Ectopic expression of phosphoenolpyruvate carboxylase inVicia narbonensisseeds: effects of improved nutrient status on seed maturation and transcriptional regulatory networks

Abstract

SummarySeed maturation responds to endogenous and exogenous signals like nutrient status, energy and hormones. We recently showed that phosphoenolpyruvate carboxylase (PEPC) overexpression in Vicia narbonensis seeds alters seed metabolism and channels carbon into organic acids, resulting in greater seed storage capacity and increased protein content. Thus, these lines represent models with altered sink strength and improved nutrient status. Here we analyse seed developmental and metabolic parameters, and C/N partitioning in these seeds. Transgenic embryos take up more carbon and nitrogen. Changes in dry to FW ratio, seed fill duration and major seed components indicate altered seed development. Array-based gene expression analysis of embryos reveals upregulation of seed metabolism, especially during the transition phase and at late maturation, in terms of protein storage and processing, amino acid metabolism, primary metabolism and transport, energy and mitochondrial activity, transcriptional and translational activity, stress tolerance, photosynthesis, cell proliferation and elongation, signalling and hormone action and regulated protein degradation. Stimulated cell elongation is in accordance with upregulated signalling pathways related to gibberellic acid/brassinosteroids. We discuss that activated organic and amino acid production leads to a wide-range activation of nitrogen metabolism, including the machinery of storage protein synthesis, amino acid synthesis, protein processing and deposition, translational activity and the methylation cycle. We suggest that a-ketoglutarate (a-KG) and/or oxalacetate provide signals for coordinate upregulation of amino acid biosynthesis. Activation of stress tolerance genes indicates partial overlap between nutrient, stress and abscisic acid (ABA) signals, indicating a common interacting or regulatory mechanism between nutrients, stress and ABA. In conclusion, analysis of PEPC overexpressing seeds identified pathways responsive to metabolic and nutrient control on the transcriptional level and its underlying signalling mechanisms.

Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation

“…Cytokinin and abscisic acid were analysed as described in Weigelt et al (2009) and Radchuk et al (2007).…”

“…For reference, ubiquitin conjugating enzymes At4g27960 (UBC9) and At5g53300 (UBC10) (Czechowski et al, 2005) were used to screen the pea EST collection, yielding three sequences (PSC34G03, PSC24O01 and PSS06B15) with amino acid identity of 93.2% (PSC34G03), 92.3% (PSC24O01) and 95.1% (PSS06B15), AtUBC9 and AtUBC10, respectively. Reference genes were validated by GENORM software (http://medgen.ugent.be/ jvdesomp/genorm) with data sets from Radchuk et al (2006Radchuk et al ( , 2007. A reference cut-off value of M < 1.5 was considered as constitutively expressed.…”

ADP-Glucose Pyrophosphorylase-Deficient Pea Embryos Reveal Specific Transcriptional and Metabolic Changes of Carbon-Nitrogen Metabolism and Stress Responses

“…Similarly, in carboxylase-overexpressing seeds with increased anaplerotic C fluxes, an improved organic acid supply can also stimulate amino acid biosynthesis (Rolletschek et al, 2004;Radchuk et al, 2007). Metabolic changes in iAGP seeds are somewhat contrary to those observed by overexpressing an amino acid transporter (Weigelt et al, 2008).…”

“…Activation of amino acid and storage protein synthesis is metabolically regulated and stimulated by carbohydrate feeding and/or supply of C precursors (Osuna et al, 2007;Radchuk et al, 2007;this work). Suc in seeds can also signal storage-associated processes , a function interacting with ABA signaling.…”

“…A key enzyme connecting C and nitrogen (N) metabolism is phosphoenolpyruvate carboxylase (Golombek et al, 2001). Its overexpression in Vicia narbonensis seeds channels more C into organic acids, stimulates amino acid biosynthesis, and increases protein content (Rolletschek et al, 2004;Radchuk et al, 2007). Accumulation of Suc, as shown for AGP-repressed Vicia (Weber et al, 2000;Rolletschek et al, 2002) and maize (Cossegal et al, 2008) seeds, causes repartitioning of C into glycolysis, tricarboxylic acid (TCA) cycle, and amino acid biosynthesis.…”