamine; (B) acetonitrile; (C) H2O. The following gradient was used: t = 0 min (100% A, 0% B, 0%C); t = 0.5 min (97% A, 3% B, 0% C); t = 17 min (95.5% A, 4.5% B, 0% C); t = 19 min (95.5% A, 4.5% B, 0% C); t = 26 min (95% A, 5% B, 0% C); t = 30 min (91% A, 9% B, 0% C); t = 34 min (90% A, 10% B, 0% C); t = 36 min (87% A, 13% B, 0% C); t = 73 min (86% A, 14% B, 0% C); t = 75 min (78% A, 22% B, 0% C); t = 77 min (0% A, 60% B, 40% C); t = 80 min (100% A, 0% B, 0% C); t = 90 min (100% A, 0% B, 0% C). The flow was 1 ml min−1. Amino acid derivatives were detected by a Jasco FP-1520 fluorescence detector (Jasco, http://www.jascoinc.com) with 300-nm excitation and 400-nm emission. Calibration was performed with 1, 10, 100 and 1000 pmol of each detected amino acid. The quantities were calculated with the waters empower software.

Determination of abscisic acid

The determination of ABA levels was performed using a modified protocol of the method described by Chiwocha et al. (2003). ABA was extracted (25 mg dry weight) twice in 750 μl ethanol, concentrated in a Speed-Vac and dissolved in 85 μl of 60% acetonitrole. (2H6)-trans,trans-ABA (20 ng) was added as a quantitative internal standard. ABA was analyzed by [LC-(−)ESI-MS/MS] using a Dionex HPLC system (Dionex, http://www1.dionex.com). The sample (10 μl) was loaded on a Phenomenex Luna 3-μm C18(2) 100A, 150 × 2-mm column, and eluted at room temperature in an isocratic mode with 65% B (A, HPLC-grade water with 0.04% v/v acetic acid; B, acetinitrile) and a flow rate of 0.15 ml min−1, and was analysed by MS/MS: 263 > 152.7 (269 > 158.7) ([M–H]–); collision gas, 2 mTorr/10.0 V; API drying gas, 300°C/21 psi; API Nebulizing gas, 50 psi; scan time, 1.0 s; SIM width, 0.7 amu; needle, −4500 V; shield, −600 V; capillary, −40 V; detector, 1575V.

GC-MS-based metabolite analysis

Metabolite analysis by GC-MS was carried out using a GC-TOF protocol optimized for Arabidopsis (Lisec et al., 2006). Relative metabolite contents were calculated as described in Roessner et al. (2001), following peak identification using TAGfinder (Luedemann et al., 2008) and spectral libraries housed in the Golm Metabolome Database (Kopka et al., 2005).

Acknowledgements

We are grateful to Katrin Blaschek and Susanne Knüpffer for their excellent technical assistance. We thank Petra Hoffmeister for plant transformation, and Ulrich Wobus and Winfriede Weschke for discussions and continuous support. We acknowledge the help of Ursula Tiemann and Karin Lipfert for figure artwork. We thank Manuela Meyer (IGS, CeBiTec, Bielefeld University) for performing the Ps6kOLI1 microarray hybridizations and the processing of microarray images. This work was supported by the European Union (Integrated project GRAIN LEGUMES), the Deutsche Forschungsgemeinschaft (WE 1641/9-1) and Sachsen-Anhalt (Innoplanta).

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