Regulation Of GhPIN3a Ubiquitination On Its Localization In Cotton Fiber And Identification Of The Related E3 Ligase

Cotton is an important commercial crop.As a natural fiber source,cotton occupies an important position in China’s textile industry.Cotton fibers are elongated single-celled structures developed by the elongation of the epidermal cells of the ovule.Providing an ideal model to study the differentiation and elongation of plant cells.The yield and quality of fiber are determined by the time and quantity of the initiation of differentiation of cotton fiber cells,and the plant hormone auxin specifically accumulates in the initiation fiber to promote the protrusion and elongation of fiber cells,which plays an important role in the development of cotton fiber.Previous studies of our group found that auxin accumulation in fiber cells at the initial stage was not achieved through in situ synthesis,but through Gh PIN3a polar mediated auxin transport.The loss of Gh PIN3a on the plasma membrane of fiber cells makes auxin unable to be excreted,but is accumulated in cells.Recently,our work found that Gh PIN3a is highly transcription in cotton fibers at the initial stage,but the protein content is very low.It is speculated that protein degradation leads to the decrease of Gh PIN3a protein content in the fibers,resulting in the disappearance of localization on the plasma membrane.However,the degradation mechanism of Gh PIN3a in fiber cells remains unclear.Eukaryotes degrade target proteins mainly through 26S proteasome and autophagy.Either pathway requires the first ubiquitination of the target protein,in which E3ubiquitin ligase specifically recognizes the target protein and regulates its protein degradation.So,is there ubiquitination of Gh PIN3a in cotton fiber,and if so,which E3ligase is involved in the regulation of this process?In order to solve the above problems,this study detected the ubiquitination level of Gh PIN3a in cotton ovules and fibers at the initial stage,analyzed the relationship between the ubiquitination level of Gh PIN3a and protein localization and degradation,and screened the up-regulated E3 ubiquitin ligase in fibers that affected the localization of Gh PIN3a.Finally,the effect of E3 ubiquitin ligase on Gh PIN3a localization and fiber development was analyzed by down-regulating the expression of E3 ubiquitin ligase,indicating that the regulation of Gh PIN3a ubiquitination is involved in the regulation of Gh PIN3a localization in fiber,and the possible regulatory genes were screened out.The main results are as follows:1.Vacuolar dependent protein degradation is involved in regulation of Gh PIN3a localization in cotton fibersTo figure out which pathways affect Gh PIN3a in cotton fibers,treatment with different inhibitors found that The ovules of Gh PIN3a:e YFP transgenic cotton were treated with BFA（50μM）,IKA（5μM）,E64d（100μM）,Con A（1μM）and FM4-64 for30min,1h and 2h on flowering day.Fluorescence observation showed that no matter which inhibitor was treated,there was co-localization with FM4-64.After BFA treatment,many BFA bodies were formed in the cell,which enhanced the cytoplasmic signal intensity,indicating that Gh PIN3a moved with vesicle transport.Moreover,plasma membrane signal was significantly enhanced after IKA treatment,indicating that clathrin-mediated endocytosis affected the localization of Gh PIN3a.Both plasma membrane and cytoplasmic signals of Gh PIN3a:e YFP were enhanced after 2h treatment with protein degradation inhibitors.The spot-like signals increased significantly in fiber cells after inhibitory protein degradation,indicating that Gh PIN3a was transported along with vesicles and affected localization through clathrin-mediated endocytosis and vacuolar mediated protein degradation pathways.The ovules of transgenic cotton Gh PIN3a:e YFP were treated with proteasome inhibitor MG132（50μM）,autophagy inhibitor E64d（100μM）,and Con A（1μM）on flowering day（12h）.Fluorescence observation showed that the plasma membrane localization signal and cytoplasmic signal of Gh PIN3a were enhanced after all inhibitor treatments,Moreover,inhibition of vacuolar mediated protein degradation was more effective than inhibition of proteasome activity.In the treated fibrocytes,the dot signal of Gh PIN3a:e YFP was significantly increased and large signal aggregation was formed.These results indicate that vacuolar mediated protein degradation is involved in the localization and regulation of Gh PIN3a in cotton fiber.2.Ubiquitination modification of Gh PIN3a is high in cotton fiberAuxin accumulation in ovule epidermis was affected by treatment with ubiquitination inhibitor Nutlin-3.Purification of Gh PIN3a:e YFP fusion protein by GFP magnetic beads,and its ubiquitination level was detected.The ovules of Gh PIN3a:e YFP transgenic cotton were cultured overnight with BFA and E64d for 3 days after flowering to inhibit protein degradation in fibers.It was found that the ubiquitination level of Gh PIN3a:e YFP was significantly increased in fibers compared with ovule.These results indicate that Gh PIN3a is highly ubiquitinated in cotton fiber.In order to further elucidate the relationship between ubiquitin modification and Gh PIN3a.The mutation16 ubiquitin binding Lys site vectors Gh PIN3a16K-R:e YFP and the fused ubiquitin binding vector Gh PIN3a:ubq:e YFP were constructed.Transient expression results of tobacco showed that the plasma membrane localization signal of Gh PIN3a^16K-R:e YFP was enhanced,and the plasma membrane localization of Gh PIN3a:ubq:e YFP was basically undetectable.These results suggest that changing the ubiquitination level of Gh PIN3a:e YFP affects the localization of the fusion protein in the plasma membrane.3.Changing Gh PIN3a ubiquitination level affects the localization of Gh PIN3a and inhibits cotton fiber developmentObserve the localization of Gh PIN3a,it was found that in Gh PIN3a^16K-R:e YFP and Gh PIN3a:ubq:e YFP transgenic cotton,whether in leaves and ovule,Gh PIN3a^16K-R:e YFP would stabilize the localization of Gh PIN3a.Gh PIN3a:ubq:e YFP The location of Gh PIN3a was not observed.The ovules of Gh PIN3a^16K-R:e YFP and Gh PIN3a:ubq:e YFP transgenic cotton 5DPA were observed,and it was found that the development of cotton ovules was seriously affected,and the abortive ratio was high.4.Screening of E3 ubiquitin ligases that impaired plasma-membrane localization of Gh PIN3a:e YFPTranscriptomics were used to compare the up-regulated genes in fibers compared with ovules at the initial stage,and 45 genes related to ubiquitin-mediated protein degradation pathway were identified.There were 13 independent E3 ubiquitin ligases involved in substrate recognition.In addition,three independent Gh RGLG genes were found to be up-regulated in fibers,which may affect the plasma membrane localization of Gh PIN3a.Expression analysis confirmed that the expression of Gh CDC was up-regulated in fibers.The effects of Gh COP1、Gh RGLG2、Gh RGLG3、Gh MIEL1 and Gh SINAT4 on Gh PIN3a:e YFP localization were observed by cloning 15 genes and transient expression of these proteins in tobacco.5.Silencing the associated E3 ubiquitin ligase and enhances plasma-membrane localization of Gh PIN3a:e YFP inhibits fiber development in cottonAfter silencing Gh COP1,Gh RGLG2,Gh RGLG3 and Gh MIEL1 in transgenic cotton Gh PIN3a:e YFP by virus-induced gene silencing technique,the intensity of plasma membrane localization of Gh PIN3a:e YFP in cotton leaves and ovular epidermal cells was significantly enhanced.Silencing Gh COP1 and Gh RGLG3 inhibited cotton fiber elongation,while down-regulation of Gh COP1 did not affect the transcription of Gh PIN3a,while down-regulation of Gh RGLG3 inhibited the transcription of Gh PIN3a.These results suggest that these E3 ubiquitin ligases are involved in the localization and regulation of Gh PIN3a in cotton fibers,thus affecting the development of cotton fibers.6.No direct interaction between Gh COP1 and Gh PIN3aYeast two-hybrid and Bi FC showed that there was no interaction between Gh COP1and Gh PIN3a.Through transient expression of tobacco,Gh COP1 was co-expressed with Gh PIN3a ^Y503Amutant endocytosis site,and it was found that the localization of Gh PIN3a was still impaired,suggesting that Gh COP1 does not regulate Gh PIN3a localization through the clastin-mediated endocytosis pathway.Above all,These results suggest that protein degradation regulates the localization of Gh PIN3a in cotton fibers,and there is a high level of ubiquitination in cotton fiber,while changing the ubiquitination level of Gh PIN3a will affect the localization of Gh PIN3a in cotton ovules and the development of cotton ovules.These E3 ubiquitin ligases,Gh COP1,Gh RGLG2,Gh RGLG3 and Gh MIEL1,participate in the localization and regulation of Gh PIN3a in cotton fiber,thus affecting the development of cotton fiber.The regulation of Gh COP1 on Gh PIN3a localization occurred before clathrin-mediated endocytosis...