SUMMARY 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.

The effect of oligochitosan O-80 treatment on metabolic changes in white radish (Raphanus sativus L.) sprouts (WRS) was investigated for its potential to enhance bioactive compounds accumulation. The seeds were germinated in deionized water containing different concentrations of oligochitosan O-80 (0 (control), 5, 10, 20 and 40 ppm). Seven-day old sprouts were harvested for metabolome analysis using liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) for phenolic compounds and glucosinolate analysis, respectively, and spectrophotometric assays to determine the total phenolic content and antioxidant capacity. Partial least squares discriminant analysis was adopted to model the data from the LC-MS and revealed that O-80 at all tested concentrations affected the metabolite profiles of the treated WRS samples. The UV chromatogram at 320 nm showed increased levels of most sinapoyl derivatives, consistent with the increased total phenolic contents. Interestingly, glucoraphasatin (4-methylthio-3-butenyl glucosinolate), a major glucosinolate detected by HPLC, was increased by 40% in the sprouts treated with 10 ppm O-80. Our results provide compelling evidence regarding the exogenous application of oligochitosan O-80 as an elicitor of bioactive metabolites in WRS.

Sulfur Deficiency Induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by down-regulating glucosinolates. Here, we identified that besides glucosinolate regulation under –S, SDI1 down-regulates 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 down-regulates 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.

A. 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 transcription is strongly induced by this condition. However, little is currently known about the cis- and trans-factors that regulate SDI expression. To identify potential transcription factors and DNA sequence element regulators of SDI expression we performed a comparative in silico analysis of their promoter sequences cataloguing known and potentially new cis-elements. We further screened an arrayed library of Arabidopsis transcription factors (TF) for binding to the SDI1 and SDI2 promoters. In total 14 candidate TF regulators of SDIs were identified with yeast-one-hybrid analyses, of which five bound to both promoters, 4 were specific to SDI1, and 5 were specific SDI2. Direct association between particular cis-elements in these promoter regions and specific TFs was established via electrophoretic mobility shift assays. SLIM1 was shown to bind SURE cis-element(s) in the proximal promoter region of both SDI1 and SDI2. The bZIP core cis-element in the proximal promoter region of SDI2 was shown to be important for bZIP16, bZIP44, and HYH binding. GBF1 was shown to bind the E-box in the proximal promoter region of SDI2. Additionally, we performed a meta-analysis of expression changes of these 14 TF candidates in a variety of conditions that alter SDI expression. These data will allow for more detailed future analysis of the molecular factors required for transcriptional regulation of SDIs under a range of physiological and metabolic conditions, apart from sulfur deficiency.

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.