Plants rapidly adapt to elevated ambient temperature by adjusting their growth and developmental programs. To date, a number of experiments have been carried out to understand how plants sense and respond to warm temperatures. However, how warm temperature signals are relayed from thermosensors to transcriptional regulators is largely unknown. To identify new early regulators of plant thermo-responsiveness, we performed phosphoproteomic analysis using TMT (Tandem Mass Tags) labeling and phosphopeptide enrichment with Arabidopsis etiolated seedlings treated with or without 3h of warm temperatures (29°C). In total, we identified 13,160 phosphopeptides in 5,125 proteins with 10,700 quantifiable phosphorylation sites. Among them, 200 sites (180 proteins) were upregulated, while 120 sites (87 proteins) were downregulated by elevated temperature. GO (Gene Ontology) analysis indicated that phosphorelay-related molecular function was enriched among the differentially phosphorylated proteins. We selected ATL6 (ARABIDOPSIS TOXICOS EN LEVADURA 6) from them and expressed its native and phosphorylation-site mutated (S343A S357A) forms in Arabidopsis and found that the mutated form of ATL6 was less stable than that of the native form both in vivo and in cell-free degradation assays. Taken together, our data revealed extensive protein phosphorylation during thermo-responsiveness, providing new candidate proteins/genes for studying plant thermomorphogenesis in the future.

植物通过调整其生长和发育程序迅速适应升高的环境温度。迄今为止，已经进行了许多实验来了解植物如何感知和响应温暖的温度。然而，温暖的温度信号如何从热传感器传递到转录调节器在很大程度上是未知的。为了识别植物热响应的新早期调节剂，我们使用 TMT（串联质量标签）标记和磷酸肽富集进行了磷酸蛋白质组学分析拟南芥经过或不经过 3 小时温暖温度 (29°C) 处理的黄化幼苗。总的来说，我们在 5,125 种蛋白质中鉴定了 13,160 个磷酸肽，具有 10,700 个可量化的磷酸化位点。其中，200 个位点（180 个蛋白质）被上调，而 120 个位点（87 个蛋白质）因温度升高而下调。GO（基因本体论）分析表明，在差异磷酸化的蛋白质中，磷酸中继相关的分子功能丰富。我们从中选择了 ATL6 (ARABIDOPSIS TOXICOS EN LEVADURA 6) 并在拟南芥 并发现 ATL6 的突变形式不如天然形式稳定 体内和在无细胞降解试验中。总之，我们的数据揭示了热响应过程中广泛的蛋白质磷酸化，为未来研究植物热形态发生提供了新的候选蛋白质/基因。