桃中两个MADS box基因的克隆与表达分析

为研究李属(Prunus sp．)果树生殖调控的相关基因,对国际公共数据库中的李属植物的EST(expressed sequence tags)序列进行了电子拼接,获得了8个MADS box基因的cDNA序列,并利用PCR技术从桃中克隆出其中的两个cDNA,分别命名为PpMDS4和PpMADS6,在GenBank中的登录号为AY705972和AY705973。PpMADS4基因长850bp,包含一个732bp的开放阅读框,编码243个氨基酸。PpMADS6基因长1190bp,包含1个768bp的开放阅读框,编码256个氨基酸。PpMADS4和PpMADS6在序列上分别与拟南芥中的AGAMOUS基因和矮牵牛中的PFG基因高度同源。RT-PCR分析表明,PpMADS4基因在桃的花瓣、心皮、果实及果仁中表达,应属于控制花器官发育的C类MADS box基因。PpMADS6基因在桃的叶、萼片、花瓣、心皮及果实中表达,应属于调控植物由营养生长向生殖生长过渡的A类MADSbox基因。     

Identification and Characterization of PpLFL, a Homolog of FLORICAULA/LEAFY in Peach (Prunus persica)

A LEAFY/FLORICAULA (LFY/FLO) homolog PpLFL (P runus p ersica L EAFY/ F LORICAULA L ike) gene was isolated from axillary buds of peach (Prunus persica (L.) Batsch. cv. Bayuecui) during flower induction period. The open reading frame of PpLFL spanned 1,248 bp, encoding a putative protein of 415 amino acid residues, which was with high similarity (50.48 %–84.69 %) to other FLO/LFY inferred proteins from different species. The spatial expression patterns of PpLFL were detected in axillary buds during the periods of flower induction by using immunohistolocalisation. The results showed that PpLFL gene was mainly expressed during flower induction time, and also in leaf and petal promordia at the SAM. For further functional analysis, the PpLFL was constitutively expressed in the Arabidopsis lfy mutant background, and the results showed that overexpression of PpLFL under the control of CaMV 35S promoter can accelerate flowering and give rise to normal flower organs. Our results suggest that PpLFL might play an important role in flower induction, and could act as a functional flower meristem identity gene in peach.     

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.

The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.     

Characterization of responses of isolated rat hepatocytes to ATP and ADP

In isolated rat hepatocytes, ATP and ADP (10(-6) M) rapidly mobilize intracellular Ca2+ and increase the concentration of free cytosolic Ca2+ ([Ca2+]i) within 1-2 s. The increase in [Ca2+]i is maximal (2.5- to 3-fold) by about 10 s and is dose-dependent, with ATP and ADP being half-maximally effective at 8 X 10(-7) and 3 X 10(-7) M, respectively. At submaximal concentrations, the rise in [Ca2+]i is transient due to hydrolysis of the agonist. The increase in [Ca2+]i in response to ATP or ADP can be potentiated by low concentrations of glucagon (10(-9) M). In addition, the [Ca2+]i rise can be antagonized in a time- and dose-dependent manner by the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. Adenosine, at concentrations as high as 10(-4) M, does not alter [Ca2+]i. AMP is ineffective at 10(-5) M, but at 10(-4) M it increases [Ca2+]i approximately 1.5-fold after a 30-s lag and at a slow rate. Conversely, high concentrations (10(-4) M) of adenosine and AMP increases cell cAMP about 2- to 3-fold. ATP and ADP, at concentrations (10(-6) M) which near-maximally increase [Ca2+]i, do not affect hepatocyte cAMP. ATP and ADP increase the cellular level of myoinositol 1,4,5-trisphosphate (IP3), the putative second messenger for Ca2+ mobilization. The increase in IP3 is dose-dependent and precedes or is coincident with the [Ca2+]i rise. There is an approximate 20% increase in IP3 with concentrations of ATP or ADP which near-maximally induce other physiological responses. It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+ and activate phosphorylase in hepatocytes due to generation of IP3. These effects may involve P2-purinergic receptors. In contrast adenosine and AMP interact with P1 (A2)-purinergic receptors to increase cAMP.     

Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.     

Genome-Wide Identification and Characterization of Nucleotide-Binding Site (NBS) Resistance Genes in Pineapple

Pineapple is a major tropical fruit and the most important crop processing CAM photosynthesis. It originated in southwest Brazil and northeast Paraguay and survived the harsh, semi-arid environment. Disease resistance genes have contributed to the survival and thriving of this species. The largest class of disease resistance (R) genes in plants consists of genes encoding nucleotide-binding site (NBS) domains. The sequenced genome of pineapple (Ananas comosus (L.) Merr.) provides a resource for analyzing the NBS-encoding genes in this species. A total of 177 NBS-encoding genes were identified using automated and manual analysis criteria, and these represent about 0.6 % of the total number of predicted pineapple genes. Five genes identified here contained the N-terminal Toll/Interleukin-l receptor (TIR) domain, and 46 genes carried the N-terminal Coiled-Coil (CC) motif. A majority of these NBS-encoding genes (84 %) contained a leucine-rich repeat (LRR) domain. A total of 130 of 177 (73 %) of these NBS-encoding genes were distributed across 20 pineapple linkage groups. The identification and characterization of NBS genes in pineapple yielded a valuable genomic resource and improved understanding of R genes in pineapple, which will facilitate the development of disease resistant pineapple cultivars.     

Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases

It has been believed that the key step in cytokinin biosynthesis is the addition of a 5-carbon chain to the N(6) of AMP. To identify cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of cytokinins, the Arabidopsis genomic sequence was searched for genes that could code for isopentenyltransferases. This resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected to detailed analysis. Overexpression of AtIPT4 caused cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when cytokinins are applied, it suggested that this gene product catalyzed cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N(6) of ATP and ADP, but not to that of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that cytokinins are, at least in part, synthesized from ATP and ADP in plants.     

Temperature dependence of ADP/ATP translocation in mitochondria

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench-back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time-dependent progress at about 10 points. The exchange rate in liver mitochondria was determined from -10 degrees C to + 10 degrees C in the presence of 20% glycol, from 0 degrees C to 25 degrees C, and from 20 degrees C to 40 degrees C under partial inhibition by carboxyatractylate. The total range between -10 degrees C to + 40 degrees C has only one temperature break at 13 degrees C. From the Arrhenius plot between -10 degrees C to + 13 degrees C, EA approximately equal to 140 kJ and above 13 degrees C, EA approximately equal to 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively. In beef heart mitochondria the exchange rate was measured between 0 degrees C and 20 degrees C, and between 15 degrees C and 30 degrees C under partial inhibition with carboxyatractylate. There is a temperature break around 14 degrees C with EA approximately equal to 143 kJ between 0 degrees C and 14 degrees C and EA approximately equal to 60 kJ from 15 degrees C to 30 degrees C. The extrapolated translocation rates at 37 degrees C are 500 and 1800 mumol min-1 (g protein)-1 for rat liver and for beef heart mitochondria respectively. The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14 degrees C in mitochondria has been reported.     

On the mechanism and functional significance of the ADP/ATP carrier (AAC) dimerization

According to previous studies, ADP/ATP carrier (AAC) can possibly exist as a monomer or in a dimer state in the inner mitochondrial membrane; however, the question on its functional oligomeric state is still open. The aim of the present work is to establish the external factors that could control the functional oligomeric state of AAC (i.e., monomer or dimer). The study is based on the results of our previous work, which revealed that the volume regulation system of mitochondria (MVRS) affects the oxidative phosphorylation (OXPHOS) system: MVRS could transfer OXPHOS system functioning in a state of supercomplex. Consequently, one may expect that the volume regulation system could also control the functional state of AAC during phosphorylation. Here, on rat liver mitochondria we show that, depending on the incubation medium tonicity, AAC functions in two different ways: either as a monomer (in hypotonic and isotonic media) or as a dimer (in a hypertonic medium). Thus, the transition between the monomeric and dimeric forms of AAC is regulated by MVRS, as well as by functioning of OXPHOS. We conclude that the structural reorganization of AAC is associated with the entire OXPHOS reorganization into a supercomplex. It was also found that dimerization of AAC can occur not only due to the action of MVRS (in hypotonic media) but also under hypoxic conditions.     

ATP and ADP hydrolysis in brain membranes of zebrafish (Danio rerio)

Nucleotides, e.g. ATP and ADP, are important signaling molecules, which elicit several biological responses. The degradation of nucleotides is catalyzed by a family of enzymes called NTPDases (nucleoside triphosphate diphosphohydrolases). The present study reports the enzymatic properties of a NTPDase (CD39, apyrase, ATP diphosphohydrolase) in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in a pH range of 7.5-8.0 in the presence of Ca(2+) (5 mM). The enzyme displayed a maximal activity for ATP and ADP hydrolysis at 37 degrees C. It was able to hydrolyze purine and pyrimidine nucleosides 5'-di and triphosphates, being insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (0.1 mM). A significant inhibition of ATP and ADP hydrolysis (68% and 34%, respectively) was observed in the presence of 20 mM sodium azide, used as a possible inhibitor of ATP diphosphohydrolase. Levamisole (1 mM) and tetramisole (1 mM), specific inhibitors of alkaline phosphatase and P1, P(5)-di (adenosine 5'-) pentaphosphate, an inhibitor of adenylate kinase did not alter the enzyme activity. The presence of a NTPDase in brain membranes of zebrafish may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in central nervous system of this specie.     

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis

Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.     

Deep Origin of Plastid/Parasite ATP/ADP Translocases

Abstract Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.     

Change in ATP and ADP content of fasting and feeding planarians (Polycelis nigra)

The ATP and ADP content of planarians subjected to starvation for two weeks followed by feeding for the same period was investigated. The ATP and ADP content during fasting increased and then, after feeding, returned to normal. The ATP/ADP ratio varied in the same way, which is consistent with the view that the adenylic nucleotide pool is implicated in the regulation of the energy metabolism of the organism.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.