The Role Of GhERF108 In Regulating Secondary Cell Wall Synthesis Of Cotton Fibers

Cotton is an important economic crop planted globally.Its cotton fiber has a very important application value in the textile industry and related industries.How to increase the output and quality of cotton fiber is one of the important problems in cotton production.Recent studies have shown that plant hormones(such as ethylene)play a key role in the growth and development of cotton fiber,but the molecular regulatory mechanism of ethylene in cotton fiber development is still unclear.A ethylene responsive factor,GhERF108,has been identified in previous studies in our laboratory.This paper mainly studies whether GhERF108 regulates the development of cotton fiber through ethylene signaling pathway,which is to provide some scientific data for molecular breeding to improve the yield and quality of cotton fiber.The main results are as follows:1.Ethylene promotes the expression of GhERF108We used the 21 days after flowering(21 DPA)of cotton ovule and fiber for culture in vitro,and then extracted the RNA of cotton fiber which treated with the ethylene synthesis precursor(ACC)and the ethylene synthesis inhibitor(AVG)at different time,and analyzed the expression of GhERF108 in cotton fiber treated with hormone for 0-120 hours by fluorescence quantitative qRT-PCR.The results showed that exogenous application of ACC could promote the expression of GhERF108 in cotton fiber,and reached the maximum value after 96 hours of induction,while the expression of GhERF108 was down-regulated by the treatment of ethylene synthesis inhibitor AVG Those results indicate that GhERF108 can respond to ethylene signal and may participate in ethylene signal pathway.2.GhEIN3 activates the expression of GhERF108Previous studies have shown that EIN3 is involved in the ethylene signaling pathway as a key regulator of ethylene response.In order to study whether GhERF108 also participates in the ethylene signal pathway through GhEIN3 pathway,the fusion expression vectors GhEIN3:eGFP,GhERF108-P1:LUC,GhERF108-P2:LUC were constructed,and then were transferred into Agrobacterium,injected into the young tobacco lower epidermis.We observed the fluorescence condition under the chemiluminescence imaging system,and detected the enzyme activity.The results showed that GhEIN3 could activate the expression of GhERF108,suggesting that GhERF108 might be involved in the ethylene signaling pathway mediated by GhEIN3.3.Ethylene promotes fiber cell elongation and secondary cell wall synthesis in cottonPrevious studies have showed that GhERF108 was highly expressed in the secondary cell wall thickening stage of cotton fiber cells.In order to study the effect of ethylene on secondary cell wall of cotton fiber cell,we took the flowering day of cotton ovule culture in vitro,and treated with ethylene,measured the length of cotton fiber,made resin sections,stained with toluidine blue,observed and counted the thickness of cotton fiber cell wall under the microscope.Those results showed that the precursor of ethylene synthesis ACC could promote the elongation and secondary cell wall thickening of cotton fiber cells,while an inhibitor of ethylene synthesis AVG could inhibit the elongation and secondary cell wall synthesis of cotton fiber cells,indicating that ethylene plays an important role in the development of cotton fiber cells.4.Phenotype analysis of GhARF108 RNAi and overexpression transgenic cottonIn order to further study the role of GhERF108 in cotton fiber development,we analyzed the phenotype of the progeny lines of transgenic cotton with GhERF108 RNAi.The results showed that the mature fiber length of T2 and T3 generation of GhERF108 RNAi transgenic cotton was shorter than that of the wild type,and the cotton peach was smaller.Moreover,the secondary cell wall thickness of the transgenic cotton fiber cells of GhERF108 RNAi was thinner than that of the wild type,but there was no obvious change in cell morphology.The analysis of the main components of the secondary cell wall of GhERF108 RNAi transgenic cotton showed that there was no significant change in the lignin content,while the cellulose content decreased,which indicated that GhERF108 could affect the secondary cell wall development of cotton.At the same time,we also constructed 35S:GhERF108 overexpression vector,and obtained transgenic cotton plants through cotton hypocotyls infected by Agrobacterium.It was found that the cotton fiber length of T1 generation of transgenic cotton was longer than that of wild type.Further research of secondary cell wall development in GhERF108 overexpression transgenic cotton is still in progress.5.GhERF108 enhances the activation of GhARF7-2 on GhLBD30 and GhMYBLl genesIn order to further study the molecular mechanism of GhERF108 on the secondary wall development of cotton fiber cells,we analyzed the interaction between GhERF108 and the GhARFs protions related to the development of secondary cell wall by using in vivo double molecule fluorescence complementary experiment.The results show that GhERF108 can interact with GhARF7-1 and GhARF7-2,but has no interaction with GhARF5-1,GhARF5-2,GhARF6-1,GhARF6-2 and GhARF6-3.In vitro pull-down test also confirmed that GhERF108 protein can interact with GhARF7-1 and GhARF7-2 protein.In addition,we analyzed the regulatory relationship between GhERF108,GhARF7-1 and GhARF7-2 on GhLBDs and GhMYBL1 related to the development of secondary cell wall by transcriptional activation experiments.The results showed that GhARF7-2 could directly activate the expression of GhLBD30 and GhMYBL1,while the regulatory analysis of GhARF7-1 on GhLBDs and GhMYBLl is still in progress.The results also showed that GhERF108 could not directly activate the expression of GhLBD30 and GhMYBL1,that is to say GhLBD30 and GhMYBL1 are not the direct downstream target genes of GhERF108.In order to further analyze whether the interaction between GhERF108 and GhARF7-2 affects the expression of GhLBD30 and GhMYBL1,the agrobacterium solutions of GhERF108,GhARF7-2 and GhLBD30/GhMYBL1 were mixed in the proportion of 1:1:1,and then injected into the lower epidermis of young tobacco for transcriptional activation.The results showed that GhER1108 could enhance the activation of GhARF7-2 on GhLBD30 and GhMYBL1.These results suggest that GhERF108 may regulate the expression of GhLBD30 and GhMYBL1 by interacting with GhARF7-2,thus regulating the secondary cell wall development of cotton.