Seed-Specific Expression of a Feedback-Insensitive Form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis Stimulates Metabolic and Transcriptomic Responses Associated with Desiccation Stress    

Abstract

and Tel-Hai College, Upper Galilee 11016, Israel (R.A.) With an aim to elucidate novel metabolic and transcriptional interactions associated with methionine (Met) metabolism in seeds, we have produced transgenic Arabidopsis (Arabidopsis thaliana) seeds expressing a feedback-insensitive form of CYSTATHIONINEg-SYNTHASE, a key enzyme of Met synthesis. Metabolic profiling of these seeds revealed that, in addition to higher levels of Met, the levels of many other amino acids were elevated. The most pronounced changes were the higher levels of stress-related amino acids (isoleucine, leucine, valine, and proline), sugars, intermediates of the tricarboxylic acid cycle, and polyamines and lower levels of polyols, cysteine, and glutathione. These changes reflect stress responses and an altered mitochondrial energy metabolism. The transgenic seeds also had higher contents of total proteins and starch but lower water contents. In accordance with the metabolic profiles, microarray analysis identified a strong induction of genes involved in defense mechanisms against osmotic and drought conditions, including those mediated by the signaling cascades of ethylene and abscisic acid. These changes imply that stronger desiccation processes occur during seed development. The expression levels of transcripts controlling the levels of Met, sugars, and tricarboxylic acid cycle metabolites were also significantly elevated. Germination assays showed that the transgenic seeds had higher germination rates under salt and osmotic stresses and in the presence of ethylene substrate and abscisic acid. However, under oxidative conditions, the transgenic seeds displayed much lower germination rates. Altogether, the data provide new insights on the factors regulating Met metabolism in Arabidopsis seeds and on the mechanisms by which elevated Met levels affect seed composition and behavior.

Dose-dependent effects of higher methionine levels on the transcriptome and metabolome of transgenic Arabidopsis seeds

“…This promoter is temporally induced during the maturation and desiccation stages of seed development when seed-storage proteins are produced (Fait et al 2011;Sundaram et al 2013). Here, we describe the transcriptomic and primary metabolite profiling of the RNAi::AtCGS seeds at the background of those previously reported for the SSE-AtD-CGS seeds (Cohen et al 2014). Such comparisons provide new insights into the mechanisms mediating the effects of elevated methionine levels on Arabidopsis seed metabolome.…”

“…Using specific sense (5 0 -GGATCCAAGGAGAA AAGGAGTGTG-3 0 and 5 0 -GAATTCGGTTT GTCGGG GACTCAG-3 0 ) and antisense (5 0 -CTCGAGGGT TTGTCGGGGACTCAG-3 0 and 5 0 -GGTACCAAGGA GAAAAGGAGTGTG-3 0 ) fragments, we created an inverted repeat RNAi cassette. These fragments were amplified using PCR and introduced into a pGEMT plasmid, inserted into an SK-intron intermediate vector (4137 bp) and finally cloned into a pPZP111 binary transformation plasmid containing the seed-specific phaseolin promoter, octopin synthase terminator and the NPTII gene for kanamycin selection as previously described (Cohen et al 2014). This plasmid was introduced into Agrobacterium tumefaciens EHA105 cells, and Arabidopsis transformation was performed by the floral dip method.…”

“…Primary metabolite profiling of the transgenic tobacco and Arabidopsis seeds showed that higher expression level of CGS in seeds resulted in overaccumulation of stress-related metabolites. Additionally, Affymetrix microarray analysis employed two selected transgenic Arabidopsis seeds (named SSE-AtD-CGS 1 and 2) that contained four and sixfold more methionine than wild-type (WT) seeds, respectively, further demonstrating that the majority of highly expressed transcripts were related to early desiccation stress responses (Cohen et al 2014). Total protein content was also elevated in these seeds accompanied by altered profiles of seed-storage proteins and fatty acids (Cohen et al 2016a).…”

“…aspartate family pathway, via its main regulatory enzyme CYSTATHIONINE c-SYNTHASE (CGS) (Karchi et al 1993;Tabe and Droux 2001). Indeed, recent studies showed that seed-specific expression of a feedback-insensitive form of the Arabidopsis thaliana CGS (AtD-CGS) leads to higher methionine contents in seeds of two soybean cultivars (Cohen et al 2016b;Song et al 2013), tobacco , and Arabidopsis (Cohen et al 2014). The expression of another form of the AtCGS also resulted in higher methionine levels in an additional two soybean cultivars, Misuzudaizu and Bert, as well as in azuki bean (Hanafy et al 2013).…”

“…mRNA analysis using quantitative real-time (qRT)-PCR qRT-PCR was used to determine the expression levels of AtCGS in WT, EV, and RNAi::AtCGS seeds. Total RNA extraction, cDNA synthesis, and qRT-PCR procedures were according to Cohen et al (2014). To normalize variance among samples, we used the constitutive AtPP2A-A3 gene as an endogenous control (Czechowski et al 2005).…”