| Cotton(Gossypium hirsutum L.)is an important worldwide economic crop and raw material for the textile industry.China cotton industry is basically critical to the development of national economy and the guarantee of citizens' lives because of the large quantity of production and demanding of cotton.However,premature senescence has been more and more serious along with the wide adoption of numerous high-yielding Bt cotton cultivars since the twenty-first century,which negatively affected cotton productivity and fiber quality.Premature senescence is a type of leaf aging.So it is extremely important to study the underlying mechanism of cotton leaf senescence to realize the normal maturity and improve yield and quality of cotton.Sink and source relationship influences leaf senescence and maturity of cotton.And sink/source ratio is a critical indicator of the sink and source relationship.Normal growth and development of cotton need a coordination of source and sink,and altered sink/source ratio could result in a series of differential expression of senescence associated genes and finally cause abnormal maturity like premeature senescence.In this study,two cotton lines L(late-senescence line)and E(early-senescence line)with the same genetic background but different sink/source ratio and leaf senescence were grown in cotton fields,and the following examination were carried out from 2014 to 2016.At first,senescence performance(maturity)and sink/source ratio of two cotton lines were compared to reveal their relationship.The leaves of L and E line were used for gene expression analysis through solexa sequencing method to screen out the differentially expressed leaf senescence associated genes.The expression patterns of some senescence related genes and transcription factors were investigated.On this basis,the ABA receptor GhPYL8 gene was cloned from two cotton lines and its function gene was studied using the VIGS.Secondly,sink/source ratio of two cotton lines was altered by removing 2 or 4 early fruiting branches at squaring and the underlying mechanism that sink/source ratio regulates leaf senescence was further studied from the perspective of sink-source relationship,endogenous hormone level and expression of senescence related genes.The main results and conclusions are as follows:1.Varying sink/source ratio resulted in different leaf senescence between two cotton lines(1)The sink/source ratio was different between L and E line.From squaring to boll-setting stage,the sink/source ratio of E line was always higher than that of the L line,and the difference was even greater at the middle and late growing stages.At 110 and 125 days after planting(DAP),the sink/source ratio of the E line was 61.5%and 21.6%higher than that of the L line,respectively.(2)The leaf senescence between L and E line was significantly different.Although the concentration of total chlorophyll(Ch1)in the main-stem leaves of both lines increased at the beginning and then decreased,the difference between them was considerable.At 110 and 125 DAP,compared with that of L line,the Ch1 content of E line was reduced by 23.5%and 19.2%;net photosynthetic(Pn)rate was reduced by 29.3%and 29.9%;the MDA content increased by 42.4%and 51%.At 95,110 and 125 DAP,the abscisic acid(ABA)content of E line leaves was increased by 11.9%,10.9%and 7.8%relative to L line,respectively;the cytokinin content was decreased by 10.5%,10.3%and 8.2%,respectively.(3)Senescence difference between two cotton lines was associated with differential expression of senescence related genes.At 88 and 106 DAP,digital gene expression profiling(DGE)were performed using the leaves of L and E.At 106 DAP,we obtained the differentially expressed genes of E and L line using leaves of two cotton lines at 88 DAP as control.And we also obtained the differentially expressed genes of E line using leaves of L line as control at 88 and 106 DAP.The DGE results showed that most differentially expressed genes were those associated with photosynthesis,metabolism and plant hormone signal transduction pathway.A large number of differentially expressed transcription factor genes were found in two cotton lines along with leaf senescence.Thirty-two of 34 differentially expressed WRKY transcription factors were determined to be up-regulated during leaf senescence,and twenty-three of 28 NAC transcription factors were up-regulated,indicating that WRKY and NAC transcription factors might play crucial roles in leaf senescence.And hormone signal transduction related genes such as ABA,JA,SA and GA signal transduction pathways were up-regulated,while a large number of CTKs,BR and Auxin related genes were down-regulated.Analysis with qRT-PCR indicated that the expression level of ABA catabolic genes CYPA707A1/A4 was down-regulated while ABA biosynthesis genes NCED5/9 were upregulated,suggesting that ABA play an important role in promoting cotton leaf senescence.(4)GhPYL8 gene is involved in the regulation of cotton senescence.Three ABA receptors(PYL)were up-regulated with the DGE results in two cotton lines along with leaf senescence.But the expression level of three PYL in the E line was lower than that in L line,suggesting that three PYL not only played a role in regulating leaf senescence,but also possibly contributed to the senescence difference between L and E line.The function of one PYL genes(GhPYL8)in two cotton lines was claimed by virus-induced gene silencing(VIGS).The chlorophyll content in GhPYL8 silencing cotton was reduced,but the MDA content was increased under dark treatment.GhPYL8 gene is involved in the regulation of cotton senescence.Gene cloning and sequence alignment found that GhPYL8 gene in the two lines contained three base differences.And only the 223rd base mutation of GhPYL8 gene in L resulted in the change of amino acid.Further studies on the involvement of GhPYL8 gene mutation in the senescence of L line may help to reveal the senescence difference between the two cotton lines.2.Removal of early fruiting branches reduced sink/source ratio and delayed leaf senescence.(1)Removal of early fruiting branches(FB)reduced sink/source ratio of L and E line.Sink/source ratio was significantly reduced under removal early fruiting branches in both years.Averaged across two years,In L line,sink/source ratio decreased by 10.1 and 17.4%with 2 FB removal and by 53.7 and 34.8%with 4 FB removal at 50 and 65 DAR(days after removal).In E line,the sink/source ratio decreased by 42.4 and 5.7%with 2 FB removal and by 80.1 and 16.4%with 4 FB removal.(2)Removal of early fruiting branches lead to delayed leaf senescence of two cotton lines.In L line,compared to none FB removal control(LRO),the Ch1 content and Pn rate increased by 8.5 and 11.6%,but MDA content decreased by 7.8%with 2 FB removal;the Pn rate and Ch1 content increased by 10.5 and 19.6%,but MDA content decreased by 14%with 4 FB removal at 65 DAR.In E line,compared to none FB removal control(ERO),the Ch1 content and Pn rate increased by 5.8%and 12.1%,but MDA content decreased by 7.9%with 2 FB removal;the Pn rate and Ch1 content increased by 19.8 and 25.1%,but MDA content decreased by 18.4%with 4 FB removal at 65 DAR.In L line,removal of 2FB decreased the ABA content by 7.2 and 10.1%while removal of 4 FB significantly decreased it by 13.7 and 15.6%at 50 and 65 DAR,respectively.In E line,ABA content was reduced by 15.9 and 13.3%with 2 FB removed and 25.1 and 31.5%with 4 FB removed.The iP + iPA content in L line increased by 7.4 and 12.9%with removal of 2 FB,and 10.4 and 16.3%with removal of 4 FB at 50 and 65 DAR.In E line,the iP + iPA content increased by 8.5%and 12.1%with removal of 2 FB,and 13.2 and 11.1%with removal of 4 FB,respectively.The leaf JA content in L line was significantly decreased by 6.8 and 9.1%at 50 and 65 DAR with removal of 2 FB,and 12.1 and 13.5%with removal of 4 FB.For E line,the JA content significantly decreased by 13.7 and 11.7%with removal of 2 FB and 20 and 24%with removal of 4 FB at 50 and 65 DAR.(3)Removal of early fruiting branches delayed leaf senescence via differential expression of the senescence associated genes.The expression of GhLHCB(Chlorophyll binding protein)was up-regulated by 1.18-1.65 folds with removal of 2 FB and 1.21-2.35 folds with removal of 4 FB from 5 to 65 DAR in the late senescence line.In the early-senescence cotton,expression of GhLHCB was up-regulated by 1.15-9.4 and 1.21-10.7 folds with removal of 2 and 4 FB from 5 to 65 DAR,respectively.The expressions of NCED2,NCED5 and NCED9 genes in L line leaves were down-regulated by 39,17 and 9.1%at 65 DAR with removal of 2 FB,and 75,87 and 95%with removal of 4 FB.The expressions of NCED2,NCED5 and NCED9 genes in E line leaves were down-regulated by 95,8 and 74%at 65 DAR with removal of 2 FB,and 75,36 and 63%with removal of 4 FB.The expression of three ABA catabolic genes(CYP707A1,CYP707A2 and CYP707A4)were increased by removal of early fruiting branches,in which their expression under with 4 FB removal was higher than in those under 2 FB removalThe expressions of CYP707A1,CYP707A2 and CYP707A4 genes in L line leaves were up-regulated by 1.2,1.2 and 4.1 folds with removal of 2 FB relative to none FB removal at 50 DAR,and 1.3,2,0 and 9.1 folds with removal of 4 FB,The expressions of CYP707A1,CYP707A2 and CYP707A4 genes in E line leaves were up-regulated by 3.5,1.6 and 1.1 folds with removal of 2 FB at 50 DAR,and 2.8,16.6 and 1.5 folds with removal of 4 FB.The expression of JA biosynthesis genes(LOX,AOS and AOC genes)in L line were down-regulated by 57,78 and 98%at 50 DAR with removal of 2 FB,and 78,91 and 98%with removal of 4 FB.The expression of LOX,AOS and AOC genes in E line were down-regulated by 56,26 and 27%at 50 DAR with removal of 2 FB,and 76,42 and 67%with removal of 4 FB.The expression level of LOX,AOS and AOC was down-regulated with removal of FB at 35 and 50 DAR.(4)Effects of removal of early fruiting branches on cotton yield varied with genotype and the number of FB removed.In late-senescence line,seed cotton yield was reduced by 8.7 and 6.4%in plants with 2 FB removed and by 15 and 12%with 4 FB removed in 2015 and 2016.In the early-senescence line,it was significantly reduced by 5.7 and 11.7%with 4 FB removed,but was increased by 10.2 and 6.3%in those with 2 FB removed in 2015 and 2016.In L line,the boll weight increased by 6.8 and 11.9%,the boll density decreased by 14.5 and 16.2%with 2 FB removal in 2015 and 2016;the boll weight increased by 3 and 11.1%and the boll density decreased by 15.9 and 20.6%with 4 FB removed.In E line,the boll weight increased by 14.9 and 8.9%,the boll density decreased by 4.1 and 2.3%with 2 FB removal in 2015 and 2016;boll weight increased by 22.7 and 8%and boll density decreased by 31.7 and 11.7%with 4 FB removed.The harvest index was reduced by 7.2 and 11.2%in L line with 2 and 4 FB removed.In E line,removal of 4 FB reduced the harvest index by 9.8%,but removal of 2 FB did not change the harvest index.Earliness(percentage of the first harvest in seed cotton weight)was decreased by 17.9 and 47%in L line with 2 and 4 FB removed.Although it was decreased by 37.9%following removal of 4 FB,removal of 2 FB did not affect the earliness in E line.In conclusion,there were significant differences in the sink/source ratio and chlorophyll content,Pn rate and MDA content of main-stem functional leaves in two cotton lines,indicating that the leaf senescence was different between two lines.Such a difference in leaf senescence was related to different sink-source ratio.The DGE results showed that chlorophyll degradated,photosynthesis-related genes were down-regulated,and the ability to assimilate decreased;macromolecules metabolism ability increased and the ability of dissimilation increased in both L and E line during leaf senescence.And most genes associated with plant hormone signal transduction pathway were involved in the leaf senescence,especially the ABA signal pathway.GhPYL8(one of ABA receptor)gene silencing lead to delayed leaf senescence,suggesting that GhPYL8 might played a positive role in regulating cotton leaf senescence.Removal of early fruiting branches decreased the sink/source ratio in two cotton lines,resulted in reduced Chl contents and Pn rate and accumulation of MDA,further suggesting that decreased sink/source ratio delayed leaf senescence.Removal of early fruiting branches resulted in differential expression of associated gene expressions,such as GhLHCB,ABA and JA biosynthesis genes,which may be attributed to increased iP+iPA and/or reduced ABA and JA contents,increased the Chl content and Pn rate and reduced the MDA content.Removal of early fruiting branches delayed leaf senescence.Removal of 2 FB in the early-senescence line delayed main-stem leaf senescence to achieve the normal maturity and increased cotton yield by 8.3%;removal of 2 FB in late-senescence line and 4 FB in both lines caused late-maturity and significantly reduced yield.It thus can be concluded that removal of early fruiting branches is an efficient practice to regulate sink/source relationship and suppress leaf senescence in cotton.However,delaying leaf senescence does not always increase cotton yield.Yield can be improved only when the sink/source ratio is appropriate and the sink/source relationship is well coordinated to realize a normal maturity. |
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