叶绿体发育和光对小麦叶谷氨酰胺合成酶基因表达的影响

利用电镜、ＤＥＡＥ－纤维素柱层析技术和小麦叶谷氨酰胺合成酶（ＧＳ）酶活性测定,研究了小麦叶片不同发育梯度的叶绿体超微结构和ＧＳ同功酶活性之间的关系。结果表明,从叶基至叶尖,随着叶绿体的成熟,净光合率增加,ＧＳ活性增加。各发育阶段离体叶绿体的３Ｈ－Ｕｒａ,３Ｈ－Ｌｅｕ 掺入试验和ＧＳ的Ｎｏｒｔｈｅｒｎ ｂｌｏｔ表明,基部是基因表达活性最高的部位。ＧＳｍ ＲＮＡ 在叶绿体发育阶段最多,而ＧＳ酶活性则在成熟叶绿体的部位最高。对黄化苗进行光照,ＧＳｍ ＲＮＡ 和ＧＳ活性明显增加,７２小时达到正常绿苗同等水平。由此说明核编码的叶绿体ＧＳ基因为光调控基因,明显促进了叶绿体ＧＳ基因的转录,而后叶绿体ＧＳ合成量增加     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

光对水稻非光合组织谷氨酰胺合成酶同工酶表达的影响

以前的研究表明,高等植物叶绿体谷氨酰胺合成酶（GS2）受光调节,但叶片胞液GS（GS1）和非光合作用组织中的GS很少受光的影响,在本报道中,笔者运用GS活性染色和Western blotting研究了光对非光合作用组织水稻根GS同工酶表达的影响,在阳光的直接照射下以及在室内不同光照强度下,可以很清楚地观察到GSra和GS rb的活性带及其蛋白质带,但是,当用尼龙网档住阳光的直接照射下,GSrb的活性带和蛋白质带消失,当阳光被尼龙网遮挡住后,其光强度仍然比室内光照强度大得多,表明光照强度不是影响GSrb表达的主要因素,当分析生长在暗处以及生长在光／暗转换下的水稻幼苗根GS同工酶变化时,仍然可以观察到GSrb的在,在所有实验条件下,GSra都未发生明显变化,这些结果提示,光对GSrb表达的影响可能是由某些光谱相互作用所产生的未知因素造成的。     

Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees.

A poplar hybrid, Populus tremula x Populus alba, was transformed with the bacterial genes for either glutathione reductase (GR) (gor) or glutathione synthetase (GS) (gshII). When the gor gene was targeted to the chloroplasts, leaf GR activities were up to 1000 times greater than in all other lines. In contrast, targeting to the cytosol resulted in 2 to 10 times the GR activity. GR mRNA, protein, and activity levels suggest that bacterial GR is more stable in the chloroplast. When the gshII gene was expressed in the cytosol, GS activities were up to 100 times greater than in other lines. Overexpression of GR or GS in the cytosol had no effect on glutathione levels, but chloroplastic-GR expression caused a doubling of leaf glutathione and an increase in reduction state. The high-chloroplastic-GR expressors showed increased resistance to photoinhibition. The herbicide methyl viologen inhibited CO2 assimilation in all lines, but the increased leaf levels of glutathione and ascorbate in the high-chloroplastic-GR expressors persisted despite this treatment. These results suggest that overexpression of GR in the chloroplast increases the antioxidant capacity of the leaves and that this improves the capacity to withstand oxidative stress.     

蔗糖对水稻幼苗叶片谷氨酰胺合成酶和1,5-二磷酸核酮糖羧化酶/加氧酶的影响

报道了在光照和暗处培养下,不同的浓度的蔗水稻幼苗叶片GS及其同工酶、1,5－二磷酸核酮糖羧化酶／加氧酶（Rubisco）的影响。无论是在光照或在暗处,蔗糖对GS活性均有抑制作用,尤其是在较高蔗糖下作用更为明显;虽然Rubisco及可溶性蛋白的水平在光照和暗处有显著的差别,但蔗糖对其未见明显影响。NativePAGE与活性染色表明,在光照下或在暗处,蔗糖对GS2的抑制蔗糖浓度升同而加强,但对GS1未有明显影响。这些结果提示,在水稻幼苗生长中,蔗糖不能象不光一样诱导叶水GS活性及其同工酶表达。     

小麦HMW-GS1Dx5基因的克隆及其特异性表达

显微切割了普通小麦钢８２－１２２（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ２ｎ＝４２）具有１Ｄｘ５＋１Ｄｙ１０亚基的１Ｄ染色体长臂端,利用ＰＣＲ扩增得到了ＨＭＷ－ＧＳ１Ｄｘ５亚基的５（端４００ｂｐ序列片段．以此作为探针从基因的组织特异性和特定发育阶段的表达两个方面研究了ＨＭＷ－ＧＳ１Ｄｘ５基因表达的规律．结果表明,干种子及萌发种子中存在此基因,而在发育的幼苗中此基因未表达．ＨＭＷ－ＧＳ１Ｄｘ５基因可能从开花初期开始表达．ＨＭＷ－ＧＳ１Ｄｘ５基因在籽粒成熟期表达,然而在营养器官如叶片中未表达,其表达存在组织特异性．ＨＭＷ－ＧＳ１Ｄｘ５基因在蜡熟期籽粒表达水平最高,其次是乳熟期籽粒．从开花１５ｄ至蜡熟期籽粒,表达趋于增加．开花１５ｄ其ｍＲＮＡ水平是蜡熟期籽粒ｍＲＮＡ的２８％,灌浆期为４０％、乳熟期为７２％、完熟期为５４％．这为进一步研究其表达调控和改善小麦品质打下基础     

Differential gene expression of chloroplast and cytosolic phosphoglycerate kinase in tobacco

Northern blot analysis of RNA extracted from leaves of increasing age and different organs, indicates that genes encoding both isoenzymes of tobacco phosphoglycerate kinase (PGK, EC 2.7.2.3) are differentially expressed in a developmental and tissue-specific manner. The genes for both chloroplast PGK (chl-PGK) and cytosolic PGK (cyt-PGK) also show light-modulated gene expression in vivo. In dark-grown developing cotyledonary leaves of tobacco both PGK mRNAs are present, but only the concentration of the chl-PGK mRNA increased on illumination. In contrast, on transfer to darkness, the concentration of both mRNAs decreased in light-grown seedlings and then increased again on resumption of illumination.     

Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

Light-independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light-independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm-like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light- and dark-grown plants in good correlation with light-independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light-grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd-GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light- and dark-grown seedlings. Fd-GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light-grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non-green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast, development whereas compartmentation would be required in species with light-independent plastid development.     

水杨酸和茉莉酸甲酯对丹参幼苗叶片显微结构、光合及非结构糖积累的影响

研究了水杨酸(SA)和茉莉酸甲酯(MeJA)处理对丹参(Salvia miltiorrhiza Bunge)幼苗叶片显微结构、叶片光合能力及幼苗中非结构糖积累的影响.结果显示:SA处理增加了丹参幼苗叶片气孔密度;叶肉细胞排列紧密、体积减小,叶肉细胞内叶绿体数目减少,但叶绿体体积增大,叶绿体基粒片层结构的数目增加;叶片中叶绿素a、b含量、叶气孔导度、蒸腾速率以及净光合速率均增加;同时,幼苗根中和叶片中酸性转化酶活性降低,幼苗地上部分蔗糖含量及可溶性糖总量显著高于对照.MeJA处理减少了叶片气孔密度,气孔发育畸形;叶肉细胞间隙增大,栅栏细胞层数减少,叶肉细胞内叶绿体数目减少,叶绿体体积减小,叶绿体基粒片层结构被破坏;叶片中叶绿素a及类胡萝卜素含量、叶片的净光合速率低于对照,叶气孔导度、蒸腾速率增强;同时,幼苗根中及叶中酸性转化酶活性增加,幼苗根中蔗糖含量及可溶性糖总量显著低于对照.可见,SA处理能促进植物叶片显微结构发育,增强叶片光合能力,抑制蔗糖降解并促进蔗糖积累;而MeJA处理则破坏了植物叶片显微结构,降低了叶片光合能力,促进了蔗糖降解并减少蔗糖积累.     

Increased glutamine synthetase activity and changes in amino acid pools in leaves treated with 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR)

Feeding 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR) through the petiole of detached young barley leaves rapidly increased activities of NADH-nitrate reductase (NR) and glutamine synthetase (GS) in leaf extracts and at least partly prevented the usual slow decrease of these enzyme activities during prolonged illumination. Further, AICAR caused drastic changes in amino acid levels: glutamine and serine levels were increased whereas glutamate and glycine were decreased, probably indicating a higher GS activity and more rapid conversion of glycine into serine. The latter may be responsible for the higher ammonium contents found in AICAR treated leaves. We tentatively suggest that GS (located in the chloroplast) and glycine decarboxylase (located in the mitochondria) are regulated in a manner similar to NR. This is discussed in the light of recent reports that 14-3-3 isoforms exist in chloroplasts and that GS binds to 14-3-3s in vitro.     

林窗对热带雨林冠层树种绒毛番龙眼幼苗生长的影响

在林窗中央、林窗边缘和林冠下3种不同光照梯度的森林生境中,研究了西双版纳季节雨林冠层树种绒毛番龙眼幼苗的早期 (种子萌发后10周内) 生长和定居后 (实生苗生长3个月以上) 的生长特点.结果表明: 绒毛番龙眼幼苗在早期生长阶段,林窗中央的株高、基径、总干质量、单株叶面积和相对生长率最大,分别为24.45 cm、3.17 mm、0.79 g、122.45 cm²和14.78×10^-3 g·d^-1.林冠下根冠比 (0.87) 高于林窗中央 (0.20) ,可能是光照和水分共同作用的结果.林窗中央较强的光照有利于定居后幼苗的生长,株高、基径、总干质量、单株叶面积、相对生长率和净同化率均在林窗中央最大,实验结束时分别达到31.48 cm、3.80 mm、2.22 g、174.52 cm²、2.29×10^-3 g·d^-1和2.54×10^-5 g·cm^-2·d^-1.幼苗死亡可能与水分胁迫密切相关,由水分胁迫引起的幼苗死亡率在林冠下最高 (26.88%),但林冠下由脊椎动物捕食引起的幼苗死亡率较低(2.93%),从而使林冠下幼苗的最终存活率最高 (70.19%).光照是影响绒毛番龙眼幼苗形态学调节的重要因素, 林窗中央不同生长阶段幼苗的比叶面积最低,但相对生长率和净同化率最大.水分胁迫和光照在幼苗定居后仍是影响幼苗生物量分配的重要因素, 林窗边缘幼苗的根冠比最高 (0.33).