干旱胁迫下小麦(Triticumaestivum L.)幼苗中ABA和IAA的免疫定位及定量分析

Immunolocalization and Quantitation of ABA and IAA in the Organs of Wheat(Triticumaestivum L.) Under Drought Stress

【目的】干旱是农业生产中存在的严重问题,是制约作物生长和限制农业种植面积进一步扩大的最主要因素,探明植物抗旱机理,将为农业增产增收提供重要保障。本试验旨在分析干旱胁迫下小麦幼苗中(根、叶鞘、叶片)脱落酸(ABA)和吲哚乙酸(IAA)的定位及含量,探索其动态变化规律,为干旱地区作物栽培的激素调控、干旱条件下小麦的化学调控提供理论依据。【方法】以冬小麦品种鲁麦21号为材料,采用温室盆栽自然干旱法,以免疫组织化学定位方法研究干旱胁迫下小麦幼苗(根、叶鞘、叶片)中内源激素ABA和IAA的定位,并用间接酶联免疫吸附测定法(icELISA)进行内源ABA和IAA含量分析。通过比较免疫组织化学定位的化学信号强度和icELISA检测的内源ABA和IAA含量,验证本试验结果的有效性。【结果】称重法控制水分持续干旱4周后,对照组(CK)小麦植株直立生长,叶片健康呈绿色,长势良好。干旱处理组(ND)小麦植株矮小,叶片面积减小,有不同程度的萎蔫和发黄现象。干旱胁迫下小麦幼苗根、叶鞘、叶片中的ABA免疫组织荧光信号均强于对照,其中叶片强弱差异最显著,叶鞘次之,各器官中保卫细胞和维管的强弱对比最为明显;IAA免疫荧光信号强度也高于对照,但相比ABA不明显;用icELISA进行定量分析,比较免疫组织化学定位的信号强度和icELISA检测的内源激素含量表明,上述免疫组织化学定位结果与icELISA的测定结果相一致,其中,根中的ABA含量为61.51 ng·g-1 FW,是对照的2.82倍(21.8 ng·g-1 FW);叶鞘中ABA含量增加幅度较大,为175.35 ng·g-1 FW,是对照的3.65倍(48.02ng·g-1 FW);叶片中ABA含量增幅最大(512.12 ng·g-1 FW),为对照的10.16倍(50.42 ng·g-1 FW)。显著性分析表明,干旱胁迫与正常供水小麦幼苗根、叶鞘、叶片中ABA含量差异均呈极显著(P<0.01)。用icELISA对IAA进行检测,正常供水时,小麦幼苗根、叶鞘、叶片中的IAA含量分别为72.81、274.46和195.75 ng·g-1 FW。水分胁迫条件下,IAA含量增加但幅度不大,分别为123.56、400.48和417.30 ng·g-1 FW。叶片中IAA上升的幅度稍大为对照的2.13倍,根、叶鞘中的IAA含量分别为对照的1.70和1.46倍。显著性分析(t检验法)显示,在干旱胁迫与正常供水条件下小麦幼苗根中IAA含量差异不显著(P>0.05),但叶片和叶鞘中IAA含量差异极显著(P<0.01)。【结论】水分胁迫下小麦幼苗根、叶鞘、叶片中ABA和IAA含量均增加,ABA含量增加幅度较大,IAA较ABA含量增加幅度较小。二者之间协调的总趋势是向着气孔关闭,促进根系生长,减缓茎叶生长速率的方向发展,可有效的避免缺水伤害。

[Objective]Drought, a serious problem existed in agricultural production, is one of the most important factors limiting agricultural growth and further expanding the crops acreage. Study of the mechanism of plant drought resistance will provide an important safeguard for the increase of agricultural production and incomes. Phytohormone plays a significant role in plant resistance to water deficiency. Based on this study we may further understand the function of phytohormone in plants in response to drought stress, and provide a theoretical basis for the regulation of plant hormone in the production and cultivation of crop in arid regions.[Method]With wheat variety Lumai No.21 as material, immunohistochemical localization was used to investigate the distribution of endogenous hormones ABA and IAA in wheat organs（root, leaf sheath and leaf blade）under drought-stress. While an indirect competitive enzyme-linked immunosorbent assay （icELISA） was applied to quantitate the content changes of ABA and IAA in wheat organs. The chemical signal intensity of the immunohistochemical localization and the endogenous hormone concentration of the icELISA were compared for confirming the effect of the results.[Result]Four weeks after drought treatment with weighing method, the wheat in control group （CK） grew uprightly, leaves were healthy and green, and the whole plant grew well. Wheat in drought treatment group （ND） grew small, leaves areas were reduced and had different degrees of wilting and yellowing phenomenon. Immunohistochemical localization was used to investigate the distribution of endogenous hormones ABA and IAA in wheat organs. The results showed that, the intensity of the staining of ABA in drought-stress wheat organs was stronger than that in wheat of control group, and that the leaf was most noticeable, especially in stomata guard cells and vascular bundle tissue, then was the leaf sheath. Similar results in IAA were obtained, but it was not as noticeable as ABA. An icELISA was applied to quantitate the content changes of ABA and IAA. The result of immunohistochemical localization above is in accord with the result of icELISA. The ABA content in the root was 61.51 ng·g-1 FW, 2.82 times that of controls （21.8 ng·g-1 FW）. The ABA content in leaf sheath was larger, 175.35 ng·g-1 FW, 3.65 times that of controls （48.02 ng·g-1 FW）. The largest rise of ABA content （512.12 ng·g-1 FW） was in leaves, 10.16 times significantly higher than controls （50.42 ng·g-1 FW）. Variance analysis showed that the ABA contents in wheat seedling root, leaf sheath and leaf were significantly different （P〈0.01） under drought stress and normal water supply. The contents of IAA in wheat seedling root, leaf sheath, leaf were 72.81, 274.46 and 195.75 ng·g-1 FW under the normal water supply, respectively. The contents of IAA under water stress condition increased but not very much, and they were 123.56, 400.48 and 417.30 ng·g-1 FW, respectively. Significant analysis （t test） showed that the difference of IAA content in wheat seedling roots under drought stress and normal water supply condition was not significant （P〉0.05）, but the difference of IAA content in leaf and leaf sheath was extremely significant （P〈0.01）. [Conclusion]The contents of ABA and IAA tended to increase in the organs of wheat when subjected to water deficiency, and the level of ABA increased obviously, while that of IAA increased gently compared with ABA. The trend of the coordination between ABA and IAA is to cause stomatal closure, promote root growth and slow the growth rate to effectively protect against drought damage.