Primary Function Analysis Of Auxin Transport Protein PtoPIN2a And PtoPIN5a In Populus Tomentosa Carr

As one of the six major phytohormones, auxin is implicated in regulating the pattern of cell division and differentiation, plant tropisms and apical dominance. PIN-FORMED proteins play an important role in polar auxin transport. It was found that PtoPINs gene family contains 15 members, but less function is known. In this study, expression patterns, subcellular localizations, protein topologys and biological functions of PtoPIN2a and PtoPIN5a were analyzed by using semi-quantitative PCR, GUS assay, phenotypic analysis of overexpression and RNAi plants. These results laid a foundation for further discovery of molecular mechanisms of PtoPINs. The major results are follows:(1) Amino acid sequence and protein topology analysis reveals that PtoPIN2a has 633 amino acids, with the molecular weight of 68 kd and isoelectric point of 9.34. PtoPIN5a contains 346 amino acids, with molecular weight of 37 kd and isoelectric point of 8.84. Both proteins contain two hydrophobic domains (each has five transmembrane helices) separated by a hydrophilic loop. Sequence aligments show that PtoPIN2a and AtPIN2 share 82% similarity, and PtoPIN5a and AtPIN5 share 71% similarity.(2) By using semi-quantitative PCR, we found that PtoPIN2a and PtoPIN5a are mainly expressed in roots. Promoter regions of PtoPIN2a and PtoPIN5a have been amplified from the genomic DNA of Populus tomentosa Carr, and the GUS reporter constructs have been generated. GUS assay shows that plant transformation vectors using the β-glucuronidase (GUS) reporter gene system and detected the expression patterns of PtoPTN2a and PtoPIN5a promoter in transgenic poplars. Histochemical GUS assay showed GUS activity driven by PtoPIN2a promoter was mainly in root tip and fraction of the root pericycle, while that driven by PtoPIN5a promoter was maily in root tip and most part of the root pericycle, which further illustrates genes have the root tissue expression specificity.(3) A fluorescent tag YFP has been tagged onto the C-termini of PtoPIN2a and PtoPIN5a. Subcellular localization of PtoPIN2a and PtoPIN5a has been determined by analyzing both the stable transgenic Arabidopsis and transisent expression tobacco leaf epidermal cells. The results show that PtoPIN2a localizes in the plasma membrane, which indicates that PtoPIN2a may play a role in auxin transport across plasm membrane. PtoPIN5a localizes in the endoplasmic reticulum, which implies that PtoPIN5a may play a role in the regulation of cellular auxin homeostasis.(4) Constructs of overexpression for PtoPIN2a and PtoPIN5a were generated. Forty eight and Fifty plants were obtained respectively. PtoPIN2a over-expressed plants exhibit increased lateral roots, while PtoPIN5a over-expressed plants exhibit decreased lateral roots, with increased adventitious root length, although the longitudinal growth is not affected. Constructs of RNAi for PtoPN2a and PtoPINSa were generated. Eighty and twenty seven RNAi lines were obtained respectively. PtoPIN2a RNAi lines are showing decreased adventitious roots and lateral roots, lost geotropism and slowed plants growth, while No obvious phenotype is observed in PtoPIN5a RNAi lines, probably due to functional redundance among PIN family genes in Populus.