Exogenous Sulfide Reverses the Alteration of Transcriptional Profiling of the des1-1 Mutant (Arabidopsis thaliana). Arabidopsis thaliana

Arabidopsis thaliana cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of cysteine. Recently, we have deeply investigated about one of the minor OASTL-like protein located in the cytosol, named DES1, highlighting some important clues about its metabolic function. We have demonstrated that DES1 catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate, instead of the biosynthesis of Cys, and thus, is a novel L-cysteine desulfhydrase (EC 4.4.1.1). The functionality of DES1 is being revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. We have performed a comparative transcriptomic analysis on leaves of the des1-1 and Col-0 wild type plants grown for 30 days under long-day conditions. The normalized data from the replicates showed differential expression of 1614 genes in the des1-1 mutant, with 701 genes down-regulated and 913 genes up-regulated by more than twofold, with a False Discovery Rate (FDR) of 7. This des1-1 transcriptional profile show a strong alteration when compared to a previous comparative transcriptomic analysis performed on leaves of the des1-1 and Col-0 wild type plants grown for 20 days under identical long-day conditions (GSE 19244). We have also performed a comparative transcriptomic analysis on leaves of the des1-1 and Col-0 wild type plants grown for 20 days and treated with sodium sulfide for 10 additional days. The comparison of the transcriptional profile of des1-1+Na2S versus Col-0+Na2S clearly shows that exogenous sulfide reversed the transcriptional level differences between the mutant and the wild type to reach similar transcriptional patterns as the array GSE19244. Our results suggest a role of sulfide as transcriptional regulator in the des1-1 mutant background. Overall design: Using the Affymetrix ATH1 GeneChips, we performed two comparative transcriptomic analyses on leaves of the des1-1 and Col-0 wild type plants grown on soil under long-day conditions. One was performed on plants grown for 30 days and the other on plants grown for 20 days and treated for 10 additional days with 200 mM Na2S. Three biological replicates were performed for each sample and hybridized to the chips. We made the comparison of des1-1 versus wild-type and des1-1+Na2S versus wild-type+Na2S to classify the differently expressed genes in the mutant plant.

拟南芥（Arabidopsis thaliana）细胞中存在多种催化半胱氨酸生物合成的O-乙酰丝氨酸(巯基)裂解酶（O-acetylserine(thiol)lyase, OASTL）。近期，我们对定位在细胞质中的一类次要OASTL样蛋白DES1展开了深入研究，获取了其代谢功能的重要线索。我们证实，DES1可催化L-半胱氨酸发生脱巯基反应，生成硫化物、氨与丙酮酸，而非参与半胱氨酸的生物合成，因此它是一种新型L-半胱氨酸脱巯基酶（EC 4.4.1.1）。

DES1的功能可通过T-DNA插入突变体des1-1与des1-2的表型进行解析。我们对长日照条件下生长30天的des1-1与Col-0野生型植株的叶片开展了比较转录组分析。重复样本的标准化数据显示，des1-1突变体中共存在1614个差异表达基因，其中701个基因表达下调、913个基因表达上调超过2倍，错误发现率（False Discovery Rate, FDR）为7。相较于此前在相同长日照条件下生长20天的des1-1与Col-0野生型植株叶片开展的比较转录组分析（GSE 19244），此次des1-1的转录组谱呈现出显著改变。

我们还对长日照条件下生长20天、额外用硫化钠处理10天的des1-1与Col-0野生型植株的叶片开展了比较转录组分析。对比des1-1+Na₂S与Col-0+Na₂S的转录谱可见，外源性硫化物可逆转突变体与野生型之间的转录水平差异，使其转录模式与GSE19244的芯片结果趋于一致。我们的研究结果表明，在des1-1突变体背景中，硫化物可作为转录调控因子发挥作用。

实验设计概述：本研究使用Affymetrix ATH1基因芯片，对土壤中长日照条件下生长的des1-1与Col-0野生型植株叶片开展了两组比较转录组分析。第一组针对生长30天的植株，第二组针对生长20天后额外用200 mM Na₂S处理10天的植株。每个样本设置3个生物学重复，并进行芯片杂交。我们通过比较des1-1与野生型、des1-1+Na₂S与野生型+Na₂S的转录谱，对突变体中的差异表达基因进行分类。