Cytokinin Inhibits Cotton Fiber Initiation By Disrupting GhPIN3a-mediated Asymmetric Accumulation Of Auxin In The Ovule Epidermis

Cotton is the most prevalent natural fiber crop,and provides the major natural fiber source for global textile industry.China is one of top cotton producer and consumer in the world.The domestic cotton production plays an important role in China's national economy.cotton fibers are differentiated from ovule epidermal cells.With the feature of highly elongated unicell,cotton fibers provide an ideal model for studying cell differentiation and polar growth.Fiber initiation,the first stage of cotton fiber development,determines number and subsequent growth of fiber cells.Thus,the fiber initiation is extraordinarily important for both yield and quality of cotton fibers.Phytohormones,such as auxin,cytokinins?CKs?,gibberellins?GAs?,brassinosteroids?BRs?and jasmonic acid?JA?,are involved in the regulation of cotton fiber initiation.Our previous study shows that indole-3-acetic acid?IAA?,the major auxin in plants,accumulates in the initiating fiber cells on the day of anthesis.Genetically enhancement of auxin biosynthesis in cotton ovule epidermis can improve both fiber yield and quality simultaneously.However,what cause the establishment of the asymmetric auxin distribution in the ovule epidermis remains unknown.The interaction between phytohormones cytokinin and auxin is found in many developmental processes of plants.However,its role in cotton fibers is largely unknown.To this end,we analyzed the dynamic change of cytokinin and auxin during cotton fiber development,and investigated effect of auxin,cytokinins and their antagonism on fiber development.Moreover,we studied origination of the auxin in the ovule epidermis and the expression profiles of auxin efflux carriers in ovules.The results indicated that polar auxin transport?PAT?mediated by auxin efflux carrier regulates auxin accumulation in ovule epidermal cells.GhPIN3a,a member of auxin efflux carrier in cotton,polarly localized to the plasma membrane of non-fiber cells,but not to that of fiber cells.This difference of protein localization results in accumulation of auxin in fiber cells.In addition,excess CKs could disturb the plasma membrane localization of GhPIN3a in non-fiber cells.Consequently,the PAT would be disrupted and the asymmetric distribution of auxin could not be built up in the ovule epidermis.As a result,the fiber initiation would be aborted.The main results are as follow.1.The antagonistic effect of CK and auxin on fiber initiation?1?Using LC-MS/MS,the dynamic change of CKs and IAA in developing ovules and fibers of the upland cotton?Gossypium hirsutum L.,cultivar‘Jimian 14'?was investigated.The level of cytokinins declined gradually in ovules and fibers after flowering.Contrary to cytokinins,IAA exhibited an increasing trend.?2?GUS reporter gene was fused with a cytokinin-inducible promoter proTCS and an auxin-inducible promoter proDR5 respectively in order to observe CK and auxin in situ.The observation confirmed the opposite change of CKs and IAA in developing ovules and fibers.?3?In vitro experiments showed that auxin promotes fiber initiation,whereas CKs inhibit the initiation.Both effects are in a dosage-dependent manner.We further down-regulated expression of GhCKX3,a cytokinin dehydrogenase gene under the control of an ovule-specific promoter proPV in cotton.A significant increase of cytokinin?ZT?content coupled with a decrease of fiber density was observed in proPV:antisenseGhCKX3transgenic ovules compared with that of wild-type on the day of anthesis,supporting the finding of inhibitory effect of CKs on cotton fiber initiation.?4?Furthermore,CK biosynthetic gene IPT was placed downstream of promoter proDR5,and auxin biosynthetic gene iaaM was placed downstream of promoter proTCS.Using these two cassettes,we analyze the in situ interaction of auxin and CK in cotton fibers.CK production induced by IAA could attenuate the promotive effect on fiber initiation.By contrast,IAA production induced by CK could relieve the inhibition effect on fiber initiation.These results indicated an antagonistic relationship between CK and auxin in fiber initiation.2.Cytokinin disrupts auxin accumulation in the ovule epidermis and inhibits cotton fiber initiationThe fiberless mutant xu142fl is a useful tool for the study of cotton fiber initiation?1?The hormone determination showed that IAA and CKs content in the mutant ovule was significantly higher than that in the wild-type ovule on the day of anthesis.?2?proDR5:GUS and proTCS:GUS reporter systems were introduced into xu142fl by crossing,respectively.Plants with the fiberless and wild-type phenotypes that harbored the proDR5:GUS or proTCS:GUS gene cassette were isolated in the F₂ generation.After staining,the outer integument and chalaza region of mutant ovules appeared stronger CK signal,which indicated by proTCS:GUS expression,than that of the wild-type ovules.In term of auxin,the mutant showed strong expression of proDR5:GUS in the nucellus of ovules but lost the signal in the ovule epidermis as compared with wild type.These results indicated that it is the shortage of auxin in the ovule epidermis,rather than in the whole ovule,accounting for the defect of fiber initiation in xu142fl.The higher level of CKs in the outer integument of the mutant might prevent the accumulation of auxin in the ovule epidermis.?3?The results of proDR5:GUS expression and fiber growth on the CK-treated proDR5:GUS ovule showed reduced auxin accumulation and inhibited fiber initiation on the ovule surface.This indicated that extra CKs could disrupt the asymmetric auxin accumulation in epidermal cells and thus inhibit fiber initiation.3.Auxin efflux carrier-mediated PAT plays a crucial role in the regulation of auxin accumulation and fiber initiation in the ovule epidermis?1?IAA starvation experiment demonstrated that de novo auxin is not the main source of auxin for the cotton fiber initiation.?2?Inhibition of auxin transport by 1-N-naphthylphthalamic?NPA?in pedicels pre-anthesis reduced the auxin level in the ovary,nucellus and ovule surface on the day of anthesis,and inhibited fiber initiation.This indicated that the auxin promoting fiber initiation was mainly imported from the outside of ovaries.?3?In vitro experiment showed that auxin efflux carrier inhibitors?NPA and BFA?inhibited auxin accumulation and fiber initiation in ovule surface,whereas auxin influx carrier inhibitor 1-NOA did not.These results indicated that auxin efflux carrier-mediated PAT plays a crucial role in the regulation of auxin accumulation and fiber initiation in the ovule epidermis.4.The difference of protein localization of GhPIN3a between non-fiber and fiber cells contributes to the asymmetric auxin distribution in cotton ovule epidermal cells?1?In situ mRNA hybridization showed that GhPIN3a was preferentially expressed in the outer integument of ovule on the day of anthesis,suggesting the probable involvement in auxin accumulation in the ovule epidermis.?2?Seed-coat specific down-regulation of GhPIN3a expression produced severe or even entire inhibition on fiber initiation in the proBAN:GhPIN3a-RNAi transgenic cotton,confirming the crucial role of GhPIN3a in fiber initiation.?3?The localization of GhPIN3a:YFP signal mainly appeared at the plasma membrane of non-fiber cells but not in the adjacent fiber cells.This result suggested that auxin can be transported within the ovule epidermis through non-fiber cells.The loss of membrane located GhPIN3a in fiber cells would block cellular efflux of auxin,and result in an inevitable accumulation of auxin in the cells.5.Cytokinin antagonizes the promotive effect of auxin on fiber cell initiation through disturbing the expression and protein localization of GhPIN3a?1?In ZT treated-ovule GhPIN3a transcription level was considerably decreased.Similar decrease of GhPIN3a transcription was also found in outer integument of xu142fl ovules,which contains abnormally high level of CK.Furthermore,GUS staining on the ovule surface of proGhPIN3a:GUS transgenic plants was considerably reduced under the treatment of ZT.These results indicated that cytokinin can inhibit the transcription of GhPIN3a.?2?Plasma membrane-located GhPIN3a:YFP in the ovule epidermis almost disappeared after the treatment of ZT.This suggested that CK not only disturbs transcription but also protein localization of GhPIN3a,and probably in turn inhibits auxin accumulation in the ovule epidermis and fiber initiation.In conclusion,the results of this study indicated that the different protein localization of GhPIN3a between non-fiber and fiber cells contributes to the asymmetric accumulation of auxin in the ovule epidermis.Excess CKs can disturb GhPIN3a-mediated PAT in the ovule epidermis through interfering both gene expression and protein localization,and thus result in the antagonistic effect on promotive effect of auxin on fiber initiation.The difference of GhPIN3a protein localization in ovule epidermal cells provides a foundation for further revealing the underlying mechanism of auxin accumulation in single cells.