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.

中文翻译：

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水稻真D-半胱氨酸脱硫酶基因的克隆与鉴定

硫化氢（H2S）被认为是第三种气体传递体，在动植物的多种信号事件中发挥着积极作用。半胱氨酸脱硫酶 (CDes)，包括将 L- 或 D-半胱氨酸降解为 H2S、丙酮酸和铵的 D- 和 L-半胱氨酸脱硫酶 (D/L-CDes)，被认为是负责植物内源性 H2S 生成的关键酶. 几种 D-CDes 与 1-氨基环丙烷-1-羧酸脱氨酶 (ACCD) 同源，同时具有 ACCD 和 D-CDes 活性，因此不是真正的特定 D-CDes。然而，人们对真正的 D-CDes 的关注很少，关于植物中这种蛋白质的信息也知之甚少。在这项研究中，从水稻中克隆并表征了一个假定的 D-CDes 转录物，该转录物编码具有 423 个氨基酸的蛋白质，具有 D-CDes 活性并命名为 OsDCD1。从 OsDCD1 重组蛋白中无法检测到 ACCD 和 O-乙酰-L-丝氨酸（硫醇）裂解酶 (OASTL) 的活性。对于 D-Cys，OsDCD1 的 Km 为 0.13 ± 0.01 mM，Vm 为 111.55 ± 1.91 单位 mg−1 蛋白质。OsDCD1 的最佳 pH 值和最佳温度分别为 8.5 和 35°C。通过定点诱变，S357E 或 S357E/T589L 的突变几乎完全消除了 OsDCD1 的 D-CDes 活性，而 T389L 突变体仅保留了 3.7% 的部分 D-CDes 活性，表明这两个氨基酸残基对维持 OsDCD1 活性。此外，水稻原生质体中的亚细胞定位分析表明，OsDCD1 定位于叶绿体而不是线粒体，这与拟南芥中的 DCD1 不同。qRT-PCR 分析进一步表明 OsDCD1 转录本的丰度受到我们使用的不同激素和化学试剂的广泛调节。总的来说，我们的研究结果证明 OsDCD1 是水稻中潜在重要的内源性 H2S 产生酶，它可能在植物生长调节剂和化学刺激物中发挥重要作用。