Developmental regulation of phospholipase D in tomato fruits

The catabolism of phospholipids initiated by phospholipase D (PLD, EC 3.1.4.4) is an inherent feature of developmental processes that include fruit growth and ripening. In cherry tomatoes (Lycopersicon esculentum Mill.), soluble and membrane-associated PLD activities increased during fruit development, which peaked at the mature green and orange stages. The increase in PLD activity was associated with a similar increase in the intensity of a 92 kDa band as demonstrated by western blot analysis. A full-length cDNA having 2430 bp and encoding a putative polypeptide with 809 amino acids, was isolated using tomato RNA, RT-PCR and 5' and 3' rapid amplification of cloned ends (RACE). Analysis of the primary and secondary structures showed the presence of the C2 domain, the PLD domain and several other features characteristic of PLD alpha. Microtom tomato plants transformed with antisense PLD alpha cDNA, were similar to untransformed plants and showed normal fruit set and development. The ethylene climacteric was delayed by over 7 d in the antisense PLD fruits, indicative of a slower ripening process. The leaves and unripened fruits of antisense PLD microtom plants possessed lowered PLD activity and PLD protein, as demonstrated by western blotting. However, during ripening, PLD activity in the transgenic fruits was maintained at a higher level than that in the untransformed control. Immunolocalization of PLD in microtom tomato fruits revealed the cytosol-membrane translocation of PLD during fruit development. The ripe fruits of antisense PLD celebrity plants possessed lowered PLD expression and activity and showed increased firmness and red colour. These results suggest that the expression of antisense PLD cDNA could be variable in different tomato varieties. The potential role of PLD in ethylene signal transduction events is discussed.     

Characterisation of the ACC oxidase activity in transgenic auxin overproducing tomato during ripening

As part of our studies on the role of auxin in regulating the ethylenebiosynthesis during fruit ripening, in this paper we describe the functionalproperties of the ACC oxidase activity extracted from transgenic tomato(Lycopersicum esculentum Mill. cv. Ailsa craig)overexpressing the tryptophan monooxygenase or iaaM protein fromAgrobacterium tumefaciens that increases the auxin levels.Maximal activity was recovered by extracting the enzyme at pH 8.0 from fruitspicked three days after the onset of the colour change. The enzyme exhibits ahalf-life of 85 min, two relative maxima at 30 and 38°C, an optimum pH of 7.9 and an apparent Km forACC of 118 M. Our results also show the first evidence of anallosteric type kinetic of the ACC oxidase activity with respect to itscosubstrate ascorbate, with an apparent Km of 12.5mM,estimated as the concentration which gave 50% Vmax.     

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings [12]. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the -glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.     

ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg／L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9．5％,番茄的贮存期在50天以上。     

Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5′-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEACO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5′-flanking sequence and the third intron of LEACO3. Received: 8 August 1996 / Accepted: 4 November 1996     

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.     

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase

Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.     

Developmental regulation of human gamma-globin genes in transgenic mice.

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.     

Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants

The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions. We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis. Antisense constructs of cDNAs for senescence-related 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation. Several transgenic lines showed higher PA contents than wild-type plants. The number and weight of seeds also increased. Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment. These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants. This shows a positive correlation between PA content and stress tolerance in plants.     

Developmental regulation for collagen II gene expression in transgenic mice

In order to evaluate the involvement of the type II collagen regulatory sequences in development, we have injected a construct containing a toxin gene under the control of the rat type II collagen promoter and enhancer. The construct, pDAS10-DTA, contained the diphtheria toxin A chain gene under the control of type II collagen sequences which had been used previously to target cartilagenous tissues in transgenics. Inspection of developing fetuses at various stages of gestation revealed a high number of aborted implants as well as abnormally developing fetuses. These abnormal fetuses were of small size, had shortened and underdeveloped limbs, cleft palates, and generally resembled a phenotype similar to chondrodystrophic mice. Histological comparisons of normal and abnormal fetuses indicated a reduced amount of extracellular matrix surrounding chondrocytes, and a disorganized appearance of the tissue. These results suggest that the expression of the toxin has occurred in chondrocytes and altered the survival and development of the transgenic mice. These results also indicate that the promoter and enhancer sequences contained in the transgene controlled the developmental expression of the type II collagen gene expression.     

Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits.

Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes. Transgenic tomato plants (Lycopersicon esculentum Mill cv. Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments. Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans. Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits. However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected. Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control. It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation.     

Developmental analysis of carbohydrate metabolism in tomato (Lycopersicon esculentum cv. Micro-Tom) fruits

Carbohydrate metabolism during the development of fruits of the tomato cultivar Micro-Tom was studied. The metabolism of the pericarp and placental tissues was found to be different. Starch was degraded more slowly in the placenta in comparison with the pericarp, whereas soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 days after flowering. Two of these, phosphoenolpyruvate phosphatase and pyruvate kinase, showed a dramatic increase in activity just before this peak, possibly indicating a role in up-regulating glycolysis to generate increased ATP that would be used during climacteric respiration. The expression of plastidial transporters was studied. Both the TPT and Glu6P transporter were expressed greatest in green fruits, before declining. The expression of the triose-phosphate transporter was greater than that of the glucose 6-phosphate transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development.