| A spontaneous programmed cell death（PCD）process is triggered when the plant enters the final stage of growth and development,which is called senescence.Senescence is a survival mechanism formed during the long evolution process to adapt to the environment,which has a significant impact on plant adaptability and biomass.Cotton is the most important cash crops in the world and the premature senescence of leaves is one of the important reasons for seed cotton yield reduction.Previous studies have shown that phytohormone ethylene and its key transcription factor ETHYLENE INSENSITIVE3（EIN3）/ETHYLENE INSENSITIVE-LIKE（EIL）in ethylene signal transduction pathway are involved in the process of plant leaf senescence.Therefore,exploring the molecular mechanism of EIN3/EIL transcription factor regulating cotton leaf senescence can lay a molecular foundation for genetic engineering to improve seed cotton yield.This paper focuses on one LINT YIELD INCREASING gene designated as GhLYI,a special truncated EIN3/EIL family member associated with increased lint yield in improved cultivars in upland cotton.Main results are presented as following.1.By bioinformatics analysis,19、19、9 and 11 EIN3/EIL family members were identified in G.hirsutum,G.barbadense,G.arboreum and G.raimondii,respectively.Allotetraploid cotton G.hirsutum had three truncated EIN3/EIL copies,but they all existed in D subgenome according to the phylogenetic tree.Such truncated EIN3/EIL gene does not exist in Arabidopsis.Gene structure,basic information and the conservative domain analysis of GhLYI protein revealed that GhLYI is different from other family members.The protein encoded by this gene had only 120 amino acids,which only contained the BDI and BDⅡof DNA binding domain within the N-terminal of the typical EIN3 protein.The protein completely lost the activation domain at the C-terminal.Transcriptome data showed that GhLYI was expressed in cotton roots,stems,leaves,flowers,ovules and fibers at different development stages,with the highest expression in stamens and-1DPA（days post anthesis）to 1DPA ovules.In the process of evolution,the variation of protein structure may lead to the emergence of this new truncated EIN3/EIL gene and the differentiation of novel functions.2.The fluorescence signal of GhLYI-GFP fusion protein was observed by laser confocal microscope and GFP signal was expressed in both cell membrane and nucleus.When cotton seedlings were treated with ethylene precursor1-aminocyclopropane-1-carboxylic acid（ACC）solution,the change trend of GhLYI expression was similar to that of control water treatment,indicating no effect on the transcriptional level.Through the construction of plant overexpression vector（p Bin GFP4-GhLYI）,GhLYI gene was ectopically expressed in wild-type and ein3eil1mutant in Arabidopsis,respectively.Ectopic expression of GhLYI in the ein3 eil1background could compensate for the ethylene insensitive phenotype upon ACC treatment.Compared with wild type,transgenic GhLYI Arabidopsis showed more obvious ethylene triple reaction phenotype with increased sensitivity to ethylene.These results indicated that GhLYI participated in ethylene signal response.In addition,the ACC content in the leaves,stems,and flower buds of transgenic cotton overexpressing GhLYI gene was lower than control,speculating that the increased sensitivity of transgenic cotton to ethylene signaling may be due to feedback inhibition of ethylene synthesis.3.Transcriptional regulation plays an important role in the mechanism of plant senescence.At present,some EIN3/EIL transcription factors involved in senescence regulation have been identified in other species,but researches in cotton are still very limited.This study revealed that the expression of GhLYI increased gradually as the aging process progressed.Compared with control materials,transgenic Arabidopsis ectopically expressing GhLYI or cotton overexpressing GhLYI showed earlier senescence.Dark treatment accelerated the senescence of detached Arabidopsis and cotton leaves.The chlorophyll content of transgenic material leaves was significantly lower than that of control,while the H2O2and oxygen free radical content were significantly higher than control.GhLYI editing cotton did not show obvious senescence reduction phenotype,which likely due to genetic compensation effects because the other two truncated EIL genes Gh＿D07G1670 and Gh＿D12G2800showed 4-fold and 3-fold increase in the expression level,respectively.In summary,GhLYI is a positive regulator of leaf senescence,promoting the premature senescence of plant leaves.By transcriptome analysis,15,287 and 10,037 differentially expressed genes（DEGs）in the leaves and ovules of GhLYI-overexpressed cottons were identified as compared with the transgenic receptor material.KEGG metabolic pathway enrichment analysis showed that up-DEGs were mainly related to plant hormone signal pathway,plant-pathogen interaction,starch and sucrose metabolism and fatty acid degradation,while the down-DEGs were mainly related to photosynthesis,flavonoid synthesis,and biosynthesis of secondary metabolites.4.CUT&Tag was used to detect GhLYI binding DNA motif in the whole genome of Arabidopsis and upland cotton.139,38 and 138 peak sequences were obtained from three biological replicates in Arabidopsis.And a total of 2,005,1,505 and 1,425binding peaks were identified respectively in three biological replicates in Upland cotton.Of them,489 genes were shared among these three replicates.By analyzing the annotation of these target genes,it is supposed that GhLYI may regulate the senescence of plant leaves mainly through genes related to photosynthesis,circadian rhythm,oxidative phosphorylation,RNA polymerase and biosynthesis of cutin,suberin and wax.It was found the highest confidence motif as ACACGTG through MEME-Ch IP analysis of the 50 bp sequence upstream and downstream of the center position of the CUT&Tag enrichment peak in Arabidopsis.A similar motif was also enriched in cotton.And another GGT/CC fragment with 9.8%proportion was also found.These findings would help us build the senescence regulation network of cotton EIN3/EIL genes.5.The senescence related gene SAG20 was existed in the results of both species,and its promoter region sequence contains a motif GGT/CCC enriched by CUT&Tag analysis,therefore,Gh SAG20 was assumed as the direct downstream target gene of GhLYI.The expression level of Gh SAG20 in transgenic cotton overexpressing GhLYI was significantly increased.Y1H,EMSA and Luciferase assays all confirmed that GhLYI could directly bind to the 42 bp DNA fragment in the promoter region of Gh SAG20 gene to activate its transcription.Under dark condition,virus induced Gh SAG20 gene silencing（VIGS）slowed down the senescence rate of detached cotton leaves and accumulated lower levels of reactive oxygen.This experiment preliminarily verifies the function of Gh SAG20,indicating that it played a role in the regulation of cotton leaf senescence.6.The above experimental proved that the truncated GhLYI protein still has DNA binding activity.Self-transcriptional activation test in yeast showed that GhLYI had no transcriptional self-activation.It is supposed that GhLYI needs other intermediate proteins to play its transcriptional regulation function.A series of candidate proteins interacting with GhLYI were obtained by yeast two-hybrid c DNA library and the zinc finger protein transcription factor Gh VOZ1 had the highest frequency of occurrence.The yeast double hybrid results of Gh VOZ1 protein segments identified its interaction domain with GhLYI protein.The luciferase complementation experiment further confirmed the interaction between these two proteins,suggesting that GhLYI may not only participate in leaf senescence,but also affect plant flowering and resistance to abiotic stress.To sum up,GhLYI was a specific truncated EIN3/EIL transcription factor in the evolution of cotton,and there was no homologous gene in Arabidopsis.It still played a regulatory role in ethylene signaling and could act as a positive factor to accelerate plant leaf senescence.Reducing the expression of this gene is expected to prolong the plant growth period and thereby increase yield.The discovery of this new gene provided high-quality genetic resources for increasing cotton yield through transgenic technology. |
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