A Small GTPase GhROP6 Facilitates GhPIN3a Internalization And Protein Degradation In Cotton Fibers

As an excellent natural fiber crop,cotton is the main source for global textile industries,and thus of great economic value.Cotton fibers are derived from ovule epidermal cells and have the feature of highly elongated unicellular structure.This provides an ideal model for studying cell differentiation and elongation.Plant hormone auxin specifically accumulates in fiber cells to promote cell protrusion from ovule surface,which affects yield and quality of cotton fibers.Our latest study reveals that the auxin efflux carrier GhPIN3 a,which is expressed in the outer integument of ovules,plays a pivotal role in establishing the concentration gradient of auxin in the ovule epidermis.When expression of GhPIN3 a was downregulated by RNAi interference,fiber initiation was remarkably inhibited.Protein localization analysis shows that GhPIN3 a polarly localizes at the plasma membrane of non-fiber cells but delocalizes in fiber cells within ovule epidermis.The loss of protein localization is believed to be the direct cause for auxin accumulation in fiber cells since GhPIN3 a mediate cellular auxin efflux.However,how does the auxin concentration gradient establish within ovule epidermis,and what regulate protein delocalization of GhPIN3 a in fiber cells remain unexplored.In this study,we study localization characteristic of GhPIN3 a in ovule epidermis and seek the reason for absence of GhPIN3 a plasma membrane localization in fiber cells through gene expression,protein localization and fiber phenotype.Meanwhile,according to the expression profile in fiber initials compared to non-fiber cells in ovule epidermal layers,we screened out a small GTP binding protein GhROP6 which is upregulated in fiber cells,as a candidate gene regulating GhPIN3 a protein localization in fibers.We examine the effect of GhROP6 on fiber development and polar localization of GhPIN3 a in fiber cells.The main results are as follow:1.Change of GhPIN3 a localization in ovule epidermal cells start from the flowering day(1)GhPIN3a::e YFP aporlarly localized at PM of ovule epidermal cells one day before anthesis.On flowering day when fiber cells initiate,GhPIN3a::e YFP polarly localized at PM of non-fiber cells,but showed no discernable signal in fiber cells.This protein-localization change is tightly associated with auxin regulated fiber cell initiation.(2)We observed GhPIN3 a localization in the different region of ovule surface at 0 DPA.The statistical results of polar orientation guided by GhPIN3a::e YFP showed that auxin flow with ovule epidermal layer mainly points to the chalaza,suggesting that GhPIN3 a in non-fiber cells routes auxin flow in ovule epidermis.2.Decreased protein level causes the loss of GhPIN3 a in fiber cellsThe loss of PM-located GhPIN3 a in fiber leads to auxin accumulation in fiber cells,so what is the reason for the absence of GhPIN3 a localization?(1)Transcription analysis shows that the expression level of GhPIN3 a in fiber cells was considerably higher than that in separated fiber-free ovule.That is to say,the transcription level does not cause the loss of GhPIN3 a localization in fiber cells.(2)When brefeldin A(BFA),an inhibitor for vesicle trafficking,was used to treat with 0 DPA ovule,the signal of GhPIN3a::e YFP appeared in fiber cells.Once additional supplement of protein synthetic inhibitor CHX,the signal disappeared again.These results indicate that GhPIN3a was translated normally in fiber cells but depleted due to some unknow reasons.(3)Protein content analysis shows that the protein level of GhPIN3 a in fiber cells was obviously lower than in separated fiber-free ovules,confirming that the low protein level causes the absence of GhPIN3 a localization in fiber cells.3.Activation of GhROP6 promotes fiber initiation while the deactivation inhibitsTo explore regulatory mechanism of GhPIN3 a localization,we hypothesized that there were some genes,which are upregulated in fiber cells,involve in the regulatory process of GhPIN3 a in fibers.A small GTP binding protein GhROP6 was targeted from the microarray result reported.To test the function of GhROP6,we first examine the effect of GhROP6 on fiber development.(1)The statistical results of 5 DPA fiber length showed that,compared with wild-type cotton,overexpression of m Cherry::GhROP6 and constitutive active mutant m Cherry::CA-ghrop6 transgenic cotton increased percentage of ovules with long fiber cells to some extent.Whereas in dominant negative mutant m Cherry::DN-ghrop6 cotton,fiber length was reduce considerably,and appeared more ovules with short fiber and even naked ovules.(2)Observation of 1 DPA ovule epidermis by SEM,results showed that there was no significant difference in the development state of m Cherry::GhROP6 and wild type,while m Cherry::CA-ghrop6 fibers developed faster than wild type,and the development delayed in m Cherry::DN-ghrop6 cotton.Taking together,we deduce that overexpressing and activation of GhROP6 have a trend to promote fiber initiation,while deactivation of GhROP6 can inhibit fiber initiation.4.GhROP6 promotes internalization of GhPIN3aThen,does GhROP6,the upregulated gene in fibers,regulate the polarity or abundance of GhPIN3 a in fiber cells?(1)In situ m RNA hybridization and tissue expression profile analysis showed that GhROP6 was highly expressed in fiber cells.(2)Fusion protein m Cherry::GhROP6 mainly localized at PM,co-localizing with GhPIN3a::e YFP.Similar expression pattern and protein localization suggested that GhROP6 and GhPIN3 a may interact with each other.(3)In transient expression,we found that m Cherry::CA-ghrop6 reduced the signal intensity of GhPIN3 a at plasma membrane,whereas m Cherry::DN-ghrop6 increased.Moreover,a great increase of bubbles-shaped GhPIN3 a was observed in the cells co-expressing both with m Cherry::GhROP6 and m Cherry::CA-ghrop6.(4)In transgenic cotton,m Cherry::GhROP6 and m Cherry::CA-ghrop6 both impaired localization of GhPIN3 a at plasma membrane and decreased the protein level of GhPIN3 a in fiber cells.m Cherry::DN-ghrop6 enhanced the fluorescense intensity of GhPIN3 a at plasma membrane and promoted the production of GhPIN3 a in fiber cells.These results indicated that GhROP6 was preferentially expressed in fiber,which reduced the localization of GhPIN3 a at PM by promoting internalization.In conclusion,the results of this study indicated that,starting from the day of flowering,the localization of GhPIN3 a was changed at PM of ovule epidermis,and the gradient of auxin concentration was established in ovule epidermis with scattered distribution of fiber cells as the center.The low protein level of GhPIN3 a leaded to the loss of GhPIN3 a localization in fiber cells.In addition,GhROP6 was largely expressed in fiber cells,which promoted the internalization of GhPIN3 a to up-regulate its degradation and regulated the fiber initiation.