Effects Of Auxin Transport Facilitator SlPIN On Tomato Pedicel Abscission

The yield and quality of crops are often affected by stress-induced abscission during protective production in China.So,clarifying the regulation mechanisms of organ abscission,is crucial to improving the yield and quality of crops.As one of the key hormones of abscission,polar auxin transport is crucial to its distribution and functioning.PIN-FORMED(PIN)proteins are the largest contributors of polar auxin transport(PAT),which play regulation roles in many developmental and environment responding processes,but its role on organ abscission is still unknown.Here,by means of bioinformatics,physiology and molecular biology,we studied the roles of key SlPINs in the regulation of PAT and abscission.Also,the potential regulation mechanisms of SlPIN transcriptional and posttranslational levels during abscission are studied subsequently in this paper.The main results are as follows:1.Bioinformatics analysis indicates that SlPIN1,SlPIN2,SlPIN3,SlPIN4,SlPIN7 and SlPIN9 belong to "long PIN",which contain a long hydrophobic loop;while SlPIN5,SlPIN6,SlPIN8 and SlPIN10 belong to "short PIN",which contain a relatively short hydrophobic loop.The ddPCR results shows 7 of 10 SlPINs are expressed in tomato flower pedicel,with SlPIN1,SlPIN3 and Sl PIN4 being the most highly expressed.The SlPINs exhibit temporal-and spatialspecific expression during pedicel abscission,which are affected by auxin and ethylene.All the SlPINs are downregulated at early stage(0~4 h),implying a decline of auxin transport.However,at late stage,some SlPINs are upregulated in the abscission zone(AZ)and the proximal part of pedicel,with continuously low expression in the distal part,indicating auxin transport is elevated in the AZ and proximal part at late stage,which contributes to AZ auxin consumption.Auxin positively affects SlPIN expression.All the pedicel SlPINs decline rapidly after flower removal and increase immediately following IAA application,indication high sensitivity to auxin depletion.In addition,with the treatment of ethylene and its inhibitor 1-MCP,we find that,by affecting SlPIN expression,ethylene may improve the AZ auxin transport and response to itself at late stage of abscission.Moreover,some SlPINs increase in proximal part at 16~24 h,implying their roles on defence layer development and pathogenesis-related defence.2.Suppressing either SlPIN1(pTRV-SlPIN1)or SlPIN4(pTRV-SlPIN4)expression accelerates the tomato pedicel abscission,while the suppressing of SlPIN3 seems not affect the auxin transport and abscission process.In the ovary,SlPIN1 and SlPIN4 mediate auxin basipetal flux to maintain the AZ auxin accumulation and prevent abscission.Down-regulating SlPIN1 and SlPIN4 induce impeded auxin transport from the ovary to the AZ,which result in lower AZ auxin concentration and premature abscission.However,SlPIN1 and SlPIN4 accelerate auxin depletion in the AZ and abscission process by facilitating auxin basipetal transport in the pedicel.After recovery AZ auxin accumulation by IAA pre-treatment,pTRVSlPIN1 and pTRV-SlPIN4 pedicels show delayed abscission process.3.Both the SlPIN1 and SlPIN4 protein levels in the AZ decrease at the early stage of abscission and increase obviously at the late stage.The experiments with MG132(a proteasome inhibitor)treatment proves that,the increased SlPIN1 and SlPIN4 proteins are responsible for the improvement of auxin basipetal flux,which induce the decline of AZ auxin and the abscission precess finally.Moreover,at late stage,the increase between gene expression and protein abundant of the two SlPINs are not consistent with each other,indicating SlPIN1 and SlPIN4 are regulated in a posttranslational way.4.Polar localization of PIN proteins is central to the control of PAT and auxin distribution,but very little information is known about the relationship between PIN localization and abscission process.Brefeldin A(BFA),which is reported to disturb PIN localization previously,changes auxin distribution and abscission process of tomato pedicel in our study,implying Polar localization of PIN protein may affect abscission.Phosphorylation disturbs the preferentially basal localization of SlPIN1 and SlPIN4,and results in nonpolar distribution of the two proteins on cell membrane.However,phosphorylation levels of SlPIN1 and SlPIN4 are significantly decreased during abscission,implying the basal localization of SlPIN1 and SlPIN4 are more stabilized to promote basipetal auxin transport and abscission process.