冯康,
李倩,
薛惠云,
李丽杰,
张志勇,
河南科技学院/河南省现代生物育种协同创新中心/河南省棉麦分子生态和种质创新重点实验室 新乡 453003
基金项目: 国家自然科学基金项目31571600
详细信息
作者简介:王果, 主要研究方向为质外体代谢组学和蛋白质组学。E-mail:18336063077@163.com
通讯作者:张志勇, 主要研究方向为作物逆境生理。E-mail:z_zy123@126.com
中图分类号:S311计量
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被引次数:0
出版历程
收稿日期:2020-02-17
录用日期:2020-04-09
刊出日期:2020-06-01
Improved methods for separating apoplastic washing fluid from roots and leaves in cotton seedlings
WANG Guo,FENG Kang,
LI Qian,
XUE Huiyun,
LI Lijie,
ZHANG Zhiyong,
Henan Institute of Science and Technology/Henan Collaborative Innovation Center of Modern Biological Breeding/Henan Key Laboratory for Molecular Ecology and Germplasm Innovation of Cotton and Wheat, Xinxiang 453003, China
Funds: the National Natural Science Foundation of China31571600
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Corresponding author:ZHANG Zhiyong, E-mail:z_zy123@126.com
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摘要
摘要: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/1 min，真空后恢复到正常压强约110 s。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。
Abstract: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.
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图1真空渗透后棉花幼苗叶片颜色的状态
三角形箭头指示真空渗透后颜色变深, 圆形箭头指示真空渗透后颜色没变化。左侧图片显示约1/2的叶片颜色变深, 定义为半绿; 右侧图片显示几乎整个叶片颜色变深, 且叶片坚挺, 定义为全绿; 如叶片变软, 定义为过绿。
Figure1.Cotton seedling leaf color status after vacuum infiltration
The triangle arrow shows the color darkening after vacuum infiltration, and the round arrow shows no color darkening. Left leaf photo shows about half leaf darkening, defined as half green; right leaf photo shows whole leaf darkening and firmness, defined as whole green; if leaf is floppy, defined as over green.
下载: 全尺寸图片幻灯片表1棉花幼苗根系和叶片不同取样方式的AWF体积、可溶性蛋白含量比值、MDH活性比值及相对电导率
Table1.AWF volume, ratios of soluble protein content and MDH enzyme activity in AWF to SWF and relative conductivity under sampling methods of organ segments and whole organ of cotton seedlings
| 器官 Organ | 取样方式 Sampling method | 真空强度/时间和离心力/时间 Vacuum intensity/time and centrifugal force/time | AWF体积 AWF volume [μL?g–1(FW)] | 可溶性蛋白含量比值 Soluble protein content ratio (%) | MDH活性比值 MDH enzyme activity ratio (%) | 相对电导率 Relative electric conductivity (%) |
| 根系 Root | 根段 Root segments | –80 kPa/1 min 800 ×g/10 min | 106.53±23.02a | 0.40±0.04a | 0.23±0.05a | 12.80±1.37a |
| 整根 Whole root | 91.06±13.61a | 0.13±0.06b | 0.05±0.01b | 10.70±0.87a | ||
| 叶片 Leaf | 叶段 Leaf segments | –80 kPa/1 min 400 ×g/5 min | 191.06±39.4a | 2.34±0.46a | 0.89±0.18a | 11.07±0.83a |
| 整叶Whole leaf | 120.00±11.5b | 0.39±0.04b | 0.09±0.02b | 1.85±0.17b | ||
| 可溶性蛋白含量比值: AWF中可溶性蛋白含量与SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性与SWF中MDH活性的比值。不同小写字母表示同一器官同列指标在不同取样方式间差异显著(P < 0.05)。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences for the same organ between two sampling methods (P < 0.05). | ||||||
下载: 导出CSV表2棉花幼苗根系和子叶-60 kPa真空渗透前后的颜色变化、增重情况及AWF稀释因子
Table2.Fresh weight increase, color change and AWF dilution factor values for whole root and leaf of cotton seedlings after -60 kPa vacuum infiltration
| 器官 Organ | 真空渗透液 Vacuum permeate | 真空时间 Vacuum time (min) | 颜色 Color | 增重 Weight increment (%) | 稀释因子 Dilution factor |
| 根系 Root | A | 1 | 不变No change | –1.22±0.09a | 1.03±0.01a |
| B | 1 | 不变No change | –1.13±0.29a | 1.05±0.02a | |
| 叶片 Leaf | A | 1 | 半绿Half green | 33.94±3.14c | 2.20±0.17c |
| B | 1 | 半绿Half green | 31.50±1.27c | 2.12±0.14c | |
| A | 2 | 全绿Whole green | 41.41±2.51a | 3.34±0.02a | |
| B | 2 | 全绿Whole green | 37.63±1.38b | 2.57±0.03b | |
| A: 50 μmol?L–1靛蓝二磺酸钠的水溶液; B: 50 μmol?L–1靛蓝二磺酸钠的磷酸缓冲液(50 mmol?L–1, pH 6.9)。不同小写字母表示同一器官同列指标在不同真空渗透液间差异显著(P < 0.05)。A: water solution containing 50 μmol?L–1 indigotindisulfonate sodium; B: phosphate solution (pH 6.9) containing 50 μmol?L–1 indigotindisulfonate sodium. Different lowercase letters in the same column indicate significant differences for the same organ between two vacuum permeates (P < 0.05). | |||||
下载: 导出CSV表3不同真空强度处理1 min后的棉花幼苗叶片AWF体积、可溶性蛋白含量比值和MDH活性比值
Table3.AWF volume, ratios of soluble protein content and MDH activity in AWF to SWF of cotton seedling leaf under different vacuum intensities for 1 minute
| 真空强度 Vacuum intensity (kPa) | AWF体积 AWF volume [μL?g–1(FW)] | 可溶性蛋白含量比值 Soluble protein content ratio (%) | MDH活性比值 MDH enzyme activity ratio (%) |
| –30 | 69.80±9.02c | 0.44±0.01c | 0.15±0.002b |
| –60 | 177.54±6.07a | 0.82±0.04a | 0.27±0.011a |
| –90 | 128.85±9.53b | 0.68±0.01b | 0.27±0.018a |
| 可溶性蛋白含量比值: AWF中可溶性蛋白含量与SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性与SWF中MDH活性的比值。同列不同小写字母表示不同真空强度间差异显著(P < 0.05)。离心力/时间为400 ×g/5 min。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among different vacuum intensities (P < 0.05). Centrifugation strength/time is 400 ×g/5 min. | |||
下载: 导出CSV表4–60 kPa强度真空处理不同时间后的棉花幼苗叶片AWF体积、可溶性蛋白含量比值和MDH活性比值
Table4.AWF volume, ratios of soluble protein content and MDH activity in AWF to SWF of cotton seedling leaf under –60 kPa vacuum intensity for different times
| 真空时间 Vacuum time (min) | 叶片颜色 Leaf color | AWF体积 AWF volume [μL?g–1(FW)] | 可溶性蛋白含量比值 Soluble protein content ratio (%) | MDH活性比值 MDH enzyme activity ratio (%) |
| 1 | 半绿Half green | 177.54±6.07c | 0.82±0.03b | 0.27±0.01c |
| 2 | 全绿Whole green | 365.23±16.90a | 0.78±0.03b | 0.71±0.04b |
| 4 | 过绿Over green | 202.10±13.20b | 1.36±0.04a | 0.95±0.05a |
| 可溶性蛋白含量比值: AWF中可溶性蛋白含量与SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性与SWF中MDH活性的比值。同列不同小写字母表示不同真空时间间差异显著(P < 0.05)。离心力/时间长度为400 ×g/5 min。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among different vacuum times (P < 0.05). Centrifugation force/time is 400 ×g/5 min. | ||||
下载: 导出CSV表5棉花幼苗叶片真空结束后恢复到正常大气压所用时间不同时的相对电导率
Table5.Relative electric conductivity of cotton seedling leaf at different pressure recovery times after the end of vacuum infiltration
| 真空强度/真空时间 Vacuum intensity (kPa)/vacuum time (min) | 恢复时间 Recovery time (s) | 相对电导率 Relative conductivity (%) |
| 101(正常大气压normal pressure)/1 | 0 | 1.16±0.18b |
| –60/1 | 110 | 1.05±0.10b |
| –60/1 | 50 | 2.49±0.29a |
| 同列不同小写字母表示差异显著(P < 0.05)。Different lowercase letter s in the same column indicate significant differences (P < 0.05). | ||
下载: 导出CSV表6不同离心力/离心时间组合条件下棉花AWF体积、可溶性蛋白含量比值和MDH活性比值
Table6.AWF volume, ratios of soluble protein content and MDH enzyme activity in AWF to SWF of cotton seedling under different regimes of centrifugal force and time
| 器官 Organ | 离心力/时间 Centrifugation force (×g/ time (min) | AWF 体积 AWF volume [μL?g–1(FW)] | 可溶性蛋白含量比值 Soluble protein content ratio (%) | MDH活性比值 MDH enzyme activity ratio (%) |
| 根系 Root | 400/5 | 69.567±3.90c | 0.013±0.001d | 0.016±0.005d |
| 400/10 | 80.809±0.86bc | 0.057±0.003cd | 0.022±0.003cd | |
| 400/20 | 81.500±6.36bc | 0.054±0.013cd | 0.022±0.002cd | |
| 800/5 | 69.041±3.62c | 0.072±0.022bcd | 0.037±0.008c | |
| 800/10 | 87.103±6.78b | 0.102±0.009bc | 0.048±0.005c | |
| 800/20 | 93.000±2.83b | 0.134±0.014b | 0.031±0.006c | |
| 1 200/5 | 88.590±1.05b | 0.124±0.006b | 0.195±0.004a | |
| 1 200/10 | 114.338±7.34a | 0.218±0.007a | 0.101±0.023b | |
| 叶片 Leaf | 400/5 | 177.538±6.07a | 0.818±0.038a | 0.267±0.011b |
| 400/10 | 186.395±8.44a | 1.040±0.025a | 0.836±0.057a | |
| 800/5 | 181.864±7.35a | 0.847±0.030a | 0.663±0.044a | |
| 可溶性蛋白含量比值: AWF中可溶性蛋白含量与SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性与SWF中MDH活性的比值。同列不同小写字母表示同一器官不同组合条件间差异显著(P < 0.05)。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among regimes for the same organ (P < 0.05). | ||||
下载: 导出CSV参考文献
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