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Cloning and Characterization of a gene Encoding True D-cysteine Desulfhydrase from Oryza sativa

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Abstract

Hydrogen sulfide (H2S) has been regarded as the third gasotransmitter and plays an active role in multiple signaling events of plants and animals. Cysteine desulfhydrases (CDes), including both D- and L-cysteine desulfhydrases (D/L-CDes) that degrade L- or D-cysteine into H2S, pyruvate, and ammonium, are considered the key enzymes responsible for endogenous H2S generation in plants. Several D-CDes are homologous to 1-aminocyclopropane-1-carboxylate deaminase (ACCD) and possess both ACCD and D-CDes activities, thus not a real specific D-CDes. However, little attention had been paid to true D-CDes and little information has been known about this protein in plants. In this study, a putative D-CDes transcript was cloned and characterized from Oryza sativa which encodes a protein with 423 amino acids possessing D-CDes activity and named as OsDCD1. Neither activities of ACCD nor O-acetyl-L-serine (thiol) lyase (OASTL) can be detected from OsDCD1 recombinant protein. For D-Cys, the Km of OsDCD1 is 0.13 ± 0.01 mM and the Vm is 111.55 ± 1.91 units mg−1 of protein. The pH-optimum and temperature-optimum of the OsDCD1 are 8.5 and 35°C, respectively. By site-directed mutagenesis, mutation of S357E or S357E/T589L almost fully abolished the D-CDes activity of OsDCD1, while the T389L mutant retained only partial D-CDes activity by 3.7%, indicating these two amino acid residues play critical roles for the maintenance of OsDCD1 activity. Besides, subcellular localization analysis in rice protoplast revealed that the OsDCD1 localizes in the chloroplast but not mitochondria, which is different from DCD1 in Arabidopsis. The qRT-PCR analysis further showed that the abundance of OsDCD1 transcript was widely regulated by different hormones and chemical reagents we used. In general, our results provided evidence that OsDCD1 is a potentially important endogenous H2S producing enzyme in rice, which may play an important role in plant growth regulators and chemical stimuli.

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Funding

This research was funded by National Natural Science Foundation of China (31670255), Fundamental Research Funds for the Central Universities (KYZ201859), and Natural Science Foundation of Jiangsu Province (BK20161447).

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Correspondence to Xianchao Yin or Yanjie Xie.

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Key Messages

• A D-CDes was characterized from rice and named as OsDCD1, which located in chloroplast.

• OsDCD1 specifically catalyze the degradation of D-Cysteine.

• The serine residue (S359) and threonine residue (T387) played critical roles for the maintenance of OsDCD1 activity.

OsDCD1 was ubiquitously expressed and respond to multiple hormones and stress conditions.

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Zhou, H., Guan, W., Zhou, M. et al. Cloning and Characterization of a gene Encoding True D-cysteine Desulfhydrase from Oryza sativa. Plant Mol Biol Rep 38, 95–113 (2020). https://doi.org/10.1007/s11105-019-01181-2

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