高原（寒）逆境草地植物叶表皮蜡质的适应性进化研究

Leaf cuticular waxes cover the outmost of plant surface and play important roles during plant growth and development. In our previous researches, we established the analyzing methods of crystal structure and chemical composition of leaf cuticular wax; analyzed the responses of leaf cuticular wax of Medicag sativa to soil water deficit, enhancing ultraviolet radiation, low air humidity, and high temperature. In the current project, the leaf cuticular waxes of alpine grassland plants from Qinghai-Tibet Plateau which were ecologically adapted to abiotic stresses in plant morphology, physiology and function, were studied. Three sampling plots will be selected according to their latitude and longitude, including Haibei and Guoluo regions of Qinghai Province, Naqu region of Tibet Autonomous Region. Three sub-plots in each sampling plot will be further selected according to altitudes, including 2000-3000m, 3000-4000m, and >4000m. Plants from same genus distributed both at Qinghai-Tibet Plateau and low altitude region (Chongqing) will be selected as control. Using the technologies of scanning and transmission electron microscopy, gas chromatography and mass spectrometry, molecular biology, and population ecology, the following three points will be analyzed, 1) the characteristics of leaf cuticular wax crystal structure and chemical composition of alpine grassland plants under abiotic stresses; 2) the relationships between environmental factors and leaf cuticular wax characteristics, and expression of wax synthase; 3)the differences of leaf cuticular wax characteristics and transcriptional-level of wax synthase between plants from same genus distributed both at Qinghai-Tibet Plateau and low altitude region. The objectives of the project are, to elucidate the responsive mechanism of plant leaf cuticular wax to abiotic stresses from Qinghai-Tibet Plateau; to reveal the relationship between the diversities of leaf cuticular wax and distribution of alpine grassland plants; to discuss the evolution of plant leaf cuticular wax to changing climate in Qinghai-Tibet Plateau at large scales. The results of the project will also provide beneficial knowledge to study the responsive and adaptive mechanisms of plants to global climate changes.

本课题以植物叶表皮蜡质为研究对象，在前期研究基础上，针对青藏高原草地植物在形态、生理和功能上对非生物逆境胁迫的生态适应性，在青藏高原三个纬度带（青海海北地区和果洛地区、西藏那曲地区），按海拔梯度分别设置采样区，并以低海拔地区(重庆北碚)同属植物为参照，采用扫描和透射式电镜技术、气相色谱-质谱技术、分子生物学以及种群生态学等研究手段，分析1）青藏高原逆境胁迫下草地植物叶表皮蜡质晶体结构与组分特征；2）环境因子与植物叶表皮蜡质特征及蜡质合成酶基因转录水平之间的关系，3）高海拔地区与低海拔地区同属植物叶表皮蜡质特征及蜡质合成酶基因转录水平。通过上述3个方面研究，在植物微形态适应层面上阐明高原草地植物叶表皮蜡质对逆境胁迫的响应机制，揭示植物叶表皮蜡质多样性与植物分布特征之间的关系，在大尺度上探讨青藏高原气候变化下植物叶表皮蜡质的进化意义，为全球气候变化下植物对环境的响应和适应机制研究提供有益资料。

植物叶表皮蜡质是覆盖在叶表的疏水保护层，由超长链脂肪酸及其衍生物组成。已知的研究表明，表皮蜡质对外界环境因子的变化较为敏感，从晶体结构到蜡质组分上均有变化，且这种变化与植物的适应有关。高原植物对逆境的适应性变化是否与表皮蜡质的适应性变化有关？为了回答这个问题，我们分析了高寒草甸54种草本植物9个纬度带上的蜡质含量、组分及晶体结构，1）发现晶体结构整体出现熔融现象，增加了对叶表的覆盖面积及厚度；2）蜡质沉积量不同环境间差异大，存在较大的可塑性；3）基于种群水平的叶角质层蜡质总量、组分含量及烷烃分布特征，与年平均气温、降雨量、干旱指数之间无显著相关关系，不能直接证明高寒草甸植物叶角质层蜡质特征变化与气候变化有关；4）但在群落水平上，我们发现蜡质平均含量及烷烃分布特征与年平均气温存在显著相关关系，如随着温度的升高，植物及土壤烷烃平均碳链长度增加，说明部分特异性物种的出现与其特殊的蜡质特征有关。5）高海拔地区菊科植物沉积较多的蜡质，特别是烷烃、酸和酯类物质含量高于低海拔植物，说明植物叶角质层蜡质为适应高海拔环境做出了适应性调整。6）考虑到高寒草甸之间尽管年平均气温存在差异，但环境压力变化不足以引起蜡质的适应性变化，进一步对分布在海拔44m到3900m环境的大车前和平车前种群进行了分析，发现环境变化梯度较大时，植物叶角质层蜡质将产生适应性变化。7）对低海拔地区草本植物的研究发现，叶角质层蜡质含量、组分及烷烃分布特征随着季节变化而产生变化，以提高其适应性。8）基于转录组高通量测序技术与qRT-PCR技术，筛选到12个草地早熟禾蜡质候选基因，如蜡质长链脂肪酸合成关键基因KCS、KCR、烷类合成关键基因CER1、一级醇类合成关键基因CER4。进一步分析发现，在逆境胁迫下，不同来源草地早熟禾蜡质基因表达量存在较大差异。9) 综合分析认为，高寒草地植物叶表皮蜡质在逆境有较高的可塑性，且蜡质含量及组分的变化是植物适应环境变化的结果，但我们尚不能证明这种适应是遗传变异还是一种表观遗传现象。