| Cotton is the main economic crop in China.With the increase of global temperature,the effects of high temperature on fiber yield and quality of cotton are becoming more severe.Therefore,it is essential to elucidate the mechanisms for cotton in responding to high temperature stress.High temperature mainly inhibits plant photosynthesis.Moreover,reactive oxygen species（ROS）are significantly accumulated and plants are damaged under hot stress.Under high temperature,expression of many genes is altered.Hot caused damage is thus eliminated or reduced in plants.Gene expression is controlled by multiple mechanisms,and DNA methylation and histone covalent modifications are among the key mechanisms.At present,the molecular and epigenetic mechanisms for model plants in response to hot stress have been reported.However,these for cotton are largely unknown.Here,the seedlings of Gossypium hirsutum were used as experimental materials,the regulatory mechanisms of molecular and epigenetic modifications of cotton in response to high temperature stress were studied after treatment at 42°C for short-term（1 h,2 h,6 h）,medium-term（1 d,3 d,5 d）and long-term（7 d,10 d）period.The main experimental results are as follows:（1）Under short term hot stress,cotton seedlings were not seriously damaged.The expression of genes encoding heat shock protein（HSP）and heat shock transcription factor（HSF）is rapidly increased.Additionally,the transcription levels of genes encoding the photosynthesis-related Ruc S（Rubisco small subunit）,Ruc L（Rubisco large subunit）and RCA（Rucisco activase）were slightly up-regulated,and the photosynthetic related parameters such as chlorophyll content and CO2 assimilation efficiency enhanced gradually.By contrast,ROS accumulation in leaves in hot-treated seedlings was not significant different from that in the control.（2）Under long-term high temperature treatment,the expression of genes encoding heat shock protein and heat shock transcription factor was further up-regulated,and that of Ruc S,Ruc L and RCA was significantly down-regulated.Moreover,leaf photosynthesis was severely inhibited,and the plant height and fresh weight of cotton seedlings were clearly lower than those of the control.Using nitro blue tetrazolium（NBT）and 3,3-diaminobenzidin（DAB）staining method,we observed that production of superoxide anion and hydrogen peroxide,and the activities of peroxidase and superoxide dismutase in leaves were markedly enhanced under long-term high temperature stress.In addition,the expression of senescence-related genes hexokinase1（HXK1）and ethylene reaction factor 1A（ERF1A）was up-regulated,and the chlorophyll content of the leaves was significantly decreased.The content of malondialdehyde was also significantly increased,and the leaves of the cotton became yellow and wilting under hot stress.（3）It was found that the amino acid sequences of HXK1 and ERF1A were highly conserved between Arabidopsis and cotton by sequence alignment.After silencing HXK1 and ERF1A genes by VIGS technology,the cotton plants exhibited evidently higher tolerant to high temperature than the control（4）The DNA methylations of the whole genome in leaves were detected by high performance liquid chromatography.The results showed that the methylation levels were decreased after high temperature treatment for 1 h and 7 d.However,those for other time points were increased or not changed.Western blot results show that the level of histone methylation H3K4me2 from leaves rose under short-term hot stress but remarkably declined under high-term stress.The content of histone acetylation H4K5ac progressively enhanced under high temperature stress,and that was also observably higher in hot-treated leaves than in the control.（5）The deconcentration regions in promoters of HSP3,Hsf A1a,Ruc S,HXK1 and RF1A were characterized by Chart-PCR.Using Chip-PCR technology,it was demonstrated that the expression of HSP3and Hsf A1a was modulated by H3K4me2 after short time hot treatment,but regulated by H4K5ac under long-term hot stress.Moreover,high temperature for short period caused the increase of Ruc S transcription,which is regulated by both H3K4me2 and H4K5ac.Under long period of high temperature stress,the level of H3K4me2 decreased,and the expression of Ruc S was downregulated.Meanwhile,the levels of H3K4me2 and H4K5ac enhanced,further stimulating the transcription of HXK1.Moreover,transcripts of ERF1A increased due to the elevation of H4K5ac level under long-term hot stress.The bisulfite sequencing revealed that there was no significant change in the DNA methylation level of the promoter regions of HSP3 and ERF1A under high temperature stress,but the DNA methylation level of Hsf A1a promoter region was decreased.Therefore,DNA methylation primarily regulates the expression of Hsf A1a but not HSP3and ERF1A.Taken together,high temperature stress can inhibit cotton seedling growth by oxidative damage and reducing photosynthesis.And these results strongly suggest that DNA methylation,H3K4me2 and H4K5ac play crucial roles in regulating the expression of HSP3,Hsf A1a,Ruc S,HXK1 and ERF1A in responding to high temperature stress,particularly long-term hot stress,thereby,improving the hot tolerance of cotton seedlings. |
