Data_Sheet_1_Genome-Wide Identification, Comprehensive Gene Feature, Evolution, and Expression Analysis of Plant Metal Tolerance Proteins in Tobacco Under Heavy Metal Toxicity.docx

Plant metal tolerance proteins (MTPs) comprise a family of membrane divalent cation transporters that play essential roles in plant mineral nutrition maintenance and heavy metal stresses resistance. However, the evolutionary relationships and biological functions of MTP family in tobacco remain unclear. In the present study, 26, 13, and 12 MTPs in three main Nicotiana species (N. tabacum, N. sylvestris, and N. tomentosiformis) were identified and designated, respectively. The phylogenetic relationships, gene structures, chromosome distributions, conserved motifs, and domains of NtMTPs were systematic analyzed. According to the phylogenetic features, 26 NtMTPs were classified into three major substrate-specific groups that were Zn-cation diffusion facilitators (CDFs), Zn/Fe-CDFs, and Mn-CDFs, and seven primary groups (1, 5, 6, 7, 8, 9, and 12). All of the NtMTPs contained a modified signature sequence and the cation_efflux domain, whereas some of them also harbored the ZT_dimer. Evolutionary analysis showed that NtMTP family of N. tabacum originated from its parental genome of N. sylvestris and N. tomentosiformis, and further underwent gene loss and expanded via one segmental duplication event. Moreover, the prediction of cis-acting elements (CREs) and the microRNA target sites of NtMTP genes suggested the diverse and complex regulatory mechanisms that control NtMTPs gene expression. Expression profile analysis derived from transcriptome data and quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed that the tissue expression patterns of NtMTPs in the same group were similar but varied among groups. Besides, under heavy metal toxicity, NtMTP genes exhibited various responses in either tobacco leaves or roots. 19 and 15 NtMTPs were found to response to at least one metal ion treatment in leaves and roots, respectively. In addition, NtMTP8.1, NtMTP8.4, and NtMTP11.1 exhibited Mn transport abilities in yeast cells. These results provided a perspective on the evolution of MTP genes in tobacco and were helpful for further functional characterization of NtMTP genes.

植物金属耐受蛋白（metal tolerance proteins, MTPs）属于一类膜定位二价阳离子转运蛋白家族，在植物矿质营养维持与重金属胁迫抗性方面发挥不可或缺的作用。然而，烟草中MTP家族的进化关系与生物学功能仍有待阐明。本研究分别在三个主要烟草属物种（普通烟草Nicotiana tabacum、林生烟草N. sylvestris和绒毛烟草N. tomentosiformis）中鉴定并命名了26、13和12个MTP家族成员。本研究对普通烟草MTPs（NtMTPs）的系统发育关系、基因结构、染色体分布、保守基序与结构域开展了系统分析。基于系统发育特征，26个NtMTPs可被划分为3个底物特异性大类：锌阳离子扩散促进因子（Zn-cation diffusion facilitators, CDFs）、锌/铁-阳离子扩散促进因子（Zn/Fe-CDFs）以及锰-阳离子扩散促进因子（Mn-CDFs），并进一步分为7个亚组（1、5、6、7、8、9和12亚组）。所有NtMTPs均含有修饰型特征序列与阳离子外排结构域（cation_efflux domain），部分成员还携带ZT二聚化结构域（ZT_dimer）。进化分析表明，普通烟草的MTP家族起源于其二倍体亲本林生烟草与绒毛烟草的基因组，并通过一次片段复制事件发生了基因扩张与丢失。此外，对NtMTP基因的顺式作用元件（cis-acting elements, CREs）与微小RNA（microRNA）靶位点进行预测，结果显示调控NtMTP基因表达的机制具有多样性与复杂性。基于转录组数据的表达谱分析与实时定量反转录聚合酶链式反应（quantitative real-time reverse transcription-PCR, qRT-PCR）结果显示，同一亚组的NtMTPs具有相似的组织表达模式，但不同亚组间的表达模式存在显著差异。此外，在重金属胁迫处理下，NtMTP基因在烟草叶片与根系中呈现出不同的响应模式。分别有19个与15个NtMTPs在叶片与根系中响应至少一种金属离子胁迫处理。此外，NtMTP8.1、NtMTP8.4与NtMTP11.1在酵母细胞中验证出锰离子转运功能。本研究结果为烟草MTP基因家族的进化研究提供了新视角，同时为后续NtMTP基因的功能鉴定提供了理论参考。