CO<Subscript>2</Subscript> exchange and structural organization of chloroplasts under hypothermia in potato plants transformed with a gene for yeast invertase

Growth, CO₂ exchange, and the ultrastructure of chloroplasts were investigated in the leaves of potato plants (Solanum tuberosum L., cv. Désirée) of wild type and transformed with a gene for yeast invertase under the control of patatin class I B33 promoter (for apoplastic enzyme) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. At a temperature of 22°C optimal for growth, the transformed plants differed from the plants of wild type in retarded growth and a lower rate of photosynthesis as calculated per plant. On a leaf dry weight basis, photosynthesis of transformed plants was higher than in control plants. Under hypothermia (5°C), dark respiration and especially photosynthesis of transformed plants turned out to be more intense than in control material. After a prolonged exposure to low temperature (6 days at 5°C), in the plants of both genotypes, the ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplasts and starch grains rose, and the area of plastoglobules decreased; in transformed plants, these changes were more pronounced. By some ultrastructural characteristics: a reduction in the cold of relative total area of sections of starch grains and plastoglobules (in percents of the chloroplast section area) and in the number of granal thylakoids (per a chloroplast section area), transformed plants turned out to be more cold resistant than wild-type plants. The obtained results are discussed in connection with changes in source-sink relations in transformed potato plants. These changes modify the balance between photosynthesis and retarded efflux of assimilates, causing an increase in the intracellular level of sugars and a rise in the tolerance to chilling.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.     

川西亚高山森林林窗不同时期土壤转化酶和脲酶活性的特征

为了解川西亚高山森林林窗对不同时期土壤生态过程的影响,于2012年6月—2013年5月期间,根据温度动态过程,对比研究了生长季节(土壤完全融化期、生长季节前期和生长季节后期)与非生长季节(冻结初期、深冻期和融化期)川西亚高山粗枝云杉(Picea asperata)人工林林窗中心、林缘和林下土壤有机层和矿质土壤层转化酶和脲酶活性变化过程。结果表明:林窗不同区域中,土壤有机层转化酶活性均高于矿质土壤层;在生长季节,土壤有机层和矿质土转化酶活性表现为:林窗中心林下林缘,而脲酶活性表现为:林窗中心林缘林下。冻结初期和深冻期林窗中心土壤转化酶活性均高于林缘和林下,而在融化期林下转化酶活性高于林窗中心和林缘;冻结初期和融化期林下土壤脲酶活性显著高于林窗中心和林缘,而在深冻期林窗不同区域土壤脲酶活性没有显著差异。林窗不同区域在不同时期对土壤转化酶和脲酶活性的响应有着深刻影响。     

‘台农1号’芒果果实发育过程中的糖分积累与相关酶活性研究

以‘台农1号’芒果为材料,测定了果实生长发育过程中淀粉、蔗糖、葡萄糖和果糖含量以及淀粉酶、蔗糖代谢相关酶———酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分与酶活性的关系进行了分析.结果显示,(1)台农1号芒果果实属于单S型生长曲线,发育前期主要积累淀粉、葡萄糖和果糖,果实成熟软化时,淀粉酶活性降至最低,淀粉水解,蔗糖快速积累.(2)酸性转化酶活性在果实整个发育过程中维持最高,完熟时略有降低;蔗糖磷酸合成酶在果实发育前期略有降低,完熟时升至最高;蔗糖合成酶和中性转化酶活性在整个发育期一直很低且较稳定.(3)淀粉含量与淀粉酶活性呈显著正相关,与SPS活性呈极显著负相关,蔗糖、葡萄糖含量均与SPS、SS呈显著、极显著的正相关;果糖含量与SS呈极显著的正相关.研究表明,芒果成熟时淀粉分解、酸性转化酶活性的降低,且蔗糖合成酶和蔗糖磷酸合成酶活性的增加是引起果实蔗糖积累的主要因子.     

Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 μm and 50 μm As₂O₃) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.     

Expression of a plant cell wall invertase in roots of Arabidopsis leads to early flowering and an increase in whole plant biomass

In order to enhance sink strength, we expressed a heterologous plant cell wall invertase (CrCIN1) under the control of a root-specific promoter (ppyk10) in Arabidopsis thaliana. Slightly elevated apoplastic invertase activity resulted in apparent phenotypic changes. Transgenic plants developed more secondary roots and subsequently, possibly because of a higher capacity to acquire nutrients, a higher shoot and whole plant biomass. Furthermore, an early flowering phenotype was detected. The data presented here demonstrate that it is possible to modulate carbohydrate metabolism by ectopic expression of cell wall invertases and thereby influence sink organ size and whole plant development.     

Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.     

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H⁺-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.