Proteomic Analysis Of Thermomorphogenic Responses In Arabidopsis

Under the background of global warming and intensified greenhouse effect,crop growth and development face great challenges.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.Hence,we adopted proteomic techniques to identify early regulators of plant thermoresponsiveness.To prevent possible interference of photosynthesis-related proteins to our proteomic analysis of thermo-responsiveness,we performed quantitative proteomics and phosphoproteomic analysis using TMT(Tandem Mass Tags)labeling and phosphopeptide enrichment with Arabidopsis etiolated seedlings treated with(29°C)or without(22°C)3h of warm temperatures.In total,in the quantitative proteomics study,7,956 of the 8,800 proteins identified could be quantified,and the expression levels of 14 proteins were up-regulated and 13 proteins were down-regulated by warm temperatures;in the quantitative phosphoproteomics study,we identified 14,191 phosphorylation sites in 5,125 proteins,among which 10,700 phosphorylation sites in 4,196 proteins had quantitative information,and the phosphorylation levels of 200 sites(corresponding to 180proteins)were upregulated,while 120 sites(corresponding to 87 proteins)were downregulated by elevated temperature.GO analysis indicated that phosphorelay-related molecular functions was enriched among the differentially phosphorylated proteins.We selected ATL6(ARABIDOPSIS TOXICOS EN LEVADURA 6),CID2(CTC-Interacting Domain2)and NFYB10(NUCLEAR FACTOR Y,subunit B10)to validate the phosphorylation sites,of which the phosphorylation levels were upregulated by elevated temperature.We expressed the native and dephosphorylation-mimicking forms in Arabidopsis and found that the mutated form of ATL6(S343A S357A)and CID2(S31A)was less stable than that of the native form both in vivo and in vitro respectively.In addition,under warm temperature conditions,ATL6 overexpression could slightly promote hypocotyl elongation in the dark and inhibit hypocotyl elongation in the light.Besides,we found that overexpression of NF-YB10 inhibits hypocotyl elongation and upregulation of thermoresposive gene,while the dephosphorylation-mimicking form NF-YB10(S21A)reinforces the process,suggesting NF-YB10 is a negative regulator of thermomorphogenesis and the regulation of phosphorylation of serine 21 is crucial to this process.Taken together,our data revealed extensive protein phosphorylation in the Arabidopsis proteome during thermo-responsiveness,providing new candidate proteins/genes for studying plant thermomorphogenesis in the future.