棉花根系和叶片质外体汁液分离方法的改进

质外体汁液(apoplastic washing fluid, AWF)在植物生长发育和抵抗生物及非生物逆境方面发挥着重要作用。目前普遍采用真空渗透离心法提取质外体汁液,但具体提取流程和条件却因植物培养条件、种类和器官等不同而不同。本试验以营养液培养的棉花幼苗为材料,改进了棉花根系和叶片AWF分离的取样方法、渗透条件和离心参数等。结果表明,相对于通常的非整体取样,改良后整体取样简单易操作且显著降低了质外体与共质体的苹果酸脱氢酶(malate dehydrogenase, MDH)活性比值,更适宜开展质外体汁液组分研究。根据真空渗透鲜重增加和AWF稀释因子,进一步证明根系不用真空渗透;而叶片需真空渗透,适宜真空强度/时间为-60 kPa/1min,真空后恢复到正常压强约110s。证明叶片颜色变深面积可作为判定真空渗透强度或时间是否适宜的一种简易可行方法。最后,AWF体积、质外体与共质体的可溶性蛋白含量比值和MDH活性比值综合分析表明,棉花根系适宜离心力大小/时间长度为800×g/10～20 min,叶片为400×g/5 min。本改进方法为棉花AWF组分,如蛋白质组学和代谢组学等研究结果的准确性和可靠性奠定了基础,为其他作物AWF分离方法的优化提供了参考。

Improved methods for separating apoplastic washing fluid from roots and leaves in cotton seedlings

Apoplast washing fluid (AWF) contains minerals, metabolites, and proteins that plays an important role in plant growth and development, as well as provides biotic and abiotic stress resistance. AWF extraction is the basis of exploring the function of AWF constituents. It is generally performed via vacuum infiltration-centrifugation technique; which varies in processes and detailed parameters depending on the plan species, organs, and culture conditions. Hydro-cultured cotton seedlings were used to investigate AWF separation processes and parameters suitable for cotton root and leaf development, and further improve methods for cotton root or leaf AWF separation. Compared with traditionally split sampling (i.e., splitting samples into segments or pieces), sampling a complete unit was simple and significantly decreased the ratio of malate dehydrogenase (MDH) activity in AWF to symplast washing fluid (SWF), which usually is used to affirm the degree of AWF substances polluted by SWF; indicating that AWF components would better to examine. Furthermore, the fresh weight increments and the AWF diluting factor after vacuum infiltration of the roots had no significant change, but significantly increased in the leaves. This indicates that vacuum infiltration is only essential for leaves, with a vacuum strength/time at -60 kPa/1 min, and about 110 s recovery from vacuum to normal atmospheric pressure. Leaf areas with dark color increased with vacuum intensity or time, which could be used as a simple indicator for determining the suitability for AWF separation. Finally, comprehensive analyses of the AWF volume, soluble protein content ratio and MDH activity ratio of AWF to SWF indicated that the suitable centrifuge forge/time was 800×g/10-20 min for the root, and 400×g/5 min for the leaf. This refined and optimized method will lay down the foundation for efficient study of AWF components such as the accuracy and reliability of proteomics and metabolomics. The approach towards establishing this method should allow it to be generally applicable to other plants.