RNA Sequencing of <i>Populus x canadensis</i> Roots Identifies Key Molecular Mechanisms Underlying Physiological Adaption to Excess Zinc

Populus x canadensis clone I-214 exhibits a general indicator phenotype in response to excess Zn, and a higher metal uptake in roots than in shoots with a reduced translocation to aerial parts under hydroponic conditions. This physiological adaptation seems mainly regulated by roots, although the molecular mechanisms that underlie these processes are still poorly understood. Here, differential expression analysis using RNA-sequencing technology was used to identify the molecular mechanisms involved in the response to excess Zn in root. In order to maximize specificity of detection of differentially expressed (DE) genes, we consider the intersection of genes identified by three distinct statistical approaches (61 up- and 19 down-regulated) and validate them by RT-qPCR, yielding an agreement of 93% between the two experimental techniques. Gene Ontology (GO) terms related to oxidation-reduction processes, transport and cellular iron ion homeostasis were enriched among DE genes, highlighting the importance of metal homeostasis in adaptation to excess Zn by P. x canadensis clone I-214. We identified the up-regulation of two Populus metal transporters (ZIP2 and NRAMP1) probably involved in metal uptake, and the down-regulation of a NAS4 gene involved in metal translocation. We identified also four Fe-homeostasis transcription factors (two bHLH38 genes, FIT and BTS) that were differentially expressed, probably for reducing Zn-induced Fe-deficiency. In particular, we suggest that the down-regulation of FIT transcription factor could be a mechanism to cope with Zn-induced Fe-deficiency in Populus. These results provide insight into the molecular mechanisms involved in adaption to excess Zn in Populus spp., but could also constitute a starting point for the identification and characterization of molecular markers or biotechnological targets for possible improvement of phytoremediation performances of poplar trees.加拿大杨（Populus x canadensis）无性系I-214在过量锌胁迫下表现出典型的指示表型；在水培条件下，其根系的金属吸收量高于地上部，且向地上器官的转运效率降低。尽管调控上述过程的分子机制仍不明晰，但这种生理适应性似乎主要由根系主导。本研究采用RNA测序（RNA-sequencing）技术开展差异表达分析，以解析根系响应过量锌胁迫的分子机制。为最大化差异表达（differentially expressed, DE）基因的检测特异性，本研究选取三种不同统计方法共同鉴定到的基因集（共61个上调基因、19个下调基因）作为验证对象，并通过实时定量聚合酶链反应（RT-qPCR）进行验证，两种实验技术的验证一致性达93%。基因本体（Gene Ontology, GO）富集分析显示，差异表达基因显著富集于氧化还原过程、物质转运及细胞铁离子稳态相关的GO条目，凸显了金属稳态在加拿大杨无性系I-214适应过量锌胁迫中的重要作用。本研究鉴定到两个可能参与金属吸收的杨属金属转运蛋白基因（ZIP2和NRAMP1）出现上调表达，同时一个参与金属转运的NAS4基因出现下调表达；此外还鉴定到4个参与铁稳态的转录因子基因（两个bHLH38基因、FIT及BTS）出现差异表达，这可能是为了缓解锌过量诱导的铁缺乏胁迫。尤为关键的是，本研究推测FIT转录因子的下调表达可能是杨属植物应对锌过量诱导铁缺乏的潜在分子机制。本研究结果不仅为解析杨属植物适应过量锌胁迫的分子机制提供了新见解，同时也可为鉴定和表征分子标记或生物技术靶点提供研究起点，有望用于提升杨树的植物修复性能。