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 homeostasis of major macronutrient metabolism needs to be tightly regulated, especially when the availability of one or more nutrients fluctuates in the environment. Both sulfur metabolism and glucose signaling are important processes throughout plant growth and development, as well as during stress responses. Still, very little is known about how these processes affect each other, although they are positively connected. Here, we showed that the crucial transcription factor of sulfur metabolism, SLIM1, is involved in glucose signaling during the shortage of sulfur. The germination rate of the slim1_KO mutant was severely affected by high glucose and osmotic stress. The expression of SLIM1-dependent genes in sulfur deficiency appeared to be additionally induced by the high concentration of either mannitol or glucose, but also by sucrose, which is not only the source of glucose but another signaling molecule. Additionally, SLIM1 affects PAP1 expression during sulfur deficiency by directly binding to its promoter. The lack of PAP1 induction in such conditions leads to much lower anthocyanin production. Taken together, our results indicate that SLIM1 is involved in the glucose response by modulating sulfur metabolism and directly controlling PAP1 expression in Arabidopsis during sulfur deficiency stress.

Under conditions of sulfur deprivation OAS accumulates which is leading to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to ROS or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived spanning stably over several hundred conditions. This allowed not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized the existing knowledge on the regulation of the OAS cluster and the co-expressed genes. As it turned out, the known sulfate deprivation related transcription factor EIL3/SLIM1 exhibits a prominent role as most genes are subject to regulation by this TF. The bearing of other transcription factors in response to OAS awaits still further investigations.

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.

Sulfur is an essential macronutrient for growth of higher plants. The entry of the sulfate anion into the plant, its importation into the plastids for assimilation, its long-distance transport through the vasculature, and its storage in the vacuoles require specific sulfate transporter proteins. In this study, mycorrhizal and non-mycorrhizal maize plants were grown for 60 days in an S-deprived substrate, whilst iron was provided to the plants in the sparingly soluble form of FePO4. On day 60, sulfate was provided to the plants. The gene expression patterns of a number of sulfate transporters as well as sulfate assimilation enzymes were studied in leaves and roots of maize plants, both before as well as after sulfate supply. Prolonged sulfur deprivation resulted in a more or less uniform response of the genes’ expressions in the roots of non-mycorrhizal and mycorrhizal plants. This was not the case neither in the roots and leaves after the supply of sulfur, nor in the leaves of the plants during the S-deprived period of time. It is concluded that mycorrhizal symbiosis modified plant demands for reduced sulfur, regulating accordingly the uptake, distribution, and assimilation of the sulfate anion.