Transcriptome Profiling Of Anther Development Under High-temperature Stress And Characterization Of GhCKI In Cotton

With the global warming, the developmental process of male reproductive organ in crops was threatened by high temperature (HT) injury and lead to male sterility, which was one of the main limited factors for crop production. In this study, transcriptome profiling of anthers from different developmental periods of treated HT-tolerance and HT-sensitive cotton lines under normal temperature (NT) and HT were analyzed by RNA-Seq. A number of anther HT response related genes were identified through differentially screen. From the transcriptome profiling, a casein kinase I (GhCKI) gene was isolated and characterized. The main results of this study were as follows:Cytological analysis revealed there were obvious differences between the anther development of two upland cotton lines (84021, HT-tolerant; H05, HT-sensitive) under under NT (28-35℃/20-28℃ day/night) and HT (35-39℃/29-31℃ day/night) conditions. The key difference between the two lines is associated with anther dehiscence; anthers from 84021 split normally, whereas those from H05 were indehiscent. Comparative transcriptome using 84021 and H05 at three key development stages of anther (tetrads stage, tapetal degradation stage, and anther dehiscence stage) under NT and HT conditions was analysed by RNA-Seq.5,194 differentially expressed genes (DEGs), which might be responsible to HTS tolerance, were identified. Among these,4,599 DEGs showed different expression patterns induced by HT in two cotton lines at different anther development stages. KEGG analysis showed that these DEGs were enriched in chromatin modifications, carbohydrate metabolism, plant hormone signaling, and endoplasmic reticulum (ER) stress pathways. The genes responsible for DNA methylation and histone modifications displayed significant changes in HT-sensitive (H05) line. The results indicated that HT active more differentially expressed genes (4004) in H05 than in 84021.According to gene expression profiling, sugar metabolism, ER stress response, and auxin metabolism and siganl transduction pathways played important roles in anther development under HT. Compared with 84021, at the analysis of the enzyme activities (AGPase, SSSase, GBSSase, SOD, and POD), the content of various substances (starch, sucrose, glucose, soluble protein, and H2O2) revealed that HT stress disordered the sugar metabolism in anthers of H05, and led to H05 sufferring from serious ER stress. Furthermore, exogenous IAA application before HT treatment resulted in indehiscence of some 84021 anthers. Meanwhile, the IAA content of anthers of 84021 and H05 at different developmental stages before and after HT treatment was measured by HPLC-MS. And the IAA content of H05 was significantly higher than 84021, which consistent with were supported by the altered expression levels of the genes responsible for auxin biosynthesis and metabolism. The results suggested higher background IAA levels made anther more sensitive to HT stress. In order to understand the mechanism of anther abortion under HT stress. we isolated and characterized a GhCKI gene from the transcriptome profile which was differentially expressed in H05. GhCKI encoded a homolog of casein kinase I (CKI). It was homologous to highly conserved serine/threonine protein kinase found in most eukaryotic cells. Expression patterns showed that the expression of GhCKI was induced by HT. It was expressed in the tapetum and microspores at earler stages in H05 after HT treatment. Overexpression of GhCKI caused anther abortion in Arabidopsis. Histological and cytological observation of the anther in GhCKI overexpression plants showed that male sterility was caused by disrupted tapetal programmed cell death (PCD), and finally resulted in delayed pollen development and anther indehiscence. Western blotting, qRT-PCR, and immunoblotting results showed that the male sterility of the GhCKI-overexpression lines was dose-dependent. RNA in situ hybridization revealed that, high expression of GhCKI in taperum cells and microspore, which led to male sterility in GACKI-overexpression Arabidopsis, was consisted with the induced expression of GhCKI in taperum cells and microspore in H05 under HT.To explore the molecular mechanisms underlying the male sterility caused by GhCKI, Affymetrix microarray was used to identify possible downstream genes and metabolic networks. The results showed that carbohydrate metabolism, ABA synthesis and metabolism, and ROS generation and scavenging pathways have significantly changed. The interaction of GhCKI and starch synthase was confirmed by yeast two hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC). GhCKI inhibited the activity of starch synthase kinase activity, resulting in the accumulation of glucose (Glc) in early stage buds. Excessive accumulation of GhCKI in early stage buds, led to feedback inhibition of assimilation and induced starch storage processes, which reduced the glucose content of late anthers. Moreover, abscisic acid (ABA) was accumulated in GhCKI-overexpression Arabidopsis, thereby disturbing the balance of reactive oxygen species (ROS) and eventually disrupting tapetal PCD, then finally leading to anther abortion or indehiscence. These results enriched the mechanism of cotton anther response to high temperature stress, providing genetic resources and theoretical basis for cotton HT tolerance research, as well as providing some important results for understanding the molecular mechanism of cotton male sterility, with the potential to facilitate the regulation of HT tolerance in crops.In this study, we also found that GhCKI participated in numerous physiological processes in cotton. Preliminary results showed GhCKI overexpression inhibited cotton somatic embryogenesis, suggested GhCKI invovled in the regulation of cotton somatic embryogenesis may be associate with cell clonal proliferation and differentiation, similar to the animals CKI play roles in Wnt pathway. In addition, we found GhCKI efficient expression in early-stage fiber, and the fiber of GhCKI silent transgenic plant was significantly inhibited. However, further study was needed to inverstigate the the regulation mechanism.