Data_Sheet_2_Environment-Driven Adaptations of Leaf Cuticular Waxes Are Inheritable for Medicago ruthenica.CSV

Cuticular waxes covering the plant surface play pivotal roles in helping plants adapt to changing environments. However, it is still not clear whether the responses of plant cuticular waxes to their growing environments are inheritable. We collected seeds of Medicago ruthenica (a perennial legume) populations from 30 growing sites in northern China and examined the variations of leaf cuticular waxes in a common garden experiment. Four wax genes, MrFAR3-1, MrFAR3-2, MrCER1, and MrKCS1, involved in biosynthesis of predominant wax classes (primary alcohol and alkane) and wax precursors, were isolated to test the contributions of genetic variations of the coding sequences (CDS) and the promoter sequences and epigenetic modifications. The plasticity responses of the cuticular waxes were further validated by two stress-modeling experiments (drought and enhancing ultraviolet B). Great variations in total wax coverage and abundance of wax classes or wax compounds were observed among M. ruthenica populations in a common garden experiment. Stress-modeling experiments further validated that M. ruthenica would alter leaf wax depositions under changed growing conditions. The transcriptional levels of the wax genes were positively or negatively correlated with amounts of cuticular waxes. However, the analysis of promoter methylation showed that the methylation level of the promoter region was not associated with their expressions. Although both promoter sequences and CDS showed a number of polymorphic sites, the promoters were not naturally selected and insignificant difference could be observed in the numbers and types of acting elements of the four wax genes among populations. In contrast, the CDS of the wax genes were naturally selected, with a number of missense mutations resulting in alterations of the amino acid as well as their isoelectric points and polarities, which could impact on enzyme function/activity. We conclude that long-term adaptation under certain environments would induce genetic mutation of wax biosynthesis genes, resulting in inheritable alterations of cuticular wax depositions.

植物表皮蜡质（cuticular waxes）覆盖于植物表面，在帮助植物适应多变环境中发挥关键作用。但目前仍不明确植物表皮蜡质对生长环境的响应是否可遗传。本研究从中国北方30个生长位点收集了花苜蓿（Medicago ruthenica，一种多年生豆科植物）的种子，并通过同质园试验（common garden experiment）分析了其叶片表皮蜡质的变异特征。我们分离得到4个蜡质合成相关基因：MrFAR3-1、MrFAR3-2、MrCER1与MrKCS1，这些基因参与主要蜡质类别（初级醇与烷烃）及蜡质前体的生物合成，以此检验编码序列（coding sequences, CDS）、启动子序列的遗传变异以及表观遗传修饰（epigenetic modifications）的贡献。我们进一步通过两类胁迫模拟实验（干旱胁迫与增强型紫外B辐射）验证了表皮蜡质的可塑性响应。同质园试验结果显示，不同花苜蓿种群的总蜡质覆盖度、蜡质类别或蜡质化合物的丰度均存在显著差异。胁迫模拟实验进一步证实，花苜蓿可在生长条件改变时调整叶片蜡质沉积量。蜡质基因的转录水平与表皮蜡质含量呈正相关或负相关。然而启动子甲基化分析表明，启动子区域的甲基化水平与基因表达并无关联。尽管启动子序列与编码序列均存在大量多态性位点，但启动子未受到自然选择，且不同种群间4个蜡质基因的顺式作用元件的数量与类型均无显著差异。与之相反，蜡质基因的编码序列受到了自然选择，大量错义突变会导致氨基酸及其等电点与极性发生改变，进而可能影响酶的功能与活性。本研究最终得出结论：特定环境下的长期适应会诱导蜡质生物合成基因发生遗传突变，从而使表皮蜡质沉积产生可遗传的改变。