Study Of The Proteinacetylation On Regulation Of Kenaf Cytoplasmic Male Sterilityand Its Related Functions

Lysine acetylation(Kac)is a universal and reversible PTM.Research shows that PTM can regulate gene expression,participate in pollen development,and thus affect plant fertility.Kenaf has strong heterosis,and Cytoplasmic male sterility(CMS)is the main way to utilize its heterosis.Information regarding Kac regulating the occurrence of CMS or anther and pollen development of kenaf have not yet been reported.Therefore,we performed a label-free based comparative acetylome analysis in kenaf anther of CMS line UG93 A and its maintain line UG93 B,identified the differentially acetylated proteins related to kenaf CMS and the metabolic pathways involved in regulating kenaf pollen development(abortion).We also analyzed the effect of acetylation on the protein.Further research on histone acetylation was also done.We cloned and identified the histone deacetylase(Hc HDACs),analyzed its gene expression pattern and subcellular localization,and studied the biological function of Hc HDA19.This study provide a better understanding of the regulation mechanism of protein acetylation.The results of this study are of great importance to reveal the regulation mechanism of acetylation on the occurrence of kenaf CMS.The main findings and conclusions of this study are as follows:1.We performed a label-free based comparative acetylome analysis ofkenaf anther of CMS line UG93 A and its maintain line UG93 B.A total of 1204 Kac sites corresponding to 672 unique proteins were identified.Further analysis showed that 56 out of 672 proteins were differentially acetylated between UG93 A and UG93B(|Fold change|?2,p<0.05),of which 92% were located in the cytoplasm and the remaining 8% in the nucleus.Bioinformatics analysis demonstrated that majority of these differentially acetylated proteins were enriched in metabolic enzyme activity,transferase activity and binding regulation,and participated in the substance and energy metabolism such as Glycolysis/gluconeogenesis,citric acid cycle(TCA),carbon metabolism,oxidative phosphorylation(OXPHOS)and amino acid biosynthesis.The 56 differentially acetylated proteins were closely related to anther and pollen development and probably involved in the occurrence of kenaf CMS.2.Energy metabolism plays an important role in anther and pollen development,and is closely related to plant fertility.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),which is closely related to energy metabolism,was choosed to study the effect of acetylation on the activity of protein.The K188 acetylation level of GAPDH in UG93 A was2.24-fold down-regulated compared with UG93 B.p PIC9 k vector was constructed to express GAPDH recombinant proteins.Enzyme activity analysis showed that the acetylation of K188 positively regulated GAPDH activity,and the decrease of acetylation level of K188 in CMS lines UG93 A resulted in lower enzyme activity.This further affected the energy metabolism process.Furthermore,the interaction between histone deacetylase SRT2 and GAPDH was demonstrated by Pull-down and Bi FC.SRT2 catalyzed the deacetylation of GAPDH,and the activity of GAPDH was negatively regulated by its acetylation level,which indicating that acetylation has an important effect on enzymeactivity.3.Histone acetylation is involved in chromatin structure remodeling and gene expression regulation.Seventeen histone lysine acetylation sites were identified from the comparative acetylome analysis of kenaf anther of CMS line UG93 A and its maintain line UG93 B.Western Blot analysis result showed that H3K9,H3K27 and H4K5 were differentially acetylated between UG93 A and UG93 B,indicating that the acetylation of H3K9,H3K27 and H4K5 were involved in the occurrence of kenaf CMS.4.Six Hc HDACs were cloned and identified in kenaf.Bioinformatics,spatiotemporal expression and subcellular localization of the six Hc HDACs were analyzed.The six Hc HDACs genes were expressed in roots,stems,leaves and anther of CMS line UG93 A and its maintain line UG93 B.This indicated that Hc HDACs were widely involved in kenaf growth and development.At the mononuclear stage and dual-core stage of anther,the expression of Hc HDA19 in UG93 A was all significantly higher than that of UG93 B.Hc HDA2 and Hc HDA8 were localized in the nucleus,Hc HDA6 and Hc HDA19 in the nucleus and cytoplasm,and Hc HDA8 in the nucleus and cell membrane.Different subcellular localization of Hc HDACs implied their different biological functions.5.CRISPR-Cas9 gene editing vector and overexpression vector of Hc HDA19 were constructed for transgenic verification.CRISPR-Cas9 gene editing vector was transformed to kenaf using acupuncture-vacuum osmotic assisted agrobacterium-mediated method,but the target sites of Hc HDA19 were not edited in the positive transgenic plants.Overexpression of Hc HDA19 had no effect on the pollen fertility of tobacco and Arabidopsis.Hc HDA19 can affect flowering time by regulating the expression of FLC.In Arabidopsis,salt stresstolerance was increased by the deacetylation of H3K27 and H3K9,which regulated the expression of genes related to salt tolerance in Hc HDA19 overexpression lines.