In silico analysis of cis-elements and identification of transcription factors putatively involved in the regulation of the OAS cluster genes SDI1 and SDI2.

Arabidopsis thaliana sulfur deficiency-induced 1 and sulfur deficiency-induced 2 (SDI1 and SDI2) are involved in partitioning sulfur among metabolite pools during sulfur deficiency, and their transcript levels strongly increase in this condition. However, little is currently known about the cis- and trans-factors that regulate SDI expression. We aimed at identifying DNA sequence elements (cis-elements) and transcription factors (TFs) involved in regulating expression of the SDI genes. We performed in silico analysis of their promoter sequences cataloging known cis-elements and identifying conserved sequence motifs. We screened by yeast-one-hybrid an arrayed library of Arabidopsis TFs for binding to the SDI1 and SDI2 promoters. In total, 14 candidate TFs were identified. Direct association between particular cis-elements in the proximal SDI promoter regions and specific TFs was established via electrophoretic mobility shift assays: sulfur limitation 1 (SLIM1) was shown to bind SURE cis-element(s), the basic domain/leucine zipper (bZIP) core cis-element was shown to be important for HY5-homolog (HYH) binding, and G-box binding factor 1 (GBF1) was shown to bind the E box. Functional analysis of GBF1 and HYH using mutant and over-expressing lines indicated that these TFs promote a higher transcript level of SDI1 in vivo. Additionally, we performed a meta-analysis of expression changes of the 14 TF candidates in a variety of conditions that alter SDI expression. The presented results expand our understanding of sulfur pool regulation by SDI genes.

Sulfur deficiency-induced genes affect seed protein accumulation and composition under sulfate deprivation.

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.

Cysteine and Methionine Biosynthetic Enzymes Have Distinct Effects on Seed Nutritional Quality and on Molecular Phenotypes Associated With Accumulation of a Methionine-Rich Seed Storage Protein in Rice.

Staple crops in human and livestock diets suffer from deficiencies in certain "essential" amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of "Push × Pull" double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum. Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.