Combining Phytate/Ca~（2＋） Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat （Triticum aestivum L.） Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.     

Ca（NO3）2和NaCl胁迫下耐盐砧木嫁接黄瓜光合特性及碳同化关键酶基因表达分析

以白籽南瓜‘青砧1号’为砧木,黄瓜作接穗,黄瓜‘津优3号’自根嫁接株为对照,研究耐盐砧木嫁接对Ca(NO3)2和NaCl胁迫下黄瓜幼苗光合特性和CO2同化特征的影响。结果表明:Ca(NO3)2胁迫下黄瓜植株光合色素含量、净光合速率(Pn)、气孔导度(Gs)、水分利用率(WUE)、光系统Ⅱ实际光化学效率(ΦPSⅡ)、光化学猝灭系数(qP)、Rubisco活性和含量、碳同化关键酶基因表达水平均显著高于等渗的NaCl胁迫;Ca(NO3)2胁迫下植株的胞间CO2浓度(Ci)呈下降趋势,而NaCl胁迫下Ci呈上升趋势;Rubisco活性显著下降导致NaCl胁迫下黄瓜幼苗Pn显著下降。不同盐胁迫下,砧木嫁接株光合色素含量、Pn、Gs、Ci、WUE、ΦPSⅡ、Rubisco活性和含量均显著高于自根嫁接株,且可上调RbcL(Rubisco大亚基)、RCA(Rubisco活化酶)和PRK(5-磷酸核酮糖激酶)的基因表达,下调RbcS(Rubisco小亚基)的基因表达。因此认为,等渗Ca(NO3)2和NaCl胁迫下砧木嫁接株能维持较高的光系统Ⅱ活性和碳同化能力。     

榨菜(Brassica juncea)和紫甘蓝(Brassica oleracea)种间周缘嵌合体光合特性研究

对榨菜和紫甘蓝种间嫁接嵌合体的光合作用、叶绿素荧光、叶绿素含量、Rubisco的活性以及Rubisco酶大亚基和小亚基基因的转录水平等进行了测定分析。研究发现,种间周缘嵌合体TCC(茎尖分生组织细胞层LI-LII-LIII=TCC,T代表榨菜,C代表紫甘蓝;)的净光合速率为18.09μmol CO2·m-2·s-1,与亲本榨菜相当,但比亲本紫甘蓝高出24.8%。嵌合体的气孔导度和胞间CO2浓度显著高于2个亲本。叶绿素荧光参数中光系统II的实际电子传递量子效率(ФPSⅡ)和光化学猝灭系数(qP)在榨菜中最高,而嵌合体和紫甘蓝的这2个参数基本一致。对叶绿素含量测定后发现,嵌合体的叶绿素a、b及总含量与榨菜比较接近,比紫甘蓝高97%。TCC嵌合体Rubisco酶的初始活性和总活性处于榨菜(最高)和紫甘蓝(最低)之间,为1.76和3.75μmol CO2·g-1·min-1。而TCC嵌合体的Rubisco酶大亚基和小亚基基因的mRNA转录丰度与榨菜和紫甘蓝相比明显增高。以上结果说明,与TCC嵌合体光合机构的层源亲本——紫甘蓝相比,其叶绿素含量升高、Rubisco酶的活性以及其大小亚基基因的转录水平的增强可能是导致其净光合速率提高的原因。可见,TCC嵌合体的异源表皮(来自榨菜)对改善其内部光合组织(来自紫甘蓝)的光合能力有很大的促进作用。     

小麦叶片Rubisco抗体的制备及其应用

利用DEAE TOYOPEARL 650M快流速阴离子交换层析技术,1d内可收集高纯度的Rubisco。以纯化的Rubisco为抗原,免疫新西兰白兔,得Rubisco抗体。以Rubisco抗体建立单向免疫扩散法,定量测定Rubisco含量,研究小麦叶片光合作用日变化过程中Rubisco含量及其活力的变化。结果表明:单向免疫扩散法可用于小麦叶片Rubisco含量的测定。     

Rubisco活化酶免疫单扩散定量分析研究

利用纯化的Rubisco活化酶，制备了该酶兔抗体，并研究了该酶免疫单扩散定量技术。结果表明，在Rubisco活化酶0～200μg/mL的浓度下，酶量与沉淀圈面积成正比，酶的粗提液经35%的硫铵沉淀后可用免疫单扩散法定量。抽穗后水稻剑叶Rubisco活化酶含量在9d内下降缓慢，随后下降迅速。     

钾元素对植物光合速率、Rubisco和RCA的影响

钾离子可提高叶肉细胞渗透势，增加植物叶片水势，减少气孔阻力，增多叶绿体内基粒，促进光合电子传递及光合磷酸化，提高光合关键酶二磷酸核酮糖羧化酶／加氧酶（Rubisco)和Rubisco活化酶（RCA）的含量和活性，明显降低量子需要量，提高光合速率。Rubisco是光合速率的关键酶，RCA对Rubisco活性有重要的调节作用。低钾主要导致Rubisco及RCA含量均降低，限制了体内Rubisco的活化和Rubisco总活性，降低了Rubisco的初始活性，使CO2固定作用低于Rubisco的最大能力，制约了光合速率和生物产量的提高。同时钾离子对Rubisco和RCA没有直接的活化作用。参50     

稻叶衰老期间光合衰退的主要内在因素

本文追踪了稻叶衰老过程中叶片光合作用，Ｒｕｂｉｓｃｏ初始羧化活力，总羧化活力及含量，可溶性蛋白和叶绿素含量的变化，以明确哪一个是叶片衰老期间光合衰退的主要原因．结果表明，尽管光合速率与上述因子都有极显著的直线正相关，但其相关系数依次降低，净光合速率与Ｒｕｂｉｓｃｏ初始羧化活力，Ｒｕｂｉｓｃｏ总羧化活力，Ｒｕｂｉｓｃｏ含量，可溶性蛋白和叶绿素含量的决定系数（ｒ￣２）分别为０．９１，０．９０，０．７８，０．７３和０．７１．在净光合速率与Ｒｕｂｉｓｃｏ初始羧化活力之间不仅有最高的决定系数，而且无论第１０叶还是剑叶的数据都较合理地分布在回归线两侧，这表明稻叶衰老期间Ｒｕｂｉｓｃｏ初始羧化活力下降是光合衰退的最直接的内在因素．     

Screening of High kcat Rubisco among Poaceae for Improvement of Photosynthetic CO2 Assimilation in Rice

Abstract

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major limitation of photosynthetic CO₂ assimilation in C₃ plants. In order to find useful Rubisco for improvement of photosynthesis in rice under elevated CO₂, we analyzed the catalytic turnover rate (k_cat) of Rubisco in Poaceae including C₃ alpine plants, C₃ cold-resistant plants and C₄ plants. Rubisco in these plants showed 1.1- to 2.8-fold higher k_cat than that in rice. However, the most of high k_cat Rubisco also showed a higher km for CO₂ (Kc) than that of rice, indicating that increase in k_cat led to decrease in the affinity for CO₂. Rubisco in Festuca ovina, Phleum pratense and Sorghum bicolor showed relatively high k_cat to Kc. Although the k_cat of Rubisco in F. ovina and P. pratense was not so high (1.5-1.6 fold relative to rice), the Kc was comparable to that in rice and the amino acid sequence of RbcL shared higher identity to that in rice than that in S. bicolor. By contrast, Rubisco of S. bicolor showed considerably high k_cat (2.5-fold relative to rice), which is considered to be the most important factor for improvement of photosynthesis. In our estimation, the expression of high k_cat Rubisco of F. ovina and S. bicolor in rice could significantly enhance CO₂ assimilation at Ci of 50 Pa, the level assumed to be reached by the middle of this century.