| Auxin controls various plant developmental responses,including shoot apical dominance,flower development and root growth.Polar auxin transport plays an important role in the dwarfing of apple interstem tree.Disruption of auxin transportation affects stamen development.This study aims at revealing the dwarfing effect of M9 interstem and pollen development of by investigating the auxin efflux carrier PIN.The research can play the certain theoretical guiding significance for breeding of dwarfing rootstock and tomato seedless fruits.Experiment materials were Baleng Crab seedling as rootstock,M9,GM256,M26,B9,MM106,SH40 and Baleng Crab as interstems,Red Fuji grafted on them as cultivar.The IAA content and the expression of MdPIN1a or MdPIN1b gene were detected in different grafted trees.It was found that IAA content was relatively higher but the expression levels of MdPIN1b was significantly lower in bark tissue of interstem than that in scion or rootstock of M9 interstem grafted combination.The expression of MdPIN1b was analyzed in a series of tangential sections across the wood-forming tissues.The distinct expression of MdPINlb was exhibited in most of the cambium and adjacent xylem and phloem,a good correlation between expression and IAA maxima could be observed for MdPIN1b.MdPIN1b contained a 1863 bp open reading frame and encoded 620 amino acids.The MdPINlb protein was localized on cell plasma membrane in tobacco/onion epidermal cells transiently expressing 35S:MdPINlb:GFP fusion protein.To investigate the mechanism for transcriptional regulation of MdPIN1b in M9 and BC rootstock,the upstream sequence of MdPIN1b from M9 and BC was isolated.M9 has two single base mutations in the promoter region of the MdPIN1b compare to BC.The mutations,-1005 bp(T/C)from the ATG start site,and-333 bp(G/A)from the ATG start site.With tobacco transformation,vector construction for site-directed mutagenesis of MdPINlb promoter helped to determine that natural allelic differences decreased promoter activity is closely associated with lower MdPIN1b expression in the M9 interstem.In order to further determine whether the promoter differences affect the polar auxin transport,we next transformed tobacco plants with a construct containing the MdPIN1b ORF sequence,driven by the MdPIN1b promoter in BC and M9,respectively.The transgenic tobacco plants were used as interstem grafted on WT plants,after 40 days,IAA content was detected in different parts of grafted seedlings.It was found that the content of IAA in grafted seedlings of BCpromoter MdPINlb-MdPINlb transgenic tobacco was significantly higher than that of M9promoterMdPINlb-MdPINlb,indicating that MdPIN1b gene promoter mutation limited the basipetal transport of auxin.In addition,we isolated the MdPIN8 gene with a short hydrophilic loop in apple genome.MdPIN8 contained a 1074 bp open reading frame and encoded 357 amino acids.Through the spatiotemporal expression of MdPIN8 in apple and SIPIN8 in tomato,MdPIN8 and SlPIN8 were found to be male gametophyte-specific genes in their respective species.The SlPIN8-RNAi transgene reduced the expression level of SIPIN8 in tomato so that the pollen development was abnormal and the rate of pollen germination was low,resulting in the formation of seedless fruit.SlPIN8 was located on the endoplasmic reticulum.SlPIN8-RNAi transgene did not change the content of auxin in tomato,and when SIPIN8 transiently overexpressed tomato protoplasts,the content of free IAA increased significantly,and the content of IAA conjugates decreased significantly,indicating that SIPIN8 involved in intracellular auxin homeostasis.MdPIN8 overexpression can increase the level of free IAA in tomato,indicating that MdPIN8 has a similar function to SlPIN8. |
