Comparative Investigation On Male Sterility Arisen By High Night And High Day Temperature In Cotton

Cotton is a highly valuable and important economic crop;its yield is highly affected when the high temperature(HT)stress happens.The effect of HT is more on the reproductive part of plants than on vegetative parts and in the reproductive part of the plant,male reproductive part is more sensitive to HT stresses.In our research in cotton,high night temperature affects more than the high day temperature and is one of the major factors in reducing cotton yield.The different HT stresses(Day and Night)may cause different male sterility phenotypes.So,the current study was conducted to unravel the possible role of high day and high night temperature in inducing male sterility.Histological,biochemical and transcriptomic analyses were performed on the anthers of cotton(H05,HT sensitive line)under HT stress.H05 was treated with HT stress at different conditions,i.e.High day and Normal night(HN),Normal day and High night(NH)and High day and High night(HH)to find out differences of HT effects on the three anther developmental stages(Tetrad stage,TS: 6-7 mm;Tapetal Degradation stage,TDS: 9-14 mm;and Anther Dehiscence stage,ADS: >24 mm),the Normal day and Normal night(NN)treatment was used as control condition.The results were summarized as followings:1)In the phenotypic study,at ADS,HT induced a significant reduction in flower size,filament length and pollen sterility in NH condition more than in HN,and HH showed the most severe phenotype disruption.In cellular study,at TDS and ADS,the microspores and pollen were more shrivel in NH treatment than in HN but less than in HH.2)In the transcriptomic study,40895 differentially expressed genes(DEGs)were screened out in the three stages of the treated anthers.At TDS and ADS,more genes were found common in HH and NH condition than in HN,and the number of genes affected in NH was more than in HN.3)In the up-regulated gene enrichment plots,at ADS,it shows that Alpha-linolenic acid metabolism possibly has a role in pollen abortion because it is highly enriched in HH and NH conditions than in HN.In HH,Linoleic acid metabolism and sphingolipid metabolism were highly enriched than in NH,showing that these pathways might have a role in anther indehiscence.Likewise,in the down-regulated enrichment at ADS,the only common pathway found in HH and NH is pyruvate metabolism.So,pyruvate metabolism may have a role in pollen abortion.4)To observe the lipid distribution and amount in anthers,TEM studies were done.In TEM,at TDS and ADS,the number of lipid drops decreased in all conditions(HH,HN & NH).Moreover,at ADS,the intine layer was vanished in NH and HH conditions.5)In the pathway analysis,four genes,ACC1,KAS1,NAD(P)-BRFSFP,AMP-DS & LFP at fatty acid biosynthesis pathway were differently expressed;three genes in fatty acid degradation pathway,ACO1,ACO2,and KAT3 were also expressed differently in the RNAseq.The differential expression of these genes was confirmed by q RT-PCR.The fatty acids(C14,C16,C18:0,C18:1,C18:2 and C18:3)were measured and found that fatty acid was decreased in all stages under HT conditions.6)Fatty acid biosynthesis and fatty acid degradation pathways enter the alphalinolenic acid pathway and end with a Jasmonic acid(JA).The expression of four genes,AOS,AOC4,ACO2,and MFP,was induced by HT at JA pathway.The expression of these genes was also confirmed by q RT-PCR.In the hormone quantification analysis,the amount of JA content was decreased in all conditions but more significant reduction was found in NH and HH than in HN.7)To further study the role of JA in HT-induced male sterility,the genes Gh AOC4(downregulated in HN condition)and Gh ACO2(shows down-regulation in NH conditions)in the JA pathway were knocked out through CRISPR technique using the protocol established in our laboratory.The regenerated plants showed that the size of the mutant flowers was smaller,anthers were indehiscence and the pollens were sterile as compared to the WT flowers,anthers,and pollens.In the cellular study,at TDS and ADS,microspores and pollen grains were found shrivel in all mutant lines anthers.8)To confirm JA affects pollen fertility,exogenous application of methyl jasmonate(Me JA)on mutant plants buds(contained TS or TDS anthers in them)was performed,and found that it can restore the sterile pollen and indehiscence anther phenotypes.To summarize,the current study revealed that lipid,fatty acid and JA may coordinately regulate plant anther responses to HT stress.The night HT affects more lipid metabolisms on the anthers and creates male sterility than the day HT.This study further improved the understanding of HT-induced male sterility in cotton,and which might provide useful reference in cotton HT-tolerance breeding.