| Male sterility caused by high temperature(HT)stress widely exists in plant,which is also an important factor for the abscission of cotton squares and bolls.Under the worse global warming situation,the increased duration and frequency of HT has extremely influenced the yield and fiber quality of cotton.Long-term HT stress affects the development of cotton anther leading to the male sterility like anther indehiscence,shorter filaments and pollen abortion.Although the male sterility caused by HT has been found earlier,the molecular basis and working principles still remain unclear.In fact,the majority of cotton varieties used in cotton production were vulnerable to HT stress,thus produce the HT resistance cotton cultivars is valuable for cotton production.In this study,small RNA sequencing was applied with anthers of two developmental stages from HT-tolerance(84021)and –sensitive(H05)cotton cultivars under NT and HT conditions.Lots of small RNAs were identified,and miRNA,a class of non-coding small RNA,was chosen for deep study.Firstly,a large amount of miRNAs who responded to HT stress were screened out,and then the sequencing accuracy was confirmed by qRT-PCR.Subsequently,the degradome sequencing technology was used for identifying the target genes of miRNA.Furthermore,to identify the function of miRNAs in cotton male sterility under HT stress,two miRNAs(miR157 and miR160)were for functional identification during the formation of male sterility under NT and HT conditions.The main results are showing as follows:Under normal growth conditions,the time from a young cotton bud(3-4 mm)to flowering nearly cost 25 days.Cotton anthers develop through TS stage need 2-3 days,and have 17-19 days to flowering;the TDS stage cost nearly a week from 6-13 days before flowering,and ADS stage lasts 2 days just before flowering.Interestingly,when TDS stage was nearly completed,the growth speed of cotton bud was stopped nearly 1day and then fast increased until flowering.For the male sterility was occurred in HT-sensitive cotton cultivar after 1 week HT stress,thus we speculate that the sensitive anther development stage might be TS and TDS stage according to this bud growth curve.A total of 16 samples(containing 2 biological repeats)from TS and TDS stages’ anthers of HT-tolerance and-sensitive cotton cultivars ‘84021’ and ‘H05’ under NT andHT conditions were used for small RNA sequencing.From small RNA sequencing data,112 known and 270 novel miRNAs were identified,and 27 miRNAs which response to HT stress were obtained according to the expression level and the ratio of fold changes between 84021 and H05.qRT-PCR was used for proving the accuracy of data from small RNA sequencing and expressing analysis of miRNAs which response to HT stress.At last,a series of HT response miRNAs were identified such as miR156/157,miR160,miR167 and miR172 etc.,which supported the database for further researches about miRNAs’ function in male sterility under HT stress.For identifying the target genes of miRNAs,degradome sequencing technology was used in our study.A total of 4 degradation libraries were constructed and 347 target genes from 76 miRNA with 528 cleavage sites were identified finally.RLM-RACE and qRT-PCR were used to confirm the data accuracy from degradome sequencing.Results showed that miRNA can cleave the target genes at predicted cleavage site and the expression trend of miRNA and its corresponding targets were opposite.A large scale of target genes related HT stress response and anther development were identified through degradome sequencing,which were benefitted for our later miRNA function analysis.In this study,overexpressed miR160 in cotton resulted in the lower expression level of its target genes including ARF10 and ARF17,and which enhanced the anther sensitivity to HT stress for anthers of miR160 overexpression cotton line showed indehiscence after HT stress for 5 days,but the pollen grains kept fertility.To further confirm the relationship between miR160 and auxin signal,exogenous IAA experiment then was performed with 84021 and H05 under NT and HT conditions.The results showed that exogenous IAA cause the male sterility of 84021 and worse performance of H05 under HT stress.And the expression of miR160 and its targets were both found effected by exogenous IAA and HT stress,indicating miR160 involved in regulation of auxin signal.Further studies confirmed that the expression of CALLOSE SYNTHASE5(Cals5),the major gene for callose biosynthesis,had no obvious changes between miR160 OE line and null control under NT or HT condition alone using anthers from TS stage,corresponding to the fertility pollen grains in anthers of miR160 OE line under HT stress for 5 d.Moreover,the expression level of JA biosynthesis related genes like DAD1,OPR3 and LOX4 were found lower than null control under HT stress,which might be the reason for anther indehiscence for miR160 OE line.Thus we speculated that overexpression of miR160 suppresses the expression level of auxin-repressed-type ARFs(ARF10,17),which causes activation of auxin signalling and suppresses JA biosynthesis,and ultimately leads to anther indehiscence under HT stress.Under normal conditions,miR157 overexpression cotton has smaller plant organ including leaf and flower buds,the anther number is decreased and shows earlier flowering.The anther of miR157 overexpression cotton lines also showed sensitivity to HT stress with anther dehiscence and pollen abortion.The expression analysis showed that miR157 was highly induced by HT stress and much higher expression level was found in miR157 overexpression cotton anthers.SPLs,the target genes of miR157 showed opposite expression trends.Besides,the expression of YUC5,auxin biosynthesis gene,was found suppressed in miR157 overexpression cotton anthers.Combining the conclusion that the activation of auxin signal in miR160 overexpression line leaded anther indehiscence under HT stress,we speculated that the auxin signal balance in anther might be essential for cotton anther fertility. |
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