Reconstitution of sugar phosphate transport systems of Escherichia coli

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.     

Genetic and genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation in Arabidopsis

Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes to cope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated an Arabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almost all aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused by overexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) in both its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-type plants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pi starvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants can be induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genes include several important Pi signaling components and those that are directly involved in Pi transport, mobilization, and distribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other both synergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compelling evidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate the signaling mechanism that controls plant responses to this particular nutritional stress.     

致病疫霉效应蛋白Pi16275的功能研究

[目的]分析致病疫霉效应蛋白Pi16275的超量表达对病原菌致病性的影响,明确Pi16275的亚细胞定位,筛选Pi16275在植物中的互作靶标蛋白及靶标蛋白在抵御病原菌侵染过程中的作用,初步揭示Pi16275在病原菌侵染植物过程中的作用机制.[方法]利用农杆菌介导的烟草瞬时表达系统在烟草叶片表皮细胞中瞬时表达Pi162...     

The expression profile of genes in rice roots under low phosphorus stress

Phosphorus (P) is one of the most essential macronutrients required for plant growth. Although it is abundant in soil, P is often the limiting nutrient for crop yield potential because of the low concentration of soluble P that plants can absorb directly. The gene expression profile was investigated in rice roots at 6, 24 and 72 h under low P stress and compared with a control (normal P) profile, using a DNA chip of 60000 oligos (70 mer) that represented all putative genes of the rice genome. A total of 795 differentially expressed genes were identified in response to phosphate (Pi) starvation in at least one of the treatments. Based on the analysis, we found that: (i) The genes coding for the Pi transporter, acid phosphatase and RNase were up-regulated in rice roots; (ii) the genes involved in glycolysis were first up-regulated and then down-regulated; (iii) several genes involved in N metabolism and lipid metabolism changed their expression patterns; (iv) some genes involved in cell senescence and DNA or protein degradation were up-regulated; and (v) some transmembrane transporter genes were up-regulated. The results may provide useful information in the molecular process associated with Pi deficiency and thus facilitate research in improving Pi utilization in crop species.     

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efficiency

SUMMARY: Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.     

一种基于亚磷酸盐及其脱氢酶的植物磷利用和杂草控制系统的建立

磷是植物生长发育所必需的大量营养元素之一。土壤中存在大量的正磷酸盐 (Pi),但由于土壤化学和微生物转化使得土壤可利用磷的浓度并不高。土壤缺磷以及杂草的抗除草剂能力已成为当前农业可持续发展的重要限制因素,所以提高植物对土壤磷的吸收利用能力或寻求可替代正磷酸盐的磷肥以及开发新型杂草控制系统已成为亟待解决的问题。自然界中亚磷酸盐 (Phi) 是含量仅次于正磷酸盐的磷源,但仅在某些细菌中能被专一性的亚磷酸盐脱氢酶 (PTDH) 氧化利用,对植物的生长发育则具有抑制作用。利用这一特性,将从土壤宏基因组中直接扩增到的假单胞菌PTDH基因PsPtx通过农杆菌侵染法转入烟草中,并通过RT-PCR、垂直板幼苗生长、显性标记和生长竞争实验分析PsPtx转基因烟草的基因表达以及在Phi胁迫条件下的特性。结果显示,PsPtx在其转基因植株的根茎叶组织中都有几乎相同水平的表达;PsPtx转基因烟草不但能解除Phi对植物的毒害作用,并将它氧化成可用的Pi作为生长发育所需的磷源,而且在Phi胁迫条件下较野生型烟草有相当明显的生长竞争优势;另外PsPtx还具备成为植物遗传转化显性选择标记的优良特质。因此,PsPtx基因编码的亚磷酸盐脱氢酶可用于开发一种基于亚磷酸盐为磷肥和除草剂的植物磷利用和杂草控制系统,为当前农作物转基因研究存在的一些重大问题提供一个有效解决方案。     

The Arabidopsis gene hypersensitive to phosphate starvation 3 encodes ethylene overproduction 1

When plants are subjected to a deficiency in inorganic phosphate (Pi), they exhibit an array of responses to cope with this nutritional stress. In this work, we have characterized two Arabidopsis mutants, hps3-1 and hps3-2 (hypersensitive to Pi starvation 3), that have altered expression of Pi starvation-induced (PSI) genes and enhanced production of acid phosphatase (APase) when grown under either Pi sufficiency or deficiency conditions. hps3-1 and hps3-2, however, accumulate less anthocyanin than the wild type when grown on a Pi-deficient medium. Molecular cloning indicated that the phenotypes of hps3 mutants were caused by mutations within the ETO1 (ETHYLENE OVERPRODUCTION 1) gene. In Arabidopsis, ETO1 encodes a negative regulator of ethylene biosynthesis, and mutation of ETO1 causes Arabidopsis seedlings to overproduce ethylene. The ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or the ethylene perception inhibitor Ag(+) suppressed all the mutant phenotypes of hps3. Taken together, these results provide further genetic evidence that ethylene is an important regulator of multiple plant responses to Pi starvation. Furthermore, we found that a change in ethylene level has differential effects on the expression of PSI genes, maintenance of Pi homeostasis, production of APase and accumulation of anthocyanin. We also demonstrated that ethylene signaling mainly regulates the activity of root surface-associated APases rather than total APase activity.     

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2′-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2′-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2′-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.     

Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.     

Phyto-adaptogens protect against environmental stress-induced death of embryos from the freshwater snail Lymnaea stagnalis.

The main purpose of the studies presented in this paper is twofold: 1) to evaluate whether phyto-adaptogens (Acanthopanax senticosus and Rhodiola rosea) are able to exert a protective action against stress-induced death of embryos of the pond snail Lymnaea stagnalis; and 2) whether a possible protective action by phyto-adaptogens can be explained by the induction of heat shock proteins. Enhancement in resistance by phyto-adaptogens was studied by applying plant extracts for a period of 20 hours to 3-day old larvae of the pond snail Lymnaea stagnalis. Subsequently they were exposed to a high and toxic dose of different environmental stressors. The following stress conditions were selected: a physical stress condition (heat shock: 43 degrees C for 4 minutes), an oxidative stress condition (superoxide radicals induced by menadione (600 microM for 2 hours)) and heavy metal-induced stress (copper (150 microM for 1 hour) or cadmium (20 microM during 1 hour)). Both Acanthopanax and Rhodiola exert a strong protective action against a lethal heat shock. These adaptogens also significantly protect against the negative effect of superoxide radicals as induced by menadione. With respect to the protective action against exposure to heavy metals a small but significant protection was observed against intoxication with copper or cadmium by the phyto-adaptogens. In summary, there appears to be a difference in efficiency in enhancing resistance to the various stress conditions used (heat shock>menadione>copper>cadmium). Based on the results presented in this paper, we can conclude that phyto-adaptogens are able to enhance the resistance against the different stress conditions tested in developing individuals of Lymnaea. Although the degree to which resistance is enhanced appears to depend on the type of stressor applied, our results confirm the definition of phyto-adaptogens as being universal enhancers of non-specific resistance against different kinds of stress conditions. With respect to the mechanism of enhanced resistance, the question was asked whether this protective action is caused by an induction of heat shock proteins (hsps), which are known to be involved in tolerance and adaptation. The phyto-adaptogens did not induce the synthesis of any of the hsps, nor did they modulate the normal heat shock induced synthesis of these stress proteins. We conclude that it is unlikely that hsps play a major role in obtaining an enhanced state of resistance provided by phyto-adaptogens.     

A soybean β-expansin gene GmEXPB2 intrinsically involved in root system architecture responses to abiotic stresses

Root system architecture responds plastically to some abiotic stresses, including phosphorus (P), iron (Fe) and water deficiency, but its response mechanism is still unclear. We cloned and characterized a vegetative β-expansin gene, GmEXPB2, from a Pi starvation-induced soybean cDNA library. Transient expression of 35S::GmEXPB2-GFP in onion epidermal cells verified that GmEXPB2 is a secretory protein located on the cell wall. GmEXPB2 was found to be primarily expressed in roots, and was highly induced by Pi starvation, and the induction pattern was confirmed by GUS staining in transgenic soybean hairy roots. Results from intact soybean composite plants either over-expressing GmEXPB2 or containing knockdown constructs, showed that GmEXPB2 is involved in hairy root elongation, and subsequently affects plant growth and P uptake, especially at low P levels. The results from a heterogeneous transformation system indicated that over-expressing GmEXPB2 in Arabidopsis increased root cell division and elongation, and enhanced plant growth and P uptake at both low and high P levels. Furthermore, we found that, in addition to Pi starvation, GmEXPB2 was also induced by Fe and mild water deficiencies. Taken together, our results suggest that GmEXPB2 is a critical root β-expansin gene that is intrinsically involved in root system architecture responses to some abiotic stresses, including P, Fe and water deficiency. In the case of Pi starvation responses, GmEXPB2 may enhance both P efficiency and P responsiveness by regulating adaptive changes of the root system architecture. This finding has great agricultural potential for improving crop P uptake on both low-P and P-fertilized soils.     

Identification and functional reconstitution of phosphate: sugar phosphate antiport of Staphylococcus aureus

Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)