| Cotton is one of the most important cash crops in the world providing raw materials to the textile industry.China is one of the leading cotton producers,and importers in the world.Mechanical picking has already become a trend of cotton production in China.Chemical defoliation plays a crucial role in the mechanical picking of cotton but,there are some problems in chemical defoliation,such as non-falling of the withered leaf,and poor defoliation due to low temperature,etc.However,the physiological and molecular responses of cotton to chemical defoliant remain unclear.Therefore,it is of great significance to clarify the mechanism of chemical defoliants promoting cotton leaf shedding to improve the application technology and the effect of chemical defoliants and subsequently cotton leaf shedding.The current study was conducted to elucidate the physiological and molecular responses to leaf abscission induced by defoliants(active ingredient=thidiazuron)through the integration of morphology,physiology,transcriptome,miRNA,degradome,metabolome,and bioinformatics analysis.To study the physiological mechanism of chemical defoliation,cotton varieties CRI 49(leaf abscission insensitive variety)and CRI 12(leaf abscission sensitive variety)were sprayed thidiazuron(0 and 100 mg/L)at eight leaf stage in the greenhouse,and subsequently,the leaf photosynthesis,carbon metabolism and antioxidant metabolism as well as in the leaf and abscission zone structure,were studied.Simultaneously,the leaves of CRI 12 were collected for transcriptome and metabolome analysis to identify the genes and metabolites responsible for leaf abscission.In addition,the leaf abscission,boll opening,yield,fiber quality and physiological indexes of CRI 49 and CRI 12 were studied in a field experiment(2019-2020).The main results are as follows:(1)After treating with thidiazuron,the leaves become dry,purple spots appeared on them,and the abscission zone rapidly started followed by leaf shedding in both CRI 49and CRI 12.Scanning electron microscope observation showed that the epidermal structure of the leaf was destroyed.A significant increase in malondialdehyde and reactive oxygen species(ROS)and imbalance in antioxidant enzyme activity was observed.At 48h of the treatment,the pigments increased,but the ratio of chlorophyll a/b,the net photosynthetic rate and photosynthate decreased.Correlation analysis showed that leaf abscission was positively correlated with malondialdehyde,ROS and pigment,while negatively correlated with leaf photosynthetic traits.The increase in ROS due to the application of thidiazuron destroyed leaf structure,and inhibited photosynthesis and carbon metabolism.(2)For transcriptome sequencing(mRNA),the samples of CRI 12 were collected at6,12,24,and 48 h after thidiazuron treatment.In total,27,534 differentially expressed genes(DEGs)were obtained.The DEGs were involved in pigments,photosynthesis-related systems,carbohydrate-binding,systemic acquired resistance,redox processes and hormone responses(ethylene,cytokinin,salicylic acid,jasmonic acid,gibberellic acid and abscisic acid,etc.).Weighted gene co-expression network analysis identified 15 gene modules related to thidiazuron-induced leaf abscission,of which 5 modules(MEpink,MEgreen,MEturquoise,MEblue and MEbrown)are highly related to photosynthetic characteristics that may play an important role in leaf shedding.(3)The combination of physiological and transcriptomic analysis suggests that 24h after treatment is important for leaf shedding response induced by thidiazuron.Therefore,we performed miRNA sequencing analysis after 24h of thidiazuron treatment,and found59 differentially expressed miRNAs,of which 34 were up-regulated and 25down-regulated(p<0.05).The combination of transcriptome,miRNA and degradome sequencing showed 36 differentially expressed miRNAs and 339 target genes(mRNAs)showed a negative regulatory relationship.GO enrichment of target gene regulated by miRNA mainly involves glycine cleavage complex;response to salicylic acid,cold,jasmonic acid;chloroplast and photosynthesis-related pathway,etc.The combined analysis of miRNA and weighted gene co-expression network analysis revealed that MEgreen(p=1.45×10-5)and MEturquoise(p=9.15×10-5)modules were significantly regulated by miRNA.The miRNA is involved in leaf abscission by regulating its target genes related to photosynthesis,photosynthetic product and transport,transcription factor of WRKY,calcium ion and ethylene.(4)The metabolome analysis of 24h with thidiazuron treatment showed that there are 48 MS2 differential metabolites,of which 32 are up-regulate accumulated and 16down-regulate.The accumulation of thidiazuron,ethylene precursors,phenylalanine and salicylic acid increased,while cytokinin and gibberellin were significantly decreased.The integrated analysis of the transcriptome and metabolome found that genes related to ethylene,cytokinin,and salicylic acid were involved in leaf abscission.It is confirmed for the first time that salicylic acid participates in the leaf abscission induced by thidiazuron.However,the interaction mechanism of ethylene,cytokinin,salicylic acid and other plant hormones in the process of leaf shedding still needs further study.(5)In the field experiment,defoliants application of the appropriate period increased the abscission and boll opening rate,but did not affect cotton yield and fiber quality.In combination with physiological and molecular mechanism study,a thidiazuron compound agent(thidiazuron and hydrogen peroxide)was formulated.Although the defoliation effect of the compound is slightly lower than that of mature defoliants,this provides a new idea(theory is transformed into practice)for the production of efficient defoliants.In summary,defoliants mainly perturbed hormone balance,caused ROS excessive accumulation,destroyed leaf structure,reduced photosynthesis and carbohydrate accumulation in leaves.Finally,the abscission zone perceived changes in the leaf blade and leaf shed. |
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