Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development⋆

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1 mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.     

Cloning and functions analysis of a pyruvate dehydrogenase kinase in Brassica napus

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), which plays a key role in intermediary metabolism. In this study, a 1,490-bp PDK in Brassica napus (BnPDK1) was isolated and cloned from Brassica cDNA library. BnPDK1 has an 1,104 open reading frame encoding 367 amino acids. Genomic DNA gel blot analysis result indicated that BnPDK1 is a multi-copy gene. RNA gel blot analysis and RNA in situ hybridization were used to determine the expression of BnPDK1 in different organs. BnPDK1 gene was ubiquitously expressed in almost all the tissues tested, having the highest expression in the stamen and the young silique. Over-expression of BnPDK1 in transgenic Arabidopsis lines would repress the PDC activity, and resulted in the decrease of seed oil content and leaf photosynthesis. These results implied that BnPDK1 was involved in the regulation of fatty acid biosynthesis in developing seeds.     

Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.     

Molecular cloning and characterization of a novel brassinolide enhanced gene OsBLE1 in Oryza sativa seedlings.

Brassinosteroids (BRs) are plant steroids essential for normal growth and development. To gain insight into the molecular mechanism by which BRs regulate the growth and development of plants, it is necessary to identify and analyze more genes that are regulated by BRs. A novel brassinolide (BL)-enhanced gene designated OsBLE1, which was originally identified by using rice (Oryza sativa L.) cDNA microarray, was cloned and characterized. Its cDNA is 598 bp long, encoding a predicted polypeptide with 81 amino acid residues. Northern blots analysis revealed that OsBLE1 expression began to increase at 6 h and reached its maximum at 12 h after BL treatment. OsBLE1 expression was most responsive to BL in lamina joint in rice seedlings; besides, IAA and GA3 also enhanced its expression. OsBLE1 expressed mainly in active tissues such as vascular bundles and root primordial. Transgenic rice expressing antisense OsBLE1 exhibits various degrees of repressed growth. Results suggest that OsBLE1 might be involved in BL-regulated growth processes in rice seedlings.     

Gibberellin-regulated XET is differentially induced by auxin in rice leaf sheath bases during gravitropic bending

The asymmetric distribution of auxin plays a fundamental role in plant gravitropism, yet little is understood about how its lateral distribution stimulates growth. In the present work, the asymmetric distribution not only of auxin, but also that of gibberellins (GAs), was observed in rice leaf sheath bases following gravistimulation. Gravistimulation induced the transient accumulation of greater amounts of both IAA and GA in the lower halves of the leaf sheath bases of rice seedlings. OsGA3ox1, a gene of active GA synthesis, was differentially induced by gravistimulation. Furthermore, 2,3,5-tri-iodobenzoic acid (TIBA), an inhibitor of auxin transport, substantially decreased the asymmetric distribution of IAA and the gradient of OsGA3ox1 expression. Externally applied GA(3) restored the gravitropic curvature of rice leaf sheaths inhibited by either TIBA or by ancymidol, a GA synthesis inhibitor. The expression of XET (encoding xyloglucan endotransglycosylase) was differentially induced in the lower halves of gravistimulated leaf sheath bases and was also up-regulated by exogenous IAA and GA(3). Both ancymidol and TIBA decreased the gradient of XET expression. These data suggest that the asymmetric distribution of auxin effected by gravistimulation induced a gradient of GAs via asymmetric expression of OsGA3ox1 in rice leaf sheath bases, and hence caused the asymmetric expression of XET. Cell wall loosening in the curvature site of the leaf sheath triggered by the expression of XET would contribute to gravitropic growth.     

Characterization of the porcine differentially expressed <Emphasis Type="Italic">PDK4</Emphasis> gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.     

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.     

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.     

A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice

Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1. BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice.     

Light regulation of leaf mitochondrial pyruvate dehydrogenase complex : role of photorespiratory carbon metabolism

Light-dependent inactivation of mitochondrial pyruvate dehydrogenase complex (mtPDC) in pea (Pisum sativum L.) leaves was further characterized, and this phenomenon was extended to several monocot and dicot species. The light-dependent inactivation of mtPDC in vivo was rapidly reversed in the dark, even after prolonged illumination. The mtPDC can be efficiently cycled through the inactivated-reactivated status by rapid light-dark cycling. Light-dependent inactivation of mtPDC was shown to be suppressed by inhibitors of photorespiratory carbon metabolism, including 2-pyridylhydroxymethane sulfonate, isonicotinic acid hydrazide, and aminoacetonitrile, and by an inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Glycine fed to pea leaf strips in the dark yielded partially inactivated leaf mtPDC, and this inactivation was blocked by inhibitors of glycine oxidation. It is concluded that the photorespiratory glycine to serine conversion that occurs in C₃ leaf mitochondria can provide the NADH to drive oxidative phosphorylation and subsequent inactivation of mtPDC. Glycine oxidation also produces ammonium ion, which has been shown to enhance the inactivation of mtPDC in vitro by stimulating the pyruvate dehydrogenase kinase that catalyzes the phosphorylation (inactivation) of the mtPDC. Thus, light-dependent, photorespiration-stimulated inactivation of the mtPDC can regulate carbon entry into the Krebs cycle during C₃ photosynthesis.     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.     

差异显示法分离赤霉素调控的水稻(Oryza sativa)cDNA(英文)

赤霉素(gibberellins)是植物生长发育过程中一类重要的调节激素。本文运用反转录和聚合酶链式反应建立了一套旨在分离差异表达cDNA的差异显示方法。以籼稻珍汕97 B为材料,将赤霉素GA_3处理后的苗期水稻与对照的cDNA片段进行比较,鉴定了15个差异cDNA,并将它们从测序胶中回收和再次扩增获得差异表达的cDNA;用其中一个差异cDNA片段DDF1为探针的Southern和northern杂交证实,DDF1所对应的基因是一个单拷贝基因,可被高浓度的GA_3诱导并获得高水平表达。     

The Regulation of Pyruvate Dehydrogenase Activity in Pea Leaf Mitochondria (The Effect of Respiration and Oxidative Phosphorylation)

The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.     

Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets

Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK.     

Tissue localization of a submergence-induced 1-aminocyclopropane-1-carboxylic acid synthase in rice

At least two 1-aminocyclopropane-1-carboxylic acid synthase genes (ACS) are implicated in the submergence response of rice (Oryza sativa). Previously, the OS-ACS5 gene has been shown to be induced during short- as well as long-term complete submergence of seedlings and to be controlled by a balance of gibberellin and abscisic acid in both lowland and deepwater rice. This study demonstrates that OS-ACS5 mRNA is localized in specific tissues and cells both during normal development and in response to complete submergence. The temporal and spatial regulation of OS-ACS5 expression is presented by in situ hybridization and histochemical analysis of beta-glucuronidase (GUS) activity in transgenic rice carrying an OS-ACS5-gus fusion. Whole-mount in situ hybridization revealed that in air-grown rice seedlings, OS-ACS5 was expressed at a low level in the shoot apex, meristems, leaf, and adventitious root primordia, and in vascular tissues of nonelongated stems and leaf sheaths. In response to complete submergence, the expression in vascular bundles of young stems and leaf sheaths was strongly induced. The results of histochemical GUS assays were consistent with those found by whole-mount in situ hybridization. Our findings suggest that OS-ACS5 plays a role in vegetative growth of rice under normal conditions and is also recruited for enhanced growth upon complete submergence. The possible implication of OS-ACS5 in root-shoot communication during submergence stress and its putative role in aerenchyma formation upon low-oxygen stress are discussed.