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.

Regulation of the phenylpropanoid pathway is critical for plant development and defense. This research investigates the transcriptional control of six Phenylalanine Ammonia-Lyase (PAL) gene homologs identified in the mulberry genome. A comprehensive in silico pipeline was employed to analyze the promoter architecture of these genes. Using the MEME suite, we identified three statistically significant conserved motifs within the 2000 bp upstream region. Subsequent TF binding prediction with FootprintDB for these motifs implicated the TCP, NAC, AP2/ERF, B3, and BBR-BPC families as potential regulators. A parallel analysis with PlantRegMap highlighted a high density of binding sites for the BBR-BPC and AP2/ERF families in the core promoter regions. A comparative analysis showed a weak correlation between the databases, underscoring the necessity of a multi-faceted predictive approach. Transcriptomic profiling under chitosan-induced conditions validated our in silico framework, suggesting the involvement of these TF families. Specifically, the data support NAC083 as a putative transcriptional activator and suggest a repressive function for members of the AP2/ERF and BBR-BPC families, providing a robust, experimentally supported model of PAL regulation.

SUMMARY: 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 SDI s 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 SDI s under a range of physiological and metabolic conditions, apart from sulfur deficiency.