Salt stress induces Kranz anatomy and expression of C4 photosynthetic enzymes in the amphibious sedge Eleocharis vivipara

Photosynthesis Research - Eleocharis vivipara Link is a unique amphibious leafless plant of the Cyperaceae. The terrestrial form develops culms with Kranz anatomy and C4-like traits, while the...     

Expression of C3 and C4 photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate,orthophosphate dikinase

Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Topology of the global regulatory network of carbon limitation in Escherichia coli

One fundamental shortcoming of biotechnological processes operating under carbon-limiting conditions is the high-energy demand (maintenance) of the cells. Although the function of the central carbon metabolism in supplying precursors and energy for biosynthesis has been thoroughly characterized, its regulation and dynamic behaviour during carbon-limited growth has not yet been revealed. The current work demonstrates a time series of metabolic flux distributions during fed-batch cultivation of Escherichia coli K-12 W3110 applying a constant feed rate. The fluxes in glycolysis, pentose phosphate pathway and biosynthesis fell significantly, whereas TCA cycle fluxes remained constant. The flux redistribution resulted in an enhanced energy generation in the TCA cycle and consequently, in a 20% lower biomass yield. The intracellular alarmones ppGpp and cAMP accumulated in large quantities after the onset of nutrient limitation, subsequently declining to basal levels. The network topology of the regulation of the central metabolic pathways was identified so that the observed metabolic and regulatory behaviour can be described. This provides novel aspects of global regulation of the metabolism by the cra, crp and relA/spoT modulons. The work constitutes an important step towards dynamic mathematical modelling of regulation and metabolism, which is needed for the rational optimization of biotechnological processes.     

C4‐type gene expression is not directly dependent on Kranz anatomy in an amphibious sedge Eleocharis vivipara Link

Eleocharis vivipara Link alters its photosynthetic mode depending on the growth environment. It utilizes C4 photosynthesis when grown under terrestrial conditions (terrestrial form) and C3 photosynthesis when grown under submerged conditions (submerged form). The photosynthetic organ (the mature internodal region of the culm) of the terrestrial form shows typical Kranz anatomy with well-developed bundle sheath cells, while the bundle sheath cells of the submerged form are not developed. In the mature internodal region of the terrestrial form, expression of the genes encoding two carboxylases, the small subunit of ribulose 1,5-bisphosphate carboxylase (RbcS) and phosphoenolpyruvate carboxylase (Ppc), occurred mainly in bundle sheath cells and in mesophyll cells, respectively, as seen in a typical C4 leaf. In the submerged form, RbcS was expressed in both bundle sheath cells and mesophyll cells, and no expression of Ppc was observed. In the immature internodal region with undeveloped bundle sheath cells, both life forms showed the same expression pattern as in C3 plants: RbcS expression was localized in mesophyll cells and no Ppc expression was observed. The C4-type expression pattern was established concomitantly with the development of bundle sheath cells during tissue maturation in the terrestrial internode. In contrast to the terrestrial form, the submerged form maintains C3-type gene expression during tissue maturation. When the terrestrial culm was submerged, a region of transition from the terrestrial form to the submerged form was established in newly sprouting culms. In this transitional region, C4-type expression of the two carboxylase genes was still maintained even though the development of bundle sheath cells was repressed. This observation suggests that the C4-type cell-specific gene expression pattern does not depend on the formation of Kranz anatomy.     

Induction of kranz anatomy and C4-like biochemical characteristics in a submerged amphibious plant by abscisic acid

The amphibious leafless sedge Eleocharis vivipara develops C4-like traits as well as Kranz anatomy under terrestrial conditions, but it develops C3-like traits without Kranz anatomy under submerged conditions. When submerged plants are exposed to aerial conditions, they rapidly produce new photosynthetic tissues with C4-like traits. In this study, experiments were performed to determine whether abscisic acid (ABA), a plant stress hormone, could induce the formation of photosynthetic tissues with Kranz anatomy and C4-like biochemical traits under water in the submerged form. When the submerged plants were grown in water containing 5 &mgr;M ABA, they developed new photosynthetic tissues with Kranz anatomy, forming well-developed Kranz (bundle sheath) cells that contained many organelles. The ABA-induced tissues accumulated large amounts of phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, and NAD-malic enzyme at the appropriate cellular sites. The tissues had 3.4 to 3.8 times more C4 enzyme activity than did tissues of the untreated submerged plants. Carbon-14 pulse and carbon-12 chase experiments revealed that the ABA-induced tissues fixed higher amounts of carbon-14 into C4 compounds and lower amounts of carbon-14 into C3 compounds as initial products than did the submerged plants and that they exhibited a C4-like pattern of carbon fixation under aqueous conditions of low carbon, indicating enhanced C4 capacity in the tissues. This report provides an example of the hormonal control of the differentiation of the structural and functional traits required for the C4 pathway.     

Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids

Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization.     

基于转录组的苹小卷叶蛾杀虫剂靶标及解毒代谢相关基因分析

【目的】建立苹小卷叶蛾Adoxophyes orana转录组数据库,挖掘杀虫剂靶标及解毒代谢相关基因。【方法】采用Illumina HiSeq~(TM) 2000高通量测序技术对苹小卷叶蛾进行转录组测序,挖掘并分析杀虫剂靶标基因;利用qPCR检测6个杀虫剂靶标基因在苹小卷叶蛾卵、幼虫、蛹和成虫各不同发育阶段的表达;挖掘并分析苹小卷叶蛾转录组中解毒代谢相关基因的代谢通路及进化关系。【结果】通过组装有效序列共获得48 610条unigene(GenBank登录号:GGMW00000000)。挖掘鉴定到155个杀虫剂靶标unigene;qPCR结果显示,1个蜕皮激素受体(ecdysone receptor, ECR)、2个乙酰胆碱酯酶(acetylcholinesterase, AChE)、1个氯离子通道蛋白(chloride channel, CLC)、1个几丁质酶(chitinase, CS)和1个鱼尼丁受体(ryanodine receptor, RyR)基因在苹小卷叶蛾不同发育阶段均存在表达差异。挖掘鉴定到69个羧酸酯酶(carboxylesterase, CarE)unigene、66个谷胱甘肽S-转移酶(glutathion S-transferase, GST)unigene和205个细胞色素P450(cytochrome P450)unigene等解毒代谢相关基因,共鉴定20个CarE unigene, 32个GST unigene和30个P450 unigene与有毒物质代谢相关的通路有关。基于氨基酸序列对具有完整ORF的unigene聚类分析结果显示:12个CarEs中9个为G类,即鳞翅目保幼激素类;分别有10个AoGSTs属于Delta和Epsilon亚家族;18个P450全部聚到CYP3集团。【结论】该研究有助于苹小卷叶蛾杀虫剂靶标基因的挖掘及抗药性的研究。     

固醇调节元件结合蛋白1及其靶基因网络

固醇调节元件结合蛋白1(Sterol regulatory element-binding protein 1, SREBP-1)是重要的核转录因子之一, 能调控内源性胆固醇、脂肪酸、甘油三酯和磷脂合成所需酶的表达, 以维持血脂动态平衡。研究表明, SREBP-1及其靶基因网络的异常可引起胰岛素抵抗、Ⅱ型糖尿病、心功能紊乱、血管并发症和肝脂肪变等一系列代谢性疾病。近年高通量组学技术的发展极大扩展了对SREBP-1靶基因及其转录调控模式的了解。文章对SREBP-1蛋白结构、活化过程、DNA结合位点及其调控的靶基因等方面的研究进展进行了综述, 并着重介绍了基于组学数据的转录调控网络的构建, 这将有助于更好的认识SREBP-1在脂类代谢中的作用, 为深入探讨脂质代谢性疾病的治疗提供新线索。     

Identification of early senescence-associated genes in rice flag leaves

Leaf senescence is one of the key stages of plant leaf development. It is a highly complex but ordered process involving expression of large scale senescence associated genes, and its molecular mechanisms still remain unclear. By using suppression subtractive hybridization, 815 ESTs that are up-regulated at the onset of rice flag leaf senescence have been isolated. A total of 533 unigenes have been confirmed by macroarray detection and sequencing. 183 of these unigenes have GO annotations, involved in macromolecule metabolism, protein biosynthesis regulation, energy metabolism, gene expression regulations, detoxification, pathogenicity and stress, cytoskeleton organization and flower development. Another 121 unigenes co-localized with previously reported known stay-green QTLS. RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone. Our results indicate that senescence is closely related to various metabolic pathways, thus providing new insight into the onset of leaf senescence mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO(2), which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquens AM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C(1) compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria.