Sucrose induction of <Emphasis Type="Italic">Arabidopsis</Emphasis> miR398 represses two Cu/Zn superoxide dismutases

MicroRNAs (miRNAs) are approximately 21-nt RNAs that reduce target accumulation through mRNA cleavage or translational repression. Arabidopsis miR398 regulates mRNAs encoding two copper superoxide dismutase (CSD) enzymes and a cytochrome c oxidase subunit. miR398 itself is down-regulated in response to copper and stress. Here we show that miR398 is positively regulated by sucrose, resulting in decreased CSD1 and CSD2 mRNA and protein accumulation. This sucrose regulation is maintained both in the presence and absence of physiologically relevant levels of supplemental copper. Additionally, we show that plants expressing CSD1 and CSD2 mRNAs with altered miR398 complementarity sites display increased mRNA accumulation, whereas CSD1 and CSD2 protein accumulation remain sensitive to miR398 levels, suggesting that miR398 can act as a translational repressor when target site complementarity is reduced. These results reveal a novel miR398 regulatory mechanism and demonstrate that plant miRNA targets can resist miRNA regulation at the mRNA level while maintaining sensitivity at the level of protein accumulation. Our results suggest that even in plants, where miRNAs are thought to act primarily through target mRNA cleavage, monitoring target protein levels along with target mRNA levels is necessary to fully assess the consequences of disrupted miRNA-mRNA pairing. Moreover, the limited complementarity required to maintain robust miR398-directed repression of target protein accumulation suggests that similarly regulated endogenous plant miRNA targets may have eluded detection.     

响应盐胁迫调控的露地菊miR398a的克隆及功能研究

miRNAs在非生物胁迫中起着重要的作用。通过前期对露地菊Small RNA高通量测序数据测得到miR398a成熟体和前体序列,命名为cgr-miR398a和cgr-MIR398a。序列对比显示,cgr-miR398a与其他植物中已经鉴定的miR398a序列高度保守;利用前期露地菊降解组数据获得miR398a预测的靶基因,cgr-miR398a根和叶中的靶基因共有18个,其中有铜/锌超氧化物歧化酶（CSD2）、铜伴侣蛋白（CCS）、类A20/AN1-l锌指家族蛋白（SAP8）等与抗性相关的基因。qPCR结果显示盐胁迫下露地菊miR398a及靶基因在不同组织部位的表达水平存在显著的负相关性。为探究cgr-miR398a响应盐胁迫的功能,克隆cgr-MIR398a并构建过表达载体转化拟南芥。结果表明,拟南芥中过表达cgr-MIR398a降低了盐胁迫下种子发芽率以及成苗期的抗盐性,说明cgr-miR398a在拟南芥响应盐胁迫中起着负调控作用。这为进一步研究露地菊mi398a的功能和露地菊的抗盐机理奠定了基础。     

Plants contain two SCO proteins that are differentially involved in cytochrome c oxidase function and copper and redox homeostasis

Two Arabidopsis thaliana genes (HCC1 and HCC2), resulting from a duplication that took place before the emergence of flowering plants, encode proteins with homology to the SCO proteins involved in copper insertion during cytochrome c oxidase (COX) assembly in other organisms. Heterozygote HCC1 mutant plants produce 25% abnormal seeds with defective embryos arrested at the heart or torpedo stage. These embryos lack COX activity, suggesting that the requirement of HCC1 during the early stages of plant development is related with its COX assembly function. Homozygote HCC2 mutant plants develop normally and do not show changes in COX2 levels. These plants display increased sensitivity of root growth to increased copper and a higher expression of miR398 and other genes that respond to copper limitation, in spite of the fact that they have a higher copper content than the wild type. HCC2 mutant plants also show increased expression of stress-responsive genes. The results suggest that HCC1 is the protein involved in COX biogenesis and that HCC2, that lacks the cysteines and histidine putatively involved in copper binding, functions in copper sensing and redox homeostasis. In addition, plants that overexpress HCC1 have an altered response of root elongation to changes in copper in the growth medium and increased expression of two low-copper-responsive genes, suggesting that HCC1 may also have a role in copper homeostasis.     

Regulation of superoxide dismutase expression by copper availability

The most abundant copper proteins in green tissues are plastocyanin (PC) in thylakoids and copper/zinc superoxide dismutase (Cu/ZnSOD) of which the major isoforms are found in the cytosol and in the chloroplast stroma. An iron superoxide dismutase (FeSOD) can also be found in the stroma. The expression of superoxide dismutases (SODs) has been studied mainly in the context of abiotic stress. However, the availability of metal cofactors may also determine SOD expression patterns. Indeed, in Arabidopsis thaliana , Cu/ZnSOD enzymes were only expressed when copper was sufficient. This observation was made for plants grown on sucrose-containing tissue culture media and regulation of SOD expression by copper has not been tested for other species. To investigate the effect of copper on SOD expression, we used a hydroponic set-up in which plants grew without any evident stress symptoms. We observed that A. thaliana , Brassica juncea , Lycopersicum lycopersicum , Zea mays and Oryza sativa , downregulated Cu/ZnSOD in response to copper limitation. Under this condition, FeSOD expression was upregulated to replace Cu/ZnSOD in the stroma in all plants except Z. mays , in which FeSOD was not detectable. Copper limitation did not affect PC accumulation in any of the plants except Z. mays . Comparisons of leaf copper contents and SOD expression suggest that Cu/ZnSOD and FeSOD expression levels are good indicators of impending copper deficiency. Plants that downregulate Cu/ZnSOD and upregulate FeSOD under copper limitation can maintain superoxide scavenging and save copper for use in PC, which is essential for photosynthesis.     

miR398在植物逆境胁迫应答中的作用

MicroRNA (miRNA)是一类新型的调控基因表达的小分子RNA, 它作为基因表达的负调控因子, 在转录后水平调节靶基因的表达。miRNA参与调控植物的生长发育, 并在多种非生物与生物胁迫响应中发挥重要作用。miR398是第一个被报道的受氧化胁迫负调控的miRNA。它通过负调控其靶基因Cu/Zn过氧化物歧化酶(Cu/Zn-superoxide dismutase, CSD)的表达, 在多种逆境胁迫响应中扮演重要角色, 如调节铜代谢平衡, 应答重金属、蔗糖、臭氧等非生物胁迫, 以及参与应答生物胁迫等。文章综述了miR398在多种逆境胁迫响应中重要的调节作用及miR398自身的转录调控。     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.