木材是树木维管形成层细胞持续分裂分化的结果。维管形成层区域生长素梯度分布对形成层活性的影响已被证实，但对其形成和维持机制还不清楚。拟南芥的研究表明生长素转运蛋白PIN对植物体内生长素聚集点的形成和梯度分布有决定作用。一种杂种白杨形成层区域的基因分析结果显示，多个PIN基因的表达与生长素径向浓度梯度分布高度一致，揭示PIN蛋白在杨树维管形成层区域生长素极性分布的形成和维持中起关键作用。我们的前期合作系统分析了毛白杨PIN家族蛋白的组成、亚细胞定位以及表达的动态变化，发现了5个在形成层区域富集表达和动态变化的PIN家族成员。本合作拟集成我们在植物细胞蛋白质定向运输、细胞极性分化和林木发育上的研究优势，进一步研究PIN蛋白在杨树维管形成层区域的精细时空表达和亚细胞定位，并确认它们在生长素极性分布和维管形成层细胞分化中的作用。本研究将为林木发育调控研究和先进的林木分子育种技术提供重要理论基础。

Wood is the end products of the division and differentiation of meristematic cells in the secondary vascular system of trees. Although the impact of polar auxin distribution across the vascular cambium zone on the cambium activity has been reported, the underlying mechanism of polar auxin distribution in the cambium remains unclear. ..Advanced studies in Arabidopsis have shown that auxin efflux carriers PIN proteins play essential roles in the formation and maintenance of auxin maximum points and gradient distribution. The auxin-responsive transcriptome analysis of a series of wood-forming tissues from the cambium zone of a hybrid aspen showed that the expression of some PIN genes is correlated with the radial auxin gradients, indicating that PIN proteins play important roles in the formation and maintenance of polar auxin distribution in the cambium zone of poplar. ..Recently, we systematically analyzed the composition, cellular distribution and expression dynamics of PIN family proteins in Populus tomentosa. We revealed that 5 PIN genes have strong expression in the cambium zone, compare to their expression in other tissues. Among these, 3 of them are expressed mainly in the cambium zone. The GUS activity of PIN promoter::GUS indicated that these PINs are dynamically expressed during the regeneration of stems in poplar. Transgenic lines over-expressing PIN showed vasculature phenotype both in the aerial and ground tissues. ..In this proposal, we will investigate the temporospatial expression in high resolution in the cambium zone during wood formation in poplar. We will also determine the polar localization of selected PINs. Using the recently generated micro vascular tissue regeneration system in early young cultured plantlets, the function of these PIN proteins in the polar auxin distribution will be analyzed in detail. We are aiming to uncover the underlying mechanism of the formation and maintenance of polar auxin distribution in the cambium zone, and ultimately, to genetically manipulate and engineer the wood formation process in trees.

木材是生物能源重要的可再生来源，而木材的形成源于树木维管形成层的分裂与分化。生长素转运蛋白PIN决定着生长素的梯度分布，在维管形成层发生和分化中起关键作用。WOX蛋白家族是植物特有的一类转录因子家族，调控植物维管形成层干细胞的维持和分化。本研究制备了生长素响应标记基因DR5的报告基因（DR5::GUS）和活体荧光标记（DR5::GFP）杨树转基因材料，并利用体外剥皮次生维管组织再生系统，研究了生长素在维管再生过程中的动态变化。研究表明，生长素浓度梯度的重建可以诱导杨树韧皮部、形成层等次生维管组织的再生，并决定形成层再生的位置。鉴定出调控杨树形成层发生和分化的PIN蛋白为PtoPIN1a、PtoPIN1b、PtoPIN1d和PtoPIN6a，并采用转基因手段初步分析了PIN1b蛋白在维管形成层干细胞的形成和木质部细胞分化的作用。分析了PtoWOX4a和PtoWUSa的组织表达模式，发现PtoWUSa主要在茎顶端分生组织区域和形成层区域表达，而PtoWOX4a在在形成层、韧皮部及维管组织中均有表达。表达模式的差异暗示二者在杨树形成层发生和分化过程中的作用不同。对转基因植株的分析表明，PtoWUSa和PtoWOX4a过表达后可以导致木质部区域增厚。本项目的开展为深入研究形成层发生和分化提供了的实验体系，为林木发育分子调控相关研究提供了理论基础，对揭示维管形成层发育分子机制具有重要意义。