Characterization of γ-Glutamyl Peptidases and γ-Glutamyl Cyclotransferases for Glutathione Degradation in Arabidopsis

Organic sulfur is stored as glutathione (GSH) in plants. In Arabidopsis, γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3) degrade cytosolic GSH, but they do not fully explain the rapid GSH turnover. Here, we demonstrate that γ-glutamyl peptidases, GGP1 and GGP3, play a substantial role in degrading GSH in the cytosol. We conducted yeast complementation assay and activity assay of recombinant proteins to identify the novel GSH degradation enzymes. The expression patterns were investigated by RT-qPCR. GSH concentrations in the mutants were also analyzed. GGP1 complemented the yeast phenotype. Recombinant GGP1 and GGP3 showed reasonable Km values considering cytosolic GSH concentration, and their activity was comparable to that of GGCTs. The GGP1 transcript was highly abundant in mature organs such as rosette leaves. The expression of GGCT2;1 was conspicuously enhanced under sulfur deficiency. GSH concentration was higher in ggp1 knockout mutants regardless of nutritional conditions; the concentration was higher in ggct2;1 knockout mutants under sulfur-deficient conditions. We propose a model wherein cytosolic GSH is degraded fundamentally by GGP1. The degradation is accelerated by GGCT2;1 under sulfur deficiency. Given the energy cost throughout the reactions, GGPs could render a more efficient route for GSH degradation than GGCTs.

Tris(methylthio)methane Produced by Mortierella hyalina Affects Sulfur Homeostasis in Arabidopsis

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Its incorporation in seedlings was shown by 34S labeling experiment. Under sulfur deficiency, TMTM downregulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

Tris(methylthio)methane Produced by Mortierella hyalina Affects Sulfur Homeostasis in Arabidopsis

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Its incorporation in seedlings was shown by 34S labeling experiment. Under sulfur deficiency, TMTM downregulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

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

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.

Structural, optical and electronic characteristics of non-stoichiometric nano cadmium sulfide

Samples of non-stoichiometric nano CdS1 − x were synthesized by a simple thermolysis method by lowering the ratio of thiourea relative to cadmium acetate as starting precursors; Cd(Ac):(1-x) thiourea (x = 0.0, 0.03, 0.05 and 0.1). X’pert HighScore Plus program manifested biphasic CdS (cubic and hexagonal) and the Rietveld analysis was utilized to match the structural and microstructure parameters of the formed samples. The possibility of formed CdS1 − xOx alloy due to the sulfur deficiency is also examined using the Rietveld method. A High-resolution transmission electron microscope imaging exhibited nano size particles with homogeneous morphology. Fourier transform infrared spectrometer was utilized to confirm the existence of O2 in CdS1 − x matrix. The bandgap energies for CdS1 − x are reduced below the values of energy gaps of CdS and CdO upon increasing the parameter (x) forming a band gap “bowing”. The photoluminescence (PL) emitted visible colors depending on the amount of sulfur deficiency and excitation wavelength used. The maximum PL intensity observed in CdS0.9 sample, confirmed the presents of oxygen inside the matrix. The influence of oxygen substitution or vacancies of sulfur on the electronic structure and optical features of CdS was also investigated applying density function calculations.