Investigating salinity tolerance in alfalfa (Medicago sativa L.): modern synchrotron and molecular approaches

我的长期研究目标是培育具有抗非生物和生物胁迫的苜蓿品种。我的短期（5年）目标是研究紫花苜蓿如何在整个植物水平上耐受土壤盐度。我认为在盐胁迫下，紫花苜蓿叶、茎和根中元素离子和有机溶质的时空分布在耐盐和敏感品种之间会有所不同。耐盐紫花苜蓿必须通过有机分子或K+和Ca2+离子进行独特的渗透调节，以减少叶片中的离子毒性。我还研究了基因表达与叶片生化变化和应激激素浓度之间是否存在联系。***为了验证我的假设，将两种耐盐性不同的苜蓿品种暴露在不同的盐溶液中，并在盐处理后定期取样。基于同步辐射（SR）的相衬x射线成像、中红外、软x射线和x射线荧光成像将在加拿大光源（sas卡通，加拿大）用于量化和定位元素离子、有机溶质，并检测它们在苜蓿组织中的时空分布。同步辐射-傅里叶变换红外光谱（SR-FTIR）将用于绘制碳水化合物和-螺旋-片状蛋白质二级结构比例的变化。本研究将对盐胁迫下紫花苜蓿叶片的基因表达进行转录组学分析，并基于RNA-seq分析和模型豆科植物“Medicago truncatula”的基因组信息，确定紫花苜蓿耐盐性的候选基因，并验证所选基因的表达。****紫花苜蓿是世界上种植最广泛的饲料豆科植物。在世界许多地区，土壤盐分是限制苜蓿产量的主要非生物因素。了解盐胁迫下紫花苜蓿组织的生化组成对研究其抗逆性生理至关重要，但在整个植物水平上的定量研究以及在组织、细胞和亚细胞水平上这些化合物的空间定位信息有限。全面了解紫花苜蓿的耐盐性是培育新品种的必要条件。****本项目旨在培养高素质人才，包括1名牧草胁迫生理学和同步加速器应用方向的硕士研究生，1名苜蓿基因组学方向的博士研究生，2名实验设计、统计学、植物生理学和基础生物技术方面的本科毕业论文学生。这一发现将促进我们对紫花苜蓿耐盐生理和遗传学的认识。这些基因的鉴定将有助于培育高耐盐苜蓿新品种。******

My long term research goal is to develop alfalfa (Medicago sativa L.) cultivars with tolerance to abiotic and biotic stresses. My short-term (5-year) objectives for this proposal are to investigate how alfalfa tolerates soil salinity at a whole plant level. I assume spatial and temporal distributions of elemental ions and organic solutes in the leaves, stems, and roots of alfalfa will vary between salt tolerant and susceptible cultivars under a salt stress. A salinity tolerant alfalfa must have a unique osmotic regulation through organic molecules or K+ and Ca2+ ions to reduce ion toxicity in the leaves. I also investigate if links exist among gene expression and biochemical changes and stress hormone's concentration in the leaves. *** To test my hypothesis, two alfalfa cultivars with contrasting tolerance to salinity will be exposed to different salt solutions and plants will be sampled periodically following salt treatments. Synchrotron radiation (SR) based phase contrast X-ray imaging, mid-infrared, soft X-ray, and X-ray Fluorescence mapping at the Canadian Light Source (Saskatoon, Canada) will be applied to quantify and localize elemental ions, organic solutes and detect their spatial and temporal distributions in alfalfa tissues. Synchrotron radiation-Fourier transform infrared (SR-FTIR) spectroscopy will be used to map changes in the ratios of carbohydrates, and -helix to -sheet protein secondary structures. A transcriptomic analysis of gene expression in alfalfa leaves in response to salt stress will be performed, and based on the RNA-seq analysis, and available genomic information of the model legume, Medicago truncatula, identify candidate genes to salinity tolerance in alfalfa and validate the expression of selected genes.**** Alfalfa is the most widely planted forage legume in the world. Soil salinity is a major abiotic factor that limits alfalfa production in many regions of the world. Knowledge of the biochemical composition of alfalfa tissues under salt stress is critical for the study of stress tolerance physiology, but there is a limited information available on quantification in whole plant level and spatial localization of those compounds at the tissue, cellular and subcellular levels. A comprehensive understanding of salinity tolerance in alfalfa is needed for development of novel cultivars.**** The proposed project has a goal to train Highly Qualified Personnel (HQP) including one M.Sc. student in forage stress physiology and synchrotron application, and one Ph.D. student in alfalfa genomics, two undergraduate thesis students in experimental design, statistics, plant physiology, and basic bio-technology skills. The findings will advance our understanding of the physiology and genetics of salt tolerance in alfalfa. The identification of genes will be useful for breeding new alfalfa varieties with high salinity tolerance.******