Plant Physiology Preview. Published on November 5, 2008, as DOI:10.1104/pp.108.129940
Running head: Effect of AGP deficiency on pea embryo metabolism Corresponding author: Hans Weber, Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben Tel.: +49 (0) 39482 5 208, Fax.: +49 (0) 39482 500, e-mail:
[email protected]. Journal Research Area: Biochemical Processes and Macromolecular Structures
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Copyright 2008 by the American Society of Plant Biologists
ADP-Glucose pyrophosphorylase deficient pea embryos reveal specific transcriptional and metabolic changes of C:N metabolism and stress responses Kathleen Weigelt1, Helge Küster2, Twan Rutten1, Aaron Fait3, Alisdair R. Fernie3, Otto Miersch4, Claus Wasternack4, R.J. Neil Emery5, Christine Desel6, Felicia Hosein1, Martin Müller1, Isolde 1
Saalbach and Hans Weber
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1
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben;
2
Institute for Genome Research and Systems Biology (IGS), Center for Biotechnology (CeBiTec),
Bielefeld
University,
D-33615
Bielefeld,
Germany;
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Max-Planck-Institut
für
Molekulare
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Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany; Leibniz-Institut für Pflanzenbiochemie, D5
06120 Halle (Saale), Germany; Biology Department, Trent University, Peterborough, ON, K9J 7B8 Canada ; 6Christian Albrechts University of Kiel, Institute of Botany, D-24098 Kiel, Germany (C.D.)
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Footnotes: This work was supported by the European Union (GRAIN LEGUMES Integrated project), the Deutsche Forschungsgemeinschaft (WE 1641/9-1) and Sachsen-Anhalt (Innoplanta). The author responsible for distribution of materials integral to the findings presented in this article in accordance
with
the
journal
policy
described
in
the
Instructions
for
Authors
(http://www.plantphysiol.org) is: Hans Weber Corresponding author: Hans Weber, email:
[email protected] Key words: Legume seed maturation, C:N partitioning, metabolic regulation, stress response, seed starch synthesis, hormonal regulation
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Abstract We present a comprehensive analysis of ADP-glucose pyrophosphorylase-repressed pea seeds using transcript and metabolite profiling to monitor effects that reduced carbon flow into starch has on C:N metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP-repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic-acid cycle and mitochondrial respiration. Enhanced provision of precursors such as acetyl-CoA and organic acids apparently support other pathways and activates amino acid and storage protein biosynthesis, as well as pathways fed by cytosolic acetyl-CoA such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher N demand depletes transient N storage pools, specifically asparagine and arginine and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species (ROS) coupled with an upregulation of energy dissipating, ROS protection and defence genes. Transcriptional activation of MAP-kinase pathways and oxylipin synthesis indicate an additional activation of stress-signalling pathways. AGP-repressed embryos contain higher levels of jasmonate derivates; however, this increase is preferentially in non-active forms. The results suggest that, although, metabolic/osmotic alterations in iAGP pea-seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress-signalling under conditions, in which excessive stress response and/or cellular damage could prematurely initiate senescence or apoptosis.
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Introduction The plastidal ADP-glucose pyrophosphorylase (AGP) catalyzes the conversion of Glc-1-P and ATP to PPi and ADP-glucose, the substrate for starch synthase, and is a key regulatory enzyme of starch biosynthesis (Preiss et al., 1991; Tetlow et al., 2004). AGP-mutants in maize and pea show dramatic decreases in seed starch content (Dickinson and Preiss 1969; Smith et al., 1989). However, unlike in leaves, flux control coefficients for AGP in legume seeds are below 0.1 (Denyer et al., 1995; Rolletschek et al., 2002) indicating low level of control for carbon flux into starch. Seed starch biosynthesis mutants often accumulate soluble sugars instead of starch leading to specific metabolic adjustments, altered water uptake, wrinkled phenotypes and increased protein contents (Casey et al., 1998; Perez et al., 1993). This metabolic shift could be of biotechnological importance in legumes, in which protein is the more valuble seed compound. The higher protein content is, at least in part, due to the increased availability of carbon acceptors in form of organic acids (Miflin and Lea, 1977), which indicates general carbon limitation for amino acid/seed protein synthesis (Weigelt et al., 2008). A key enzyme connecting carbon and nitrogen metabolism is PEP carboxylase (Golombek, 2001). Its over-expression in V. narbonensis seeds channels more carbon into organic acids, stimulates amino acid biosynthesis and increases protein content (Rolletschek et al., 2004; Radchuk et al., 2007). Accumulation of sucrose, as shown for AGP-repressed Vicia (Weber et al., 2000, Rolletschek et al., 2002) and maize seeds (Cossegal et al., 2008), causes re-partitioning of carbon into glycolysis, TCA-cycle and amino acid biosynthesis. Apart from its metabolic role sucrose acts as signal molecule regulating gene expression and seed maturation (Osuna et al., 2007; Borisjuk et al., 2002; Weber et al., 2005). In maturing seeds sucrose is present at high concentrations, promotes cell enlargement, endopolyploidisation and induces starch and protein storage in pea (Wang and Hedley 1993), V. faba (Barratt and Pullen 1998, Weber et al., 1996) and wheat (Jenner et al., 1991). Pea seeds overexpressing a surose transporter show stimulated storage protein biosynthesis on the level of transcripts, proteins and protein bodies. It has been concluded that sucrose functions both as a signal and fuel to stimulate storage protein accumulation (Rosche et al., 2002, 2005). In addition, sucrose signalling has been demonstrated to interact with that of ABA (Finkelstein et al., 2002). Possibly, sucrose increases ABA sensitivity or levels (Smeekens, 2000) or ABA modulates the response to sugar signals. Several ABA-biosynthesis (aba) 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 (Rolland et al., 2006; Radchuk et al., 2006). Decreased ABA levels affect the proper metabolism of sucrose in SnRK1-repressed pea (Radchuk, Emery, Weber, unpublished; Radchuk et al., 2006). Whilst sucrose limitation is detrimental to the plant an excess of sugar can lead to stress responses either caused directly by the sugar source itself, its metabolism (Price et al., 2004) or via indirect effects such as increased water uptake and hyperosmotic stress (Rolletschek et al., 2002). Such stress conditions can initiate complex multiple responses involving hormonal, metabolic and transcriptional changes (Sanchez et al., 2008; Shulaev et al., 2008; Price et al., 2004).
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The aim of this study was to analyse the effects of reduced carbon flux into starch on seed metabolic pathways by in depth phenotypic analysis of pea seeds with reduced AGP using a combination of transcript and metabolite profiling. The results indicate that decreasing the carbon flux through the starch pathway, on the one hand, leads to re-partitioning of carbon into alternative pathways and stimulates storage protein synthesis, but on the other hand, produces a number of pleiotropic, mainly stress-induced, effects as a consequence of increased sugar accumulation and metabolism. The findings on iAGP-3 seeds highlights the importance of a balanced C:N status for proper seed development and further gives insight into the sugar-mediated regulation of seed maturation. Potential abilities and strategies of legume seeds are discoverd to cope with detrimental stress situations. Thus seed models like the iAGP-3 line with altered pathways contribute substantially to a better understanding of “normal” seed metabolism and towards a more profound knowledge of seed biochemistry. Results Generation of AGP-deficient pea lines by RNA interference (RNAi) Three homozygous pea (Pisum sativum) lines (iAGP-1, iAGP-2 and iAGP-3) containing single inserts of the AGP-RNAi (small subunit) construct were selected by Southern gel blot and segregation analysis. Suppl. Fig. 1 and Table I show that the lines behave identically. Northern gel blot analysis performed on embryos at 15, 20, 25, 30 and 35 days after pollination (DAP) revealed strongly decreased AGP-mRNA-levels and AGP-activity from 20 DAP onwards. Starch levels between 20 and 35 DAP were only reduced by approximately 50 % (Suppl. Fig. 1) indicating a low control coefficient for pea seed AGP on starch synthesis, in accordance with what has already been reported for AGPreduced Vicia seeds (Weber et al., 2000). In transgenic seeds mRNA-levels of major storage protein vicilin were increased after 20 DAP, whereas that of legumin decreased down to 40 % of wildtype level (Suppl. Fig. 1D, E). Supplementary Table I presents mature seed composition of all three lines. The wrinkled seed phenotypes are shown in Suppl. Fig. 2. AGP-deficient pea seeds reveal a shift in seed composition Analysis of seed composition in mature dry iAGP-3 seeds revealed more total nitrogen, carbon, albumins, globulins, lipids and sucrose on the per g dry weight level. Seed starch was reduced by 40 to 50 % of wildtype levels and the residual starch was altered in composition containing considerably less amylose. Seed dry weight was decreased by ~ 20 % (Tab. I). Analysis of seed composition and developmental parameters were performed at 20, 25, 30 and 35 DAP. AGP-activity was lower at all stages analysed (Fig. 1A), whereas sucrose synthase-activity was higher at 20 and 30 DAP (Fig. 1B). Starch accumulation was lower reaching only 40 to 50 % of wildtype levels (Fig. 1C), however, iAGP-3 seeds exhibited 50 % higher sucrose levels at all stages (Fig. 1D). Total seed N and C percentages were higher at 25, 30 and 35 DAP (Fig. 1E, F). Seed fresh weight was higher at 30 and 35 DAP (Fig. 1G), whereas seed dry weight was decreased at all stages (Fig. 1H). Consistently, iAGP-3 seeds have higher water content at all stages (Fig. 1I) most likely due to the higher sugar content.
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AGP-deficient pea embryos reveal altered cell morphology Following staining for starch and proteins, cell morphology was analysed by light microscopy in iAGP3 and wildtype embryos at 20, 25, 30 and 35 DAP. At 20 DAP transgenic cells contain elongated starch grains that are tiny (Fig. 3A) compared to the wildtype (Fig. 3B). Almost no storage protein vacuoles are visible in iAGP-3 cells (Fig. 3C) compared to the wildtype (Fig. 3D). At 25 DAP the transgenic cells contain fewer and smaller starch grains (Figs. 3E, F), whereas storage protein vesicles are larger but less densely filled (Fig. 3G, 3H). At 30 DAP starch grains were again less numerous in iAGP-3 cells and exhibited light brown colour (Fig. 3I), whereas wildtype grains were larger and of dark blue colour (Fig. 3J). Moreover, in transgenic cells storage vesicles were larger and appeared to be less dense (Fig. 3K) than the wildtype (Fig. 3L). A similar pattern with regard to grain size and colour between transgenics (Fig. 3M) and wildtype (Fig. 3N) occurred at 35 DAP. However, there is no difference in the size of the storage protein vacuoles at this timepoint, although, they appear to be less densely packed in iAGP-3 cells (Fig. 3O) than the wildtype (Fig. 3P). Transcript profiling of iAGP-3 embryos We analysed differential gene expression between phytochamber-grown embryos at 20, 25, 30 and 35 DAP, using microarrays enriched in seed-expressed genes (Weigelt et al., 2008). Transcript levels are given as ratios between wildtype and iAGP-3 embryos (fold up- or downregulated). Genes were regarded as differentially expressed if they varied by a factor of at least 2.0 fold with statistical differences (P
