Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato

The ripening of a fleshy fruit represents the summation of an array of biochemical processes that are regulated by interactions between developmental programs and environmental inputs. Analysis of tomato (Solanum lycopersicum) mutants and inhibitor studies indicate that ethylene is necessary for full development of the ripening program of climacteric fruit such as tomato, yet ethylene alone is not sufficient. This suggests that an interaction between ethylene and nonethylene (or developmental) pathways mediates ripening. In this study, we have examined the physiological basis for ripening inhibition of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato. Our data suggest that this inhibition is due to ethylene insensitivity in mutant fruit. Further investigation of ethylene responses in Gr and Nr-2 plants also revealed weak ethylene insensitivity during floral senescence and abscission and, during inhibition of root elongation, a phenotype associated with the triple response. However, ethylene-induced inhibition of hypocotyl elongation and petiole epinasty are normal in Gr and Nr-2, suggesting that these loci regulate a subset of ethylene responses. We have mapped both dominant mutations to a 2-cM overlapping region of the long arm of chromosome 1 of tomato, a region not previously linked to any known ethylene signaling loci. The phenotypic similarity and overlapping map location of these mutations suggest Gr and Nr-2 may be allelic and may possibly encode a novel component of the ethylene response pathway.     

Inhibitor-resistant early ethylene production during tomato fruit development

In addition to the ethylene formed at the onset of tomato fruit ripening, three peaks of ethylene are produced during earlier periods of in vitro development of tomato flower to fruit. This is the first report characterizing ethylene production during early development of tomato fruit. Previous reports from this laboratory showed that VFNT Cherry tomato calyces are transformed into fruit tissue when cultured in vitro at lower temperatures (16–23 °C). Early ethylene production was also measured in these ripening calyces, as well as in fruit and calyces of other tomato cultivars cultured in vitro. Calyces from Ailsa Craig and rin tomato flowers, which are not transformed into fruit tissue at these lower temperatures, also form ethylene during early periods of in vitro culture, but to a much smaller extent. Unlike ethylene formed at the onset of fruit ripening, the earlier peaks are resistant to the inhibitors, aminovinylglycine (AVG) and CoCl₂. The data suggest that ethylene produced during earlier periods of tomato fruit development is formed by an alternative biosynthetic pathway.     

Genome-wide identification and expression analysis of extensin genes in tomato

Extensins (EXTs) are major protein components in plant cell walls that play crucial roles in higher plants. The function of EXTs has been reported in several plants but is limited in tomato, especially in fruit ripening. In this study, we identified 83 EXTs in tomato, and divided them into seven groups. The gene intron-exon structure and protein-motif composition of SlEXTs were similar within each group but different among groups. SlEXT genes showed different expression patterns in roots, leaves, flowers and fruits, and some SlEXT gene expressions in flowers could be regulated by treatments of auxin, gibberellic acid and jasmonic acid. In particular, SlSEXT8 had higher and increased expression during tomato fruit ripening, and its expression could be induced by ethylene, suggesting SlSEXT8 may be involved in tomato fruit softening. The result provides insights into the function of EXTs, and will facilitate to further study EXT roles in tomato fruit ripening.     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant

The carbon dioxide and ethylene concentrations in tomato fruit ( Lycopersicon esculentum cv. Castelmart) and their stage of ripeness (characteristic external color changes) were periodically measured in fruit attached to and detached from the plant. An external collection apparatus was attached to the surface of individual tomato fruit to permit non-destructive sampling of internal gases. The concentration of carbon dioxide and ethylene in the collection apparatus reached 95% of the concentration in the fruit after 8 h. Gas samples were collected every 24 h. A characteristic climacteric surge in carbon dioxide (2-fold) and ethylene (10-fold) concentration occurred coincident with ripening of detached tomato fruit. Fruit attached to the plant exhibited a climacteric rise in ethylene (20-fold) concentration during ripening, but only a linear increase in carbon dioxide concentration. The carbon dioxide concentration increases in attached fruit during ripening, but the increase is a continuation of the linear increase seen in both attached and detached fruit before ripening and does not exhibit the characteristic pattern normally associated with ripening climacteric fruit. In tomato fruit, it appears that a respiratory climacteric per se, which has been considered intrinsic to the ripening of certain fruit, may not be necessary for the ripening of "climacteric" fruit at all, but instead may be an artifact of using harvested fruit.     

Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening

Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 ± 8 nmoles per minute per milligram protein at the mature green stage to 13.4 ± 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO₂ formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred.     

Ripening of fleshy fruit: Molecular insight and the role of ethylene

Development and ripening in fruit is a unique phase in the life cycle of higher plants which encompasses several stages progressively such as fruit development, its maturation, ripening and finally senescence. During ripening phase, several physiological and biochemical changes take place through differential expression of various genes that are developmentally regulated. Expression and/or suppression of these genes contribute to various changes in the fruit that make it visually attractive and edible. However, in fleshy fruit massive losses accrue during post harvest handling of the fruit which may run into billions of dollars worldwide. This encouraged scientists to look for various ways to save these losses. Genetic engineering appears to be the most promising and cost effective means to prevent these losses. Most fleshy fruit ripen in the presence of ethylene and once ripening has been initiated proceeds uncontrollably. Ethylene evokes several responses during ripening through a signaling cascade and thousands of genes participate which not only sets in ripening but also responsible for its spoilage. Slowing down post ripening process in fleshy fruit has been the major focus of ripening-related research. In this review article, various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described. Role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is also included. Taking clues from the studies in tomato as a model fruit, few case studies are reviewed.     

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxin.     

The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers

We investigated the function of the tomato (Lycopersicon esculentum) E8 gene. Previous experiments in which antisense suppression of E8 was used suggested that the E8 protein has a negative effect on ethylene evolution in fruit. E8 is expressed in flowers as well as in fruit, and its expression is high in anthers. We introduced a cauliflower mosaic virus 35S-E8 gene into tomato plants and obtained plants with overexpression of E8 and plants in which E8 expression was suppressed due to co-suppression. Overexpression of E8 in unripe fruit did not affect the level of ethylene evolution during fruit ripening; however, reduction of E8 protein by cosuppression did lead to elevated levels during ripening. Levels for ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC oxidase mRNA were increased approximately 7-fold in fruit of plants with reduced E8 protein. Levels of ACC synthase 2 mRNA were increased 2.5-fold, and ACC synthase 4 mRNA was not affected. Reduction of E8 protein in anthers did not affect the accumulation of ACC or of mRNAs encoding enzymes involved in ethylene biosynthesis. Our results suggest that the product of the E8 reaction participates in feedback regulation of ethylene biosynthesis during fruit ripening.