Preliminary Analysis Of PtPIF And PtPIN1s Gene Function In Populus

A subfamily of basic helix-loop-helix transcription factors,Phytochrome Interacting Factor(PIF)plays a key role in the light signaling of plant.Arabidopsis thaliana contains 7PIFs in its genome.The role of PIFs in regulating plant growth and development including seed germination,hypocotyl elongation,shade avoidance and leaf senescence.The regulation of auxin distribution and concentration is one of the mechanisms for PIFs to modulate the growth and development of plants.Perennial woody plants,compared with annual herbaceous plants,have some unique development processes and may exhibit difference in the optical signal response.Therefore,researches on the function of PIF gene family members in Populus contribute to reveal the mechanism of trees.The molecular characteristics and expression profiles of PIF family members in different tissues and were stressed under cold,drought and NaCl were analyzed with real-time quantitative PCR.In addition,PIFs regulate plant growth through affecting synthesis and distribution of auxin.The PIN-FORMED(PIN)proteins are a plant-specific family of auxin-efflux carriers localized at the plasma membranes,and their determination of the distribution of intercellular auxin possibly under the regulation of PIFs,will influence plant growth and development.Populus trichocarpa has 15 PINs and PtPINs share conserved topology and transmembrane domains,and found to be strongly expressed in the roots,leaves,and cambium in Populus.To explore the differences of expression and function between PtPIN1 s,we examined the expression pattern in different tissues,in three stages of adventitious root generation and under cold,drought and NaCl stresses.The main results are summarized as the followings:1.Bioinformatics analysis showed that there were at least 10 PtPIF members in Populus and PtPIF genes were conserved during evolution of the species,all of them contained bHLH domain.Promoters of the PtPIF genes contain a lot of stress related cis-elements.This indicates that PIFs may be in response for many environmental fluctuations,as well as light.2.PtPIF had distinct expression pattern across different tissues and different abiotic stress conditions.PtPIF1,PtPIF8 a and PtPIF8 b were expressed mainly in the mature leaves,PtPIF3 a and PtPIF9 b were expressed mainly in the young leaves.The result of qRT-PCR confirmed that PtPIF10 was up-regulated,while PtPIF1,Pt PIF4/5a,PtPIF8 b and PtPIF9 b were inhibited by cold treatment,the expression of PtPIF8 a increased and those of PtPIF3 b and PtPIF4/5b decreased under drought stress,the expression of PtPIF3 b and PtPIF9 a raised,and PtPIF4/5a decreased under NaCl treatment.These results suggest that different PIFs may involved in different environment responses.3.To investigate whether PtPIFs can recognize G-box,the three repeats of the G-box and mutant G-box were constructed separately into pHIS2 vector,and PtPIF3 a were cloned into pGADT7-Rec2 vector.Yeast one hybrid assay revealed that PtPIF3 a can bind to G-box,but failed in binding to the mutants.This binding ability suggests that PIFs may regulate PINs through the G-box element.4.PtPIN1 s can be divided into two groups.One is PtPIN1 a and PtPIN1 b,another is PtPIN1 c and PtPIN1 d.Histochemical GUS staining showed that the different expression of this two group in adventitious root tip.This difference was displayed in the stage of callus formation,and not affected by exogenous hormones.Pt PIN1 s also showed distinct expression pattern under different abiotic stress conditions.PtPIN1 c was up-regulated,while PtPIN1 b was inhibited under cold treatment.The abundance of PtPIN1 b and PtPIN1 c were increased and PtPIN1 d decreased under drought stress.PtPIN1 d was up-regulated under NaCl treatment.These differences of PINs in responses to stresses indicate that the evolution and functions of PINs in woody plants are diversified.This study has analyzed the molecular characteristics and expression profiles of PIF family members in different tissues and under stresses like cold,drought and NaCl.Smilarly,different expression patterns of PtPIN1 s in adventitious rooting and under various abiotic stresses were also verified.The specific binding between PIF3 a and G-box element leads to the suggestion that the interaction of PIF and PIN through G-box.These results contribute to thefunctional analysis of PtPIFs and PtPIN1 s in regulation of plant growth and development under different conditions in Populus.