Preliminary Research On The Mechanism Of PINA Protein Regulating Protonema Cells Development In Physcomitrella Patens

As auxin polar transporter,PIN proteins play important role in regulating plant growth and development.At present,the research of PIN proteins mainly focuses on the tissues and organs level of seed plant,and little on the cellular level.There are four PINs in Physcomitrella patens,namely PINA-PIND.The homology between PINA and At PIN1 is the highest.So far two papers have reported the function of PIN proteins as auxin efflux carriers in Physcomitrella patens.In PINA overexpressing Physcomitrella,the content of extracellular auxin increased and extracellular auxin content decreased in pinapinb.Leaves become narrow and long,caulonema formation ealier and sporophyte abnormal in pinapinb.However the phenotype of pina knockout mutant protonema was not reported.We obtained PINA knockout mutant(pina)by PEG mediated transformation.The protonema phenotype of pina was analyzed.Besides abnormal leaf shape which was similar to the pinapinb,the protonema of pina had distinct phenotypes,including,shorter protonema cell,few caulonema formation from protonema and the cell division manners.According to Tom’s research,overexpressing PINA enhances extracellular IAA level.In our study,the content of intracellular auxin in pina protonema was significantly higher than WT.It’s confirmed that PINA is an auxin export carrier.So,the intracellular auxin increased and extracellular auxin decreased for pina protonema cell.Are the changes of auxin levels in both inside and outside of protonema cell related to the phenotype of pina protonema cells? Treated with Yucasin,an auxin synthesis inhibitor,auxin biosynthesis could be decreased in Physcomitrella protonema(reported by Hu Yongyue,unpublished).The development of protonema cells with altered auxin level was observed.Protonema development was observed under different concentrations and combinations of IAA and Yucasin treatment.The short cell-phenotype of pina protonema could not be recovered by Yucasin but could be recovered by 1 μM IAA.This result indicated that the decrease of extracellular auxin content is the main reason for short cell of pina protonema.In other experiments,there were some special IAA concentration requirements.Exogenous of IAA should be lower than 1 μM in order to antagonize Yucasin’s inhibition for cell elongation.For pina mutant,more Yucasin treatment need less exogenous IAA to antagonize.When treated with Yucasin,more IAA was required to recover pina short cell-phenotype.These results indicate that difference of intracellular and extracellular auxin concentration may be the key factor affecting the normal elongation of protonema cells in pina mutant.As an axin efflux carrier,PINA protein may affect the growth of protonema cells by regulating the difference of auxin concentration between inside and outside of cells.It has been reported that ABA could regulate root apical meristem development through inhibiting At PINs’ expression in Arabidopsis thaliana.Does ABA have a similar mechanism in Physcomitrella patens? ABA could down-regulate PIND expression and enhance GH3 expression in the protonema of Physcomitrella to our previous results.Tom’s results also showed that PIND overexpression increased auxin exportation in protonemata of Physcomitrella.Therefore,it could be speculated that the application of ABA could cause both increase of intracellular auxin and decrease of extracellular auxin.The short cell phenotype of ABA treatment is similar to pina mttant.Short cell phenotype of ABA treatment could be partially weakened by exogenous IAA and be completely eliminated by polar IAA application.These results suggested that ABA inhibiting protonema cells elongation through extracellular auxin regulation.Moreover,ABA may regulate protonema cell elongation through reducing the expression of PIND and leading decrease of extracellular auxin.In a word,it is possible that PINA protein regulates the elongation of protonema cells through changing the auxin level between intracellular and extracellular.ABA may regulate protonema cell elongation through reducing extracellular auxin.These results provide a new direction for further studying the function and mechanism of PIN proteins at the cellular level.