The Efflux Carrier Protein PtoPIN1d Mediates The Transport Of Auxin In Rays And Affects The Maturation Of The Xylem Of Populus Tomentosa

Poplar is an important economic and energy plant,and also a model plant for studying wood development.The thickening of the wood is mainly determined by the secondary development of the stem.The vascular cambium differentiates outwards to form phloem and inwards to form xylem.The development of the secondary xylem is continuous.The differentiated xylem cells experience expansion,secondary cell wall thickening and programmed cell death sequentially.Auxin,a privital plant hormone involved in wood formation,exhibits a level peak in cambium layers.The concentration of auxin decrease gradually in cells toward both the bark and the pith.So far,how the auxin gradient is established is still unknown.Previous studies have reported that there are 15 homologs of PIN-formed protein,which facilitate auxin efflux in poplar.Some of them,such as PtoPIN1a,PtoPIN1b and PtoPIN1d,are expressed in the cambium and may possibly mediate the establishem of the auxin gradient in poplar stems.Our previous study reveal that PtoPIN1a and PtoPIN1b direct polar auxin transport within the cambium.Up-regulation of PtoPIN1a and PtoPIN1b expression enhances activity of cambium division,xylem proliferation as well as thickening.In studying roles of PtoPIN1d,we noticed an interesting phenotype that maturation of the xylem cells was affected in transgenic Populus tomentosa.Thus,in this study,we analyze the role of PtoPIN1d in guiding auxin transport in stems and in influencing xylem maturation.The main results are as follows.1.Up-regulation of PtoPIN1d promotes development of the cambium and xylem.The fusion protein of PtoPIN1d::eYFP was expressed in in Populus tomentosa under control of the own promoter of PtoPIN1d.The transgenic Populus tomentosa showed a significant increase in width as well as in layer number of the cambium and xylem,consistent with the previous observation in transgenic Populus tomentosa expressing PtoPIN1a or PtoPIN1b.By contrast,the width and layer number of the cambium and xylem were significantly decreased when expression of PtoPINs was interfered specifically in cambium layers.These results indicate that,PtoPIN1d plays a redundance role as PtoPIN1a and PtoPIN1b in positively affecting development of the cambium and xylem of populus tomentosa.2.Up-regulation of PtoPIN1d specifically inhibits maturation of xylem cells.Some immature xylem cells were observed in the region of mature xylem cells in transgenic poplar upregulating PtoPIN1d.Phloroglucinol staining also showed that the lignin deposition of xylem cells was inhibited in the trnagenic plants.We then observed the thickness of xylem cells by scanning electron microscopy.No matter in xylem fibers and vessel cells was cell wall thickness significant reduced in transgenic plants.Meanwhile,genes related to xylem secondary wall synthesis were largely repressed.However,upregulation of PtoPIN1a and PtoPIN1b had no such effect on xylem maturation.These results suggested that the up-regulation of PtoPIN1d expression specifically inhibited xylem maturation3.PtoPIN1d::eYFP specifically mediates polar auxin transport in xylem rays.To reveal how PtoPIN1d affects xylem maturation,we observed protein localization of PtoPIN1d::eYFP in poplar stems.In stems PtoPIN1d::eYFP has a preferentially expression in parenchymal cells of primary xylems.Basal-side localization of PtoPIN1d::eYFP at plasma membrane(PM)directs the basipetal auxin transport in stems.In addition,we also detected PtoPIN1d::eYFP localization at PM of cortex,cambium and ray cells.PtoPIN1d::eYFP localized at apical side of cortex cells,and direct acropetal auxin transport there.In cambiums,PtoPIN1d::eYFP exhibited two different polar localizations.PtoPIN1d::eYFP mainly localized at two anticlinal PM in most cambium cells,while exhibits additional localization at the periclinal PM towards the pith in the ray protocells.Similar polar localization behaviors were also observed in PtoPIN1a::eYFP and PtoPIN1b::eYFP.These results suggest that PtoPIN1d,as well as PtoPIN1a and PtoPIN1b,mediate auxin transport in cambium rings,where auxin flows inwards through ray protocells and then is distributed through the other cambium cells.However,we found that the polar localization of PtoPIN1d::eYFP in ray protocells also appeared in ray cells in xylems,different from that of PtoPIN1a::eYFP and PtoPIN1b::eYFP.This specific PtoPIN1d::eYFP localization indicate that PtoPIN1d mediated auxin transport to xylems and may consequently affect maturation of xylem cells.4.Up-regulation of PtoPIN1d in poplar changed the auxin level in xylemIAA determination showed that the upregulation of PtoPIN1d increase auxin level in transgenic stems.We used auxin transport inhibitor NPA to treat up-regulation of PtoPIN1d of transgenic plant.NPA treatment impaired the promovtive effect of PtoPIN1d upregulation on cambium and xylem development.Moreover,xylem maturation in the transgenic plant recovered to that of wild type.Phloroglucinol staining result showed that NPA treatment reduced the inhibitory effect on xylem maturation caused by upregulation of PtoPIN1d.In another hand,we genetically blocked auxin signalling pathway in xylems by expressing a mutated repressor IAA9 m under the control of pro PtoSND1.The introduction of pro PtoSND1::IAA9m also impaired the promotive effect of PtoPIN1d upregulation on cambium and xylem development.In particular,the expression of IAA9 m restored xylem maturation to wild type level.These results indicate that PtoPIN1d mediates auxin accumulation in xylems,and thereby influences xylem maturation through auxin auxin signalling pathway.5.Downregulation of PtoPIN1d in poplars inhibited cambium and xylem development,but had no significant effect on xylem maturationWe analyzed the effect of down-regulating the expression of PINs in cambium on the stem development of poplar.The results showed that down-regulation of PtoPINs expression affected the development of cambium and xylem,suggesting that poplar PtoPINs gene did participate in cambium division and xylem differentiation of poplar stem,and played a positive regulatory role.However,compared with the wild type,the transgenic plants with down-regulated PtoPINs had no significant effect on xylem maturation.6.Up-regulation of PtoPIN1d promotes development of xylem rays.A large number of coupled rays were observed in the transgenic poplar up-regulating PtoPIN1d,rather than PtoPIN1a or PtoPIN1b.Moreover,number of rays were also increased in PtoPIN1d::eYFP poplar.When PtoPINs expression was supressed in cambiums,number of rays were significantly reduced.In addition,NPA treatment reduced number of rays in PtoPIN1d up-regulating plants,suggesting that auxin transport mediated by PtoPIN1d affects the formation of rays.Taken together,all these results indicate that PtoPIN1d specifically localizes at ray cells to promote formation of rays,which benefits auxin transport to xylem and consequently influences xylem maturation through auxin signaling pathway.