Differential Response Of Cotton To Waterlogging At Different Growth And Development Stages And The Underlying Physiological Mechanism

Waterlogging is a major hazardous abiotic stress which has become increasingly frequent due to global warming.Cotton is poorly adapted to waterlogging stress and usually suffers from waterlogging stress at boll-setting stage.However,in recent years,waterlogging events has been greatly exacerbated by climate change,which may occur at any growth and development stage of cotton.The effects of waterlogging at boll-setting on cotton growth and yield are well documented,but it is not determined how cotton plants deferentially respond to waterlogging at different growth stages,and the underlying mechanism also remains unclear.In this study,the experiments were conducted using a commencial cotton(Gossypiym hirsutum L)variety K638 in an electrically powered rain-shelter at Experimental Station of Shandong Cotton Research Center at Linqing in 2014 and 2015.Cotton was subjected to 10-20 day-waterlogging at squaring,flowering or boll-setting stage.The effects of waterlogging at each growth stage on cotton yield and yield components were studied.On this basis,the physiological mechanism was investigated by examining the changes in photosynthesis,activity of antioxidant enzyme,anaerobic enzymes,levels of endogenous hormones and carbon and nitrogen metabolism in stressed cotton plants.In addition,the leaves of 15-d waterlogging treatment and non-waterlogged control at flowering stage were used for gene expression analysis through solexa sequencing method to screen out the differentially expressed genes of cotton leaves under waterlogging stress.GO function terms and KEGG enrichment pathway responded to waterlogging stress were analyzed.The differentially expressed hormone-related genes and transcription factors were identified.And the expression of genes related to anaerobic metabolism,hormone signal and several ERF genes were detected by real-time quantitative PCR.Moreover,the indoor-simulated waterlogging experiment was also carried out to study the morphological changes of cotton roots under waterlogging stress.The main results and conclusions are as follows:1.Differential response of cotton yield and yield components to temporal waterlogging stress.Cotton yield and yield components were significantly affected by waterlogging duration.Averaged across the two years,after waterlogged for 10,15 and 20 d,the lint yield was reduced by 31.2,37.6 and 47.4%relative to non-waterlogging control;biological yield was reduced by 20.5,25.5 and 30.2%;harvest index was reduced by 13.2,20.0 and 28.8%;and the number of bolls per ground area(boll density)was reduced by 33.8,36.0 and 46.0%.Cotton yield and yield components were also significantly affected by waterlogging at squaring,flowering and boll-setting stage(different growth stages).Averaged across the two years,after waterlogging at squaring,flowering and full-boll stage,the lint yield was reduced by 58.3,39.7 and 17.4%relative to non-waterlogging control;biological yield was reduced by 49.8,25.3 and 1.9%;harvest index was reduced by 15.2,30.6 and 15.7%;boll density was reduced by 53.5,40.5 and 18.8%.These data indicated that 10?20 day-waterlogging stress at different growth stages had a marked inhibitory effect on cotton yield.On the one hand,cotton yield varied with waterlogging duration.The longer the duration,the larger the yield loss.On the other hand,yield reduction was more pronounced with waterlogging at squaring than at flowering or boll-setting stage,showing different adaptability of cotton to waterlogging at different growth stages.Younger cotton plants were more sensitive to waterlogging than fully grown and developed plants.Such a difference in sensitivity was due to more reductions in the biological yield and the harvest index under waterlogging at squaring than that at flowering and boll-setting stages.Thus,more reductions in biological yield and harvest index under earlier waterlogging than later waterlogging were important contributory factors to more pronounced inhibition of boll density and lint yield.2.Differentiall physiological responses of cotton to temporal waterlogging stress.Physiological traits of cotton were significantly affected by duration of waterlogging.Averaged across the two years,after being waterlogged for 10,15 and 20 d,relative to non-waterlogging control,concentration of total chlorophyll(Chl t)in the main stem leaves was reduced by 29.8,38.8 and 43.1%;net photosynthetic(Pn)rate was reduced by 28.2,43.3 and 55.3%;the MDA content in cotton leaves increased by 72.6,72.0 and 119%;the ADH activity was increased by 71.2,64.3 and 94.8%;the soluble protein content was decreased by 21.7,30.2 and 47.1%;similarly,the soluble sugar content was also reduced by 15.0,23.6 and 26.6%.Cotton plants accumulated 3.7,4.1 and 3.8 times more H2O2 after 10,15 and 20 d-waterlogging stress.These physiological traits were also significantly affected by waterlogging at squaring,flowering and boll-setting stage.Averaged across the two years,after waterlogged at squaring,flowering and full-boll stage,relative to non-waterlogging control,Chl t content in the main stem leaves was reduced by 45.9,43.0 and 24.7%;leaf Pn rate in waterlogged cotton was reduced by 65.4,32.4 and 29.8%;the H2O2 content in the main-stem leaves of waterlogged cotton was increased by 12.8,3.7 and 2.5 times;the MDA content in cotton leaves was increased by 65.1,32.7 and 29.5%;the ADH activity was increased by 43.2,46.4 and 40.9%;the soluble protein content was decreased by 39.2,20.0 and 35.6%;the soluble sugar content was also reduced by 29.4,13.1 and 20.9%.After 15 d-waterlogging at squaring,flowering and boll-setting stage,GA level in waterlogged cotton leaves was decreased by 68.1,57.0 and 22.0%;IAA decreased by 59.2,45.7 and 15.8%;ABA level increased by 59.2,44.3 and 18.3%.respectively.We found that waterlogging resulted in accumulation of H2O2 and MDA in the main-stem leaves and reductions in Chl contents and net photosynthetic rate.GA and IAA contents decreased and plant growth and development were also reduced after waterlogging.These changes might be contributory factors to reduction in cotton yield.Moreover,earlier waterlogging caused greater decreases in IAA and GA,Pn,soluble sugar and soluble protein,which were consistent with the difference in yield loss.And earlier waterlogging resulted in more accumulation of ABA,H2O2 and MDA in main-stem leaves than later waterlogging,which means earlier waterlogging imposed a greater damage to cell membranes than later waterlogging and resulted in greater reduction in plant growth as indicated by larger loss in biomass and biological yield.Thus,waterlogging inhibits cotton growth and yield through disturbance of many physiological functions.The younger plants were more poorly adapted to waterlogging than the fully grown or developed plants and the differential yield loss was attributed to the differential physiological changes.3.Differential expression of genes related to photosynthesis,anaerobic metabolism,ABA synthesis or other pathways explained the differentiall response of cotton to various timing of waterlogging.The results of DGE showed that 794 genes were up-regulated and 1018 genes were down-regulated in 15-d waterlogged cotton leaves compared with non-waterlogged control.KEGG pathway analysis indicated that most genes related to photosynthesis,flavonoid biosynthesis,oxidative phosphorylation,amino acid metabolism and biosynthesis as well as circadian rhythm pathways were differentially expressed.Many genes related to plant hormones and transcription factors were differentially expressed under waterlogging stress.Twenty-one of 27 differentially expressed hormone-related genes were up-regulated in waterlogged cotton compared with non-waterlogged control.Thirty-four waterlogging-regulated TFs were identified with 26 up-regulated and 8 down-regulated under waterlogging stress condition.These TFs including 13 ERFs,15 MYBs and 6 WRKYs with 9 ERF,13 MYB and 4 WRKY were up-regulated.As indicated by qRT-PCR analysis,after 10-,15-and 20-d waterlogging at squaring stage,the expression level of GhLHCB was down-regulated by 8.9,18.1 and 37.2 folds of non-waterlogging control;GhADH2a was up-regulated by 16.1,33.6 and 54.2 folds.After 10-,15-and 20-d waterlogging at flowering stage,the expression level of GhLHCB was down-regulated by 1.2,27.5 and 42.7 folds of non-waterlogging control;GhADH2a was up-regulated byl.8,3.1 and 4.4 folds.After 10-,15-and 20-d waterlogging at boll-setting stage,the expression level of GhLHCB was down-regulated by 1.4,9.2 and 11.4 folds of non-waterlogging control;GhADH2a was up-regulated by 0.9,1.6 and 2.0 folds.In addition,after 15 d-waterlogging at squaring,flowering and boll-setting stage,the ABA biosynthesis genes,9-cis-epoxycarotenoid dioxygenase(GhNCED2)was up regulated by 40.9,22.2 and 1.9 folds;GhNCED5 was up regulated by 37.6,16.6 and 8.2 folds.Earlier waterlogging resulted to greater down-regulation of GhLHCB expression and up-regulation of GhNCED expression than later waterlogging.Thus,gene expression differences from the solexa sequencing data and qRT-PCR method may illuminate the performance of cotton under waterlogging at the molecular level.It is suggested that differential regulation of genes might be an important reason for the different physiological responses to varied timing of waterlogging,and finally leads to various growth and yield penalties.4.Three approaches of cotton adaption to waterlogging conditionsWhen subject to waterlogging,the cotton plant undergoes a series of physiological and biochemical and molecular changes,leading to reduced energy loss through regulation of the growth and development;cotton plants also try to avoid waterlogging stress to a certain extent by generating adventitious roots;after removal of waterlogging,cotton plant accelerates its growth and development to compensate for the damage caused by waterlogging.When cotton plants suffered from various timing of waterlogging,the molecular changes were considered to be the first signal.Differential expression of genes controlling biochemical processes such as photosynthesis,glycolysis,fermentation,mitochondrial electron transport pathways and hormone synthesis or perception resulted in associated physiological changes.These variations finally lead to changes in cotton plant growth,biological yield and economic yield,suggesting that the response of cotton to various timing of waterlogging is a gradual process with different approaches and mechanisms.The combined physiological and molecular mechanism of waterlogging responses in cotton has yielded a series of approaches for cotton to protect itself from yield loss through adapting to waterlogging conditions.These waterlogging-adaptive approaches include escaping approach,quiescent adaptive approach and regeneration-compensation approach.The quiescence adaptive approach is a tolerant mechanism,characterized by adjustment of many processes of metabolism,which can minimize energy expenditures through a hormonal signaling cascade with higher levels of ABA and reduced GA,limited growth and conservation of carbohydrates.This approach was driven by the changed expression of genes associated with photosynthesis,anaerobic metabolism,phytohormone and some transcription factors.It occurs predominantly in cotton plants from waterlogging events and is the key trait for the adaptation in cotton under waterlogging.The escaping approach works on maintaining the root respiration by developing aerenchyma or adventitious roots,which consequently contributes to less yield loss under waterlogged conditions.As one of the plant species with indeterminate growth habit,cotton possessed a regeneration&compensation approach.By this approach,the recoveries of cotton growth and plant biomass would appear soon after waterlogging-removal through accelerating growth and growing new plant parts to compensate for the damage caused by waterlogging.Moreover,the recovery extents depend on waterlogging duration and timing.Through these adaptive approaches,cotton can adapt to the waterlogging environment or at least alleviate plant growth and yield loss.In conclusion,this study ascertained the differential response of cotton to waterlogging at different growth and development stages,revealed the underlying physiological mechanism and recommended three approaches of cotton adaption to waterlogging conditions.Our results provided the theoretical guidance and reference for genetic breeding of waterlogging-tolerant cotton variety and applying agronomic measures to alleviate waterlogging damage.