水稻根系果胶去甲酯化促进细胞壁磷再利用的机制探究

Study on Mechanism of Demethylation of Pectin Promoting Reutilization of Cell Wall Phosphorus in Rice(Oryza sativa) Root

在缺磷条件下,水稻根系细胞壁中的果胶组分能促进细胞壁磷的再利用,而其中的潜在机制仍有待进一步的研究。选取粳稻品种Nipponbare(Nip)和籼稻品种Kasalath(Kas)作为试验材料,研究了在缺磷条件下,水稻内源磷可利用水平的变化及其差异,并探究了产生这种差异的原因。结果表明:在缺磷处理后,水稻体内的可溶性磷含量迅速降低,而Nip根系和地上部的可溶性磷含量均一直高于Kas。同时Nip根系中释放出了更多的细胞壁磷,说明相对于Kas而言,Nip的内源磷再利用能力更强。缺磷胁迫时,与Kas相比,Nip可通过提高根系中的果胶甲酯酶活性,维持较低的果胶甲酯化度。体外试验又表明,甲酯化度越低的果胶,活化难溶态磷的能力越强。综上,缺磷胁迫下,水稻可通过提高根系果胶甲酯酶活性,将细胞壁的果胶甲酯化度维持在较低水平,从而促进细胞壁磷的释放来增加体内的可溶性磷含量,以供其他部位再利用。

【Objective】Plants can survive under the stress of phosphorus（P）-deficiency by coordinating inorganic phosphorus（Pi） acquisition, translocation from roots to shoots and redistribution within the plant. Studies in the past have demonstrated that cell-wall pectin contributes greatly to reutilization of rice cell-wall Pi under the stress of P-deficiency, but not how to. Therefore, it calls for further studies for clarification. 【Method】Two rice cultivars, ‘Nipponbare＇（Nip） and ‘Kasalath＇（Kas）, were selected as the subjects in the experiment to explore changes in endogenous phosphorus availability in the plants and difference between the two growing under the stress of phosphorus deficiency, and their specific reasons. Rice seedlings were cultured in the normal solution for two weeks and then moved into a nutrient solution with（＋P） or without（-P） P for cultivation for a week. Roots and shoots of the seedlings were sampled and pulverized in a pestle with a mortar in liquid nitrogen, separately and by cultivar too. A portion of each pulverized sample was prepared into cell walls. To determine availability of endogenous phosphorus, inorganic phosphate（Pi） was extracted from fresh roots and shoots, separately, with 5%（v/v） sulfuric acid solution, and cell wall P was extracted fromdried cell wall with 2 mol·L^-1 HCl solution. Concentration of phosphorus in the extracts was determined with the Mo-Sb colorimetric method. Then, in the late-on in vitro P analysis experiment, pectins different in methyl esterification degree were used to study relationship between the degree of methyl esterification degree of pectin and ability of the pectin to release hard-to-dissolve phosphorus. To follow changes in methyl esterification and activity of pectin methyl esterase（PME） in samples of the seedlings under the stress of P deficiency, root samples were treated with high-salt buffer solution to extract PME, which is able to trigger hydrolysis of pectin into methanol, which is then oxidized into formaldehyde. The activity of PME can be scaled according to the color reaction of formaldehyde with the Purpald reagent. Pectin in root was extracted from dried cell wall with hot water. Content of uronic acid in the pectin hydrolysates was determined with the colorimetric method, and methyl ester in the pectin hydrolysates was measured to characterize content of carbomethoxy in pectin. Then methyl-esterifcation degree of pectin was calculated based on the contents of uronic acid and carbomethoxy. 【Result】The present study shows that soluble phosphorus in rice was reduced rapidly when the plant suffered phosphorus deficiency. The content of soluble phosphorus in roots kept declining for 5 days and then leveled off, while the content in shoots started to decline obviously some time later, and the downward trend went on even after the 7^th day. However, Nip was always found to be higher than Kas in content of soluble phosphorus in the roots and shoots of the plants, and released more cell wall phosphorus from its root, which, indicates that Nip is higher than Kas in ability to recycle its endogenous phosphorus. The in vitro experiment shows that the lower the methyl esterification degree of the pectin, the higher the capacity of the pectin of activating hard-to-solve phosphorus. Once coming under the stress of P deficiency, Nip kept methyl esterification of pectin low in degree by improving the activity of pectin methyl esterase, while Kas did not vary much in activity of pectin methyl esterase and hence unable to lower pectin methyl esterification degree of the root. 【Conclusion】All the findings in this study suggest that under the stress of phosphorus deficiency, rice may keep cell wall pectin low in methyl esterification degree by improving the activity of pectin methyl esterase, thus promoting P release from root cell wall to raise the content of endogenous soluble phosphorus for use by other organs of the plant.