植物丙酮酸磷酸双激酶（PPDK）研究进展

丙酮酸磷酸双激酶（PPDK）是C4植物和景天科酸代谢（CAM）植物光合作用的关键酶,催化形成固定CO2的初始分子受体磷酸烯醇式丙酮酸（PEP）。本文重点比较了植物的ppDK基因结构及分子进化关系,综述了PPDK在C4植物和C3植物中的功能、PPDK的调控机理、PPDK在胁迫条件下的功能以及转PPDK基因等在近年来的研究进展。     

重组丙酮酸磷酸双激酶与荧光素酶偶联催化ATP-AMP循环反应

丙酮酸磷酸双激酶（pyruvate phosphate dikinase, PPDK; EC 2.7.9.1）能够可逆催化磷酸烯醇式丙酮酸（phosphoenolpyruvate, PEP）、单磷酸腺苷（adenosine monophosphate, AMP）和焦磷酸盐（pyrophosphate, PPi）生成三磷酸腺苷（adenosine triphosphate, ATP）、无机磷酸盐（orthophosphate, Pi）和丙酮酸（pyruvate）.以热玫瑰小双孢菌基因组DNA为模板,PCR扩增得到了编码PPDK的基因,将此基因片段插入表达载体pET24a (+),在大肠杆菌中表达C端融合His-Tag的重组PPDK.与我们先前表达的N端融合His-Tag的PPDK相比,酶的活性提高了20倍,提示该酶的N端对活性十分重要.重组PPDK单体分子量为98 kD.经过镍亲和层析和超滤后,重组PPDK基本达到电泳纯.重组PPDK与荧光素酶偶联能够形成1个ATP-AMP循环反应,在该循环反应中,荧光素酶催化ATP生成的AMP和PPi能够被PPDK重新转化成ATP,产生一个持续稳定的信号.     

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.     

Pyruvate orthophosphate dikinase gene expression in developing wheat seeds

The amount of pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) protein in wheat (Triticum aestivum L. var Cheyenne) grains was determined at different stages of development by the protein blot method. The variation in PPDK protein with time in developing wheat grains was similar to that of the enzyme's activity reported by Meyer et al. (1982 Plant Physiol 69: 7-10). The variation in levels of PPDK mRNA with seed development was determined by analysis of polypeptides immunoprecipitated by anti-PPDK serum from in vitro translation products of extracted seed RNA. This mRNA variation was similar to that of the in vivo enzyme levels and the correlation is consistent with the regulation of PPDK gene expression by the level of its mRNA.

The highest level of PPDK in developing wheat seeds occurs later than the highest levels of both ribulose bisphosphate carboxylase (EC 4.1.1.39) and of chlorophyll, which are located in the green pericarp tissue. PPDK was located in both endosperm and pericarp tissue of the seeds. The tissue location and developmental profile of seed PPDK are consistent with a metabolic role of providing phosphoenolpyruvate as a substrate for recapturing respiratory CO₂ in the seed, and possibly for amino acid interconversions during development.

     

Immunological Studies on Pyruvate Orthophosphate Dikinase in C3 Plants

Pyruvate orthophosphate dikinase (PPDK) was detected in someC₃ plants, wheat, barley, rice and tobacco, by protein blottingusing an antibody against maize PPDK, although the amounts weremuch lesser than those of C₄ plants. The PPDK activity in immaturegrains of rice was specifically immunoprecipitated by the anti-(maize)PPDK antibody. The molecular weight of the subunit of PPDK inall tested C₃ plants was similar (ca. 95 kD) to that of maizePPDK, and the fragment patterns of the C₃ PPDKs in peptide mappingwere also similar to that of maize PPDK. These results suggestthat C₃ PPDKs have a primary structure similar to that of maizePPDK. In order to obtain information about the expression of PPDKin C₃ plants, changes in the enzyme activity and in the amountof PPDK protein were investigated during the greening of riceseedlings. PPDK, which was found in the etiolated seedlings,decreased temporarily in an early stage of greening and thenincreased. The mechanism of this variation is discussed. ¹ To whom correspondence should be addressed. (Received December 9, 1986; Accepted March 12, 1987)     

Cytosolic pyruvate,orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content

The protein content of seeds determines their nutritive value, downstream processing properties and market value. Up to 95% of seed protein is derived from amino acids that are exported to the seed after degradation of existing protein in leaves, but the pathways responsible for this nitrogen metabolism are poorly defined. The enzyme pyruvate,orthophosphate dikinase (PPDK) interconverts pyruvate and phosphoenolpyruvate, and is found in both plastids and the cytosol in plants. PPDK plays a cardinal role in C₄ photosynthesis, but its role in the leaves of C₃ species has remained unclear. We demonstrate that both the cytosolic and chloroplastic isoforms of PPDK are up‐regulated in naturally senescing leaves. Cytosolic PPDK accumulates preferentially in the veins, while chloroplastic PPDK also accumulates in mesophyll cells. Analysis of microarrays and labelling patterns after feeding ¹³C‐labelled pyruvate indicated that PPDK functions in a pathway that generates the transport amino acid glutamine, which is then loaded into the phloem. In Arabidopsis thaliana, over‐expression of PPDK during senescence can significantly accelerate nitrogen remobilization from leaves, and thereby increase rosette growth rate and the weight and nitrogen content of seeds. This indicates an important role for cytosolic PPDK in the leaves of C₃ plants, and allows us to propose a metabolic pathway that is responsible for production of transport amino acids during natural leaf senescence. Given that increased seed size and nitrogen content are desirable agronomic traits, and that efficient remobilization of nitrogen within the plant reduces the demand for fertiliser applications, PPDK and the pathway in which it operates are targets for crop improvement.     

植物丙酮酸磷酸双激酶研究进展

丙酮酸磷酸双激酶(pyruvate orthophosphate dikinase,PPDK)作为C4光合途径中一个非常重要的限速酶,其功能已经清楚,但在C3植物中以及逆境条件下的作用尚不明确。在阐述PPDK基本生物学特征的基础上,重点介绍了PPDK在C4植物和C3植物中的功能、活性调控、基因工程以及PPDK对逆境胁迫应答的研究进展,以期为植物抗逆基因挖掘及抗逆种质创制提供参考。     

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

Stabilization of pyruvate, pi dikinase regulatory protein in maize leaf extracts

The objective of this study was to determine the biochemical basis for genetic variability in pyruvate,Pi dikinase (PPDK) activity among inbred lines of maize (Zea mays L.). Although in vitro PPDK activity varied more than 5-fold among eight maize inbreds, immunochemical determinations of the proportion of leaf soluble protein as PPDK revealed no significant differences among the inbreds. Genetic differences in the stability of PPDK activity in crude homogenates over 5 hours were not evident, but PPDK from some inbreds could not be activated in vitro. In vitro PPDK activation in crude homogenates could be restored by addition of casein (1% w/v) to homogenization media, and to a lesser extent, by gentle homogenization in a mortar. The major effect of casein appeared to be on processes other than proteolysis, as casein exerted its effects during tissue homogenization, rather than later. During homogenization, PPDK did not lose its ability to undergo in vitro activation; instead, it was instability of the regulatory protein responsible for PPDK activation that was the cause of the lack of PPDK activation in homogenates prepared without casein.     

Screening marine fungi for inhibitors of the C4 plant enzyme pyruvate phosphate dikinase: unguinol as a potential novel herbicide candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

Cell-specific expression of pyruvate, pi dikinase : in situ mRNA hybridization and immunolocalization labeling of protein in wheat seed

Pyruvate, Pi dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway. However, its metabolic role in C3 plants remains uncertain. Northern blot analyses of PPDK mRNAs from wheat leaves and seeds probed with maize PPDK cDNA indicates the presence of organ-specific mRNAs. Immunofluorescent labeling of protein in wheat seed demonstrate that the PPDK polypeptide and the ribulose-1, 5-bisphosphate carboxylase small subunit polypeptide are localized predominantly in the aleurone layer and the chlorophyllous pericarp tissue, respectively. This differential distribution of the two polypeptides in wheat seed is paralleled by the differential localization of the their mRNAs as revealed by in situ hybridization. These results suggest a distinct role of cytoplasmic PPDK in seeds, which is different from the well established role in C4 photosynthesis.     

Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds

Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C₄ plants. The enzyme is also ubiquitous in C₃ plant tissues, although a precise non-photosynthetic C₃ function(s) is yet to be validated, owing largely to its low abundance in most C₃ organs. The single C₃ organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C₃ PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development.     

The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both bifunctional and interact with the catalytic and nucleotide-binding domains of pyruvate, orthophosphate dikinase

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C(4) photosynthesis and is also found in C(3) plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.     

High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C₄ photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C₄ photosynthesis of maize under cold conditions.     

Cold stability of pyruvate,orthophosphate dikinase of Flaveria brownii

The nucleotide sequences of the complementary DNA of pyruvate, P_i dikinase (PPDK) from Flaveria bidentis, a C₄ plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C₄-like plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.