cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.     

水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶（G6PDH）是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。木研究首次从水稻（Oryza sativa L.）幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8．5,分子量66kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体。多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81％、87％、83％。进化关系说明水稻OsG6PDH2与拟南芥（AtG6PDH3）、马铃薯（StG6PDH1）处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT／DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将OsG6PDH2的完整开放阅读框构建到大肠杆菌表达载体pET30a（＋）中,pET30a（＋）-OsG6PDH2在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

Four rice genes encoding NADP malic enzyme exhibit distinct expression profiles

In plants, the NADP malic enzymes (NADP-MEs) are encoded by small gene families. These NADP-ME gene families are relatively well described in C4 plants but not well studied in C3 plants. In this study, we investigated the NADP-ME gene family in a model C3 monocot plant (rice, Oryza sativa) based on its recently released genomic DNA sequence. We found that the rice NADP-ME family is composed of four members, one plastidic NADP-ME and three cytosolic versions. Although the rice NADP-ME genes identified share a high degree of similarity with one another, one cytosolic NADP-ME (OscytME3) contains several unique amino acid substitutions within highly conserved amino acid regions. Phylogenetic analysis showed that OscytME3 might be derived from a different evolutionary branch than the other three rice genes. Expression analysis of the four rice NADP-ME genes indicated that each had a different tissue-specific and developmental profile, although all four responded to stress stimuli.     

Micronutrient availability to cereals from calcareous soils

Summary On several alkaline calcareous soils, Zn and Cu deficiency occurred mainly in lowland rice (Oryza sativa L.) and was rarely found in wheat (Triticum aestivum L.). Zinc and Cu requirement of plants was not responsible as the critical Zn and Cu contents in tops of the two plant species were almost similar i.e. 17.4, 6.5 and 14.5, 5.6 ppm respectively. Neither did rice absorb Zn and Cu less efficiently. On the contrary, their rates of absorption in rice were double than in wheat. They were 22.2, 6.3 and 10.2, 3.3 ng atoms/g fresh root/h respectively in the two plant species. Flooded soil conditions appeared to be responsible for Zn and Cu deficiency in rice as their deficiency was found mainly in plant samples collected from continuously flooded fields. The mechanism is not known.Both Zn and Cu inhibited uptake of each other in wheat on most of the soils. In rice, only applied Zn depressed Cu uptake but Cu had generally little effect on Zn uptake. Little Cu inhibition of Zn uptake in lowland rice seems to be related to flooded soil conditions. The mechanism is yet to be known. The antagonising element accentuated the deficiency of the other element both in wheat and rice and severely reduced their yields on soils marginal to deficient in Zn or Cu supplies. It is recommended that their soil availability status should be thoroughly considered before their fertilizers are applied. re]19750515     

OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm

Pyruvate kinase (PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice (Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK (PKp). OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein-protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.     

Cloning and Expression Pattern of a Gene Encoding a Putative Plastidic ATP/ADP Transporter from Helianthus tuberosus L.

Herein, we report the cloning and molecular characterization of a full cDNA encoding a putative plastidic ATP/ADP transporter, designated HtAATP, for Helianthus tuberosus L. The ATP/ADP translocator protein was isolated from the tuber-cDNA library of H. tuberosus for the first time. The predicted HtAATP protein was judged as a plastidic ATP/ADP translocator protein from its high homology at the amino acid sequence level to the two Arabidopsis thaliana plastidic ATP/ADP translocator proteins AATP1 and AATP2 （84.8% and 79.9% identity, respectively）. Amino acid sequence analysis of the primary structure of HtAATP revealed that it belonged to the plastidic ATP/ADP transporter family. Hydropathy prediction indicated that HtAATP gene product is a highly hydrophobic membrane protein that contains 10 transmembrane domains to form a spanning topology. Southern blotting analysis showed that the HtAATP gene is a single-copy gene in the H. tuberosus genome. Tissue distribution analysis showed that the HtAATP gene is prominently expressed in sink tissues. A stable expression pattern in tubers at different developmental stages implies an active involvement of HtAATP during carbohydrate formation.     

Characterization of an ascorbate peroxidase in plastids of tobacco BY-2 cells

In higher plants, ascorbate peroxidase (APX; EC 1.11.1.11), the major H₂O₂-scavenging enzyme, occurs in several distinct isoenzymes that are localized in cytosol and various cell organelles. Here, we have purified and characterized an APX from the soluble fraction of plastids of non-photosynthetic tobacco BY-2 cells. The plastidic APX was a monomer with a molecular weight of 34 000. The enzymatic properties of the plastidic APX, including the rapid inactivation by H₂O₂ in ascorbate-depleted medium, were highly comparable with those of the chloroplastic stromal APX of spinach and tea leaves. However, the other chloroplastic APX isoenzyme, the thylakoid-membrane bound APX, was not detected in the plastids of the BY-2 cells. The N-terminal amino acid sequence of the plastidic APX was completely identical with the deduced amino acid sequence of a previously identified cDNA sequence of tobacco chloroplastic APX. When a green fluorescence protein gene tagged with the chloroplast-targeting signal sequence of APX was expressed in the BY-2 cells, the fluorescence protein exclusively localized into plastids, and not into mitochondria. We conclude that plastidic APX in non-photosynthetic tissues is the same as the chloroplastic APX that occurs in leaves.     

有色稻米Fe、Zn、Cu和Mn含量及与种皮颜色相关分析

用AAS方法测定了弥勒县相同生态条件下种植的27份有色稻和34份普通稻糙米4种矿质元素含量,并对有色米和普通米Fe、Zn、Cu和Mn含量进行了比较研究。结果表明,有色稻米4种矿质元素含量明显高于无色稻米,其差异均达显著水平,其含量高低依次为Zn>Fe>Cu>Mn;对黑、褐、红、黄、绿5种不同种皮颜色的稻米4种矿质元素含量进行比较研究,发现稻米Fe含量(mg/kg)依次为黑>绿>褐>红>黄,Zn含量(mg/kg)依次为绿>红>黑>褐>黄,Cu含量(mg/kg)依次为黑>褐>红>黄>绿,Mn含量(mg/kg)依次为褐>黑>红>黄>绿;并且Fe和Mn含量在不同颜色稻米间差异均达显著水平,与有色米种皮颜色密切相关,而Zn和Cu差异不显著,与有色米种皮颜色关系不大。黑米和褐米富Fe、Zn、Cu和Mn,绿米富Fe和Zn,红米富Zn和Cu,黄米4种矿质元素含量较低,Fe、Cu和Mn均低于普通稻米。     

长期施用猪粪红壤稻田土壤Cu、Zn累积规律

为揭示长期施用猪粪红壤稻田土壤Cu、Zn累积规律,以设立于1981年的红壤稻田有机肥定位试验为载体,选取PM1(早稻施猪粪和紫云英)、PM2(早稻施紫云英+晚稻施猪粪)、GMS(早稻施紫云英+晚稻秸秆还田)和NPK(早稻施化肥)等处理为对象,分析了不同试验年限土壤全量和有效态Cu、Zn含量。结果表明:长期施用猪粪显著提高了土壤Cu、Zn含量;连续施用猪粪30 a后,土壤全量Cu、Zn含量分别增加了7.69—9.52 mg/kg和22.42—35.46 mg/kg;生物有效性显著增加,有效态Cu、Zn含量占全量Cu、Zn的比例分别由15%和5%增加到51%和27%。猪粪年度内的施用时间对土壤Cu的累积没有显著影响,早稻施用猪粪加剧了土壤Zn的累积。土壤铜、锌累积分为两个差异显著的阶段,1981—2002年为缓慢增长期,2002—2010年为快速增长期,这可能与2002年后施用的猪粪中Cu、Zn含量增高有关。以研究的结果推算,红壤稻田鲜猪粪施用量在9.5 t hm-2a-1以下,50 a内不会造成土壤Cu、Zn含量超标。     

Effects of Cadmium and Mixed Heavy Metals on Rice Growth in Liaoning,China

Joint effects of Cd and other heavy metals (Pb, Cu, Zn and As) on the growth and development of rice plants and the uptake of these heavy metals by rice were studied using the pot-culture method combined with chemical and statistical analyses. The results showed that the growth and development of rice plants were strongly influenced by the double-element combined pollution. There was an average decrease in the height of rice plants of 4.0–5.0 cm, and grain yield was decreased by 20.0–30.0%, compared with the control. The uptake of Cd by rice plants was promoted due to the interactions between Cd and the other heavy metals added to the soil. The Cd concentration in roots, stems/leaves and seeds increased 31.6–47.7, 16.7–61.5 and 19.6–78.6%, respectively. Due to interactions, uptake of Pb, Cu and Zn by roots and stems/leaves was inhibited, accumulation of Pb, Cu and Zn in seeds was increased, uptake of As by roots was promoted and uptake of As by stems/leaves was inhibited. In particular, the upward transporting ability of the heavy metals absorbed by rice plants was significantly increased.     

Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves : Occurrence of NADH-Dependent Glutamate Synthase Protein and Activity in the Unexpanded, Nongreen Leaf Blades

To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the synthesis of glutamate from the glutamine that is transported from senescing source tissues through the phloem in the nongreen sink tissues in rice leaves.     

重金属对水稻和小麦DNA甲基化水平的影响

和对照相比,0．025（或0.05)-0.1mmol/L的Cu^2 （或0．05）－1．0mmol/L的Cd^2 或Hg^2 导致水稻（或小麦）叶DNA中的5－甲基胞嘧啶百分含量大幅度上升;当Cu^2 浓度＞0.1mmol/L时,小麦和水稻叶DNA中5－甲基胞嘧啶的百分含量随Cu^2 浓度的增高略有下降,但仍高于对照。0.1-1.0mmol/L的Cu^2 ,Cd^2 和Hg^2 也导致小麦穗DNA为5－甲基胞嘧啶的百分含量随Cu^2 ,Cd^ 和Cd^2 能使小麦和水稻根系DNA中5－甲基胞嘧啶的百分含量显著高于对照,而0.1-1.0mmol/L的Hg^2 以及1．0mmol/L的Cu^2 和Cd^2 则造成小麦和水稻根系DNA中5－甲基胞嘧啶的百分含量显著低于对照。     

The physical relationship of barley chromosome 5 (1H) to the linkage groups of rice chromosomes 5 and 10

 Using a recently developed polymerase chain reaction (PCR)-mediated approach for physical mapping of single-copy DNA sequences on microisolated chromosomes of barley, sequence-tagged sites of DNA probes that reveal restriction fragment length polymorphisms (RFLP) localized on the linkage maps of rice chromosomes 5 and 10 were allocated to cytologically defined regions of barley chromosome 5 (1H). The rice map of linkage group 5, of about 135 cM in size, falls into two separate parts, which are related to the distal portions of both the short and long arms of the barley chromosome. The markers on the rice map of chromosome 5 were found to be located within regions of the barley chromosome which show high recombination rates. The map of rice chromosome 10, of about 75 cM in size, on the other hand, is related to an interstitial segment of the long arm of chromosome 5 (1H) which is highly suppressed in recombination activity. For positional cloning of genes of this homoeologous region from the barley genome, the small rice genome will probably prove to be a useful tool. No markers located on rice chromosomes were detected within the pericentric Giemsa-positive heterochromatin of the barley chromosome, indicating that these barley-specific sequences form a block which separates the linkage segments conserved in rice. By our estimate approximately half of the barley-specific sequences of chromosome 5 (1H) show a dispersed distribution, while the other half separates the conserved linkage segments. Received: 29 February 1996 / Accepted: 28 June 1996     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.