番茄果实中乙烯与多聚半乳糖醛酸酶的关系

乙烯与多聚半乳糖醛酸酶(PG)都是果实成熟过程中关键的调节因子.一方面,在有乙烯合成缺陷的转反义ACS番茄和乙烯感受缺陷的Nr突变体番茄果实中PG基因表达量都明显下降,PG酶活性明显降低;用外源乙烯(100 μL/L)处理绿熟期番茄果实使PG基因的表达明显增强,而1-甲基环丙烯(1-MCP,1 μL/L)处理转色期番茄果实明显抑制PG基因表达.另一方面,转反义PG基因番茄果实乙烯释放量在授粉后低于其野生型,番茄乙烯受体基因LeETR4和乙烯反应因子LeERF2基因表达量比野生种低.PG降解果胶的产物D-GA(100 mg/L)促进未熟期番茄果实中的乙烯生成和LeETR4、LeERF2基因的表达.     

Ethylene and fruit ripening

The latest advances in our understanding of the relationship between ethylene and fruit ripening are reviewed. Considerable progress has been made in the characterisation of genes encoding the key ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) and in the isolation of genes involved in the ethylene signal transduction pathway, particularly those encoding ethylene receptors ( ETR ). These have allowed the generation of transgenic fruit with reduced ethylene production and the identification of the Nr tomato ripening mutant as an ethylene receptor mutant. Through these tools, a clearer picture of the role of ethylene in fruit ripening is now emerging. In climacteric fruit, the transition to autocatalytic ethylene production appears to result from a series of events where developmentally regulated ACO and ACS gene expression initiates a rise in ethylene production, setting in motion the activation of autocatalytic ethylene production. Differential expression of ACS and ACO gene family members is probably involved in such a transition. Finally, we discuss evidence suggesting that the NR ethylene perception and transduction pathway is specific to a defined set of genes expressed in ripening climacteric fruit and that a distinct ETR pathway regulates other ethylene-regulated genes in both immature and ripening climacteric fruit as well as in non-climacteric fruit. The emerging picture is one where both ethylene-dependent and -independent pathways coexist in both climacteric and non-climacteric fruits. Further work is needed in order to dissect the molecular events involved in individual ripening processes and to understand the regulation of the expression of both ethylene-dependent and -independent genes.     

The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene.

Fruit ripening represents a complex system of genetic and hormonal regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) mutant is insensitive to the plant growth regulator ethylene (M.B. Lanahan, H.-C. Yen, J.J. Giovannoni, H.J. Klee [1994] Plant Cell 6:521-530). We report here ethylene sensitivity over a range of concentrations in normal and Nr tomato seedlings and show that the Nr mutant retains residual sensitivity to as little as 1 part per million of ethylene. Analysis of ripening-related gene expression in normal and mutant ethylene-treated fruit demonstrates that Nr exerts its influence on development at least in part at the level of ethylene-inducible gene expression. We have additionally used cloned tomato and Arabidopsis sequences known to influence ethylene perception as restriction fragment length polymorphism probes, and have identified a tomato locus linked to Nr that hybridizes to the Arabidopsis ETR1 gene at low stringency, suggesting the possibility that Nr may be homologous to ETR1.     

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.     

Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit

Perception of the plant hormone ethylene is essential to initiate and advance ripening of climacteric fruits. Since ethylene receptors negatively regulate signaling, the suppression is canceled upon ethylene binding, permitting responses including fruit ripening. Although receptors have autophosphorylation activity, the mechanism whereby signal transduction occurs has not been fully determined. Here we demonstrate that LeETR4, a critical receptor for tomato (Solanum lycopersicum) fruit ripening, is multiply phosphorylated in vivo and the phosphorylation level is dependent on ripening stage and ethylene action. Treatment of preclimacteric fruits with ethylene resulted in accumulation of LeETR4 with reduced phosphorylation whereas treatments of ripening fruits with ethylene antagonists, 1-methylcyclopropene and 2,5-norbornadiene, induced accumulation of the phosphorylated isotypes. A similar phosphorylation pattern was also observed for Never ripe, another ripening-related receptor. Alteration in the phosphorylation state of receptors is likely to be an initial response upon ethylene binding since treatments with ethylene and 1-methylcyclopropene rapidly influenced the LeETR4 phosphorylation state rather than protein abundance. The LeETR4 phosphorylation state closely paralleled ripening progress, suggesting that the phosphorylation state of receptors is implicated in ethylene signal output in tomato fruits. We provide insights into the nature of receptor on and off states.     

An Antisense Gene Stimulates Ethylene Hormone Production during Tomato Fruit Ripening

The ripening of many fruits is controlled by an increase in ethylene hormone concentration. E8 is a fruit ripening protein that is related to the enzyme that catalyzes the last step in the ethylene biosynthesis pathway, 1-aminocyclopropane-1-carboxylic (ACC) oxidase. To determine the function of E8, we have transformed tomato plants with an E8 antisense gene. We show here that the antisense gene inhibits the accumulation of E8 protein during ripening. Whereas others have shown that reduction of ACC oxidase results in reduced levels of ethylene biosynthesis, we find that reduction of the related E8 protein produces the opposite effect, an increase in ethylene evolution specifically during the ripening of detached fruit. Thus, E8 has a negative effect on ethylene production in fruit.     

The never ripe mutation blocks ethylene perception in tomato.

Seedlings of tomato fruit ripening mutants were screened for their ability to respond to ethylene. Ethylene induced the triple response in etiolated hypocotyls of all tomato ripening mutants tested except for one, Never ripe (Nr). Our results indicated that the lack of ripening in this mutant is caused by ethylene insensitivity. Segregation analysis indicated that Nr-associated ethylene insensitivity is a single codominant trait and is pleiotropic, blocking senescence and abscission of flowers and the epinastic response of petioles. In normal tomato flowers, petal abscission and senescence occur 4 to 5 days after the flower opens and precede fruit expansion. If fertilization does not occur, pedicel abscission occurs 5 to 8 days after petal senescence. If unfertilized, Nr flowers remained attached to the plant indefinitely, and petals remained viable and turgid more than four times longer than their normal counterparts. Fruit development in Nr plants was not preceded by petal senescence; petals and anthers remained attached until they were physically displaced by the expanding ovary. Analysis of engineered 1-aminocyclopropane-1-carboxylate (ACC) synthase-overexpressing plants indicated that they are phenotypic opposites of Nr plants. Constitutive expression of ACC synthase in tomato plants resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. There were no obvious effects on senescence in leaves of ACC synthase overexpressers, suggesting that although ethylene may be important, it is not sufficient to cause tomato leaf senescence; other signals are clearly involved.     

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.     

Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon,a chemical precursor of ethylene

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.     

Control of ethylene-mediated processes in tomato at the level of receptors

The plant hormone ethylene controls many aspects of development and response to the environment. In tomato, ethylene is an essential component of flower senescence, organ abscission, adventitious root initiation, and fruit ripening. Responses to ethylene are also critical for aspects of biotic and abiotic stress responses. Clearly, much of the control of these events occurs at the level of hormone synthesis. However, it is becoming apparent that levels of the ethylene receptors are also highly regulated. The tomato ethylene receptors are encoded by a family of six genes. Levels of expression of these genes are spatially and temporally controlled throughout development. Further, a subset of the receptor genes respond to external stimuli. Genetic and biochemical evidence supports a model in which the ethylene receptors act as negative regulators of downstream responses; in the absence of ethylene, receptors actively suppress expression of ethylene responsive genes. Consistent with this model, a reduction in the overall level of receptor increases ethylene responsiveness of a tissue while higher expression of receptor decreases ethylene sensitivity. Evidence to support this model will be presented.     

Analysis of the ethylene response in the epinastic mutant of tomato

Ethylene can alter plant morphology due to its effect on cell expansion. The most widely documented example of ethylene-mediated cell expansion is promotion of the "triple response" of seedlings grown in the dark in ethylene. Roots and hypocotyls become shorter and thickened compared with controls due to a reorientation of cell expansion, and curvature of the apical hook is more pronounced. The epinastic (epi) mutant of tomato (Lycopersicon esculentum) has a dark-grown seedling phenotype similar to the triple response even in the absence of ethylene. In addition, in adult plants both the leaves and the petioles display epinastic curvature and there is constitutive expression of an ethylene-inducible chitinase gene. However, petal senescence and abscission and fruit ripening are all normal in epi. A double mutant (epi/epi;Nr/Nr) homozygous for both the recessive epi and dominant ethylene-insensitive Never-ripe loci has the same dark-grown seedling and vegetative phenotypes as epi but possesses the senescence and ripening characteristics of Never-ripe. These data suggest that a subset of ethylene responses controlling vegetative growth and development may be constitutively activated in epi. In addition, the epi locus has been placed on the tomato RFLP map on the long arm of chromosome 4 and does not demonstrate linkage to reported tomato CTR1 homologs.     

Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato

In this study we investigated the role of ethylene in the formation of lateral and adventitious roots in tomato ( Solanum lycopersicum ) using mutants isolated for altered ethylene signaling and fruit ripening. Mutations that block ethylene responses and delay ripening – Nr ( Never ripe ), gr ( green ripe ), nor ( non ripening ), and rin ( ripening inhibitor ) – have enhanced lateral root formation. In contrast, the epi ( epinastic ) mutant, which has elevated ethylene and constitutive ethylene signaling in some tissues, or treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC), reduces lateral root formation. Treatment with ACC inhibits the initiation and elongation of lateral roots, except in the Nr genotype. Root basipetal and acropetal indole-3-acetic acid (IAA) transport increase with ACC treatments or in the epi mutant, while in the Nr mutant there is less auxin transport than in the wild type and transport is insensitive to ACC. In contrast, the process of adventitious root formation shows the opposite response to ethylene, with ACC treatment and the epi mutation increasing adventitious root formation and the Nr mutation reducing the number of adventitious roots. In hypocotyls, ACC treatment negatively regulated IAA transport while the Nr mutant showed increased IAA transport in hypocotyls. Ethylene significantly reduces free IAA content in roots, but only subtly changes free IAA content in tomato hypocotyls. These results indicate a negative role for ethylene in lateral root formation and a positive role in adventitious root formation with modulation of auxin transport as a central point of ethylene–auxin crosstalk.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998