Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.     

Regulation of ripening and opportunities for control in tomato and other fruits

Fruits are an important part of a healthy diet. They provide essential vitamins and minerals, and their consumption is associated with a reduced risk of heart disease and certain cancers. These important plant products can, however, be expensive to purchase, may be of disappointing quality and often have a short shelf life. A major challenge for crop improvement in fleshy fruit species is the enhancement of their health‐promoting attributes while improving quality and reducing postharvest waste. To achieve these aims, a sound mechanistic understanding of the processes involved in fruit development and ripening is needed. In recent years, substantial insights have been made into the mechanistic basis of ethylene biosynthesis, perception and signalling and the identity of master regulators of ripening that operate upstream of, or in concert with a regulatory pathway mediated by this plant hormone. The role of other plant hormones in the ripening process has, however, remained elusive, and the links between regulators and downstream processes are still poorly understood. In this review, we focus on tomato as a model for fleshy fruit and provide an overview of the molecular circuits known to be involved in ripening, especially those controlling pigment accumulation and texture changes. We then discuss how this information can be used to understand ripening in other fleshy fruit‐bearing species. Recent developments in comparative genomics and systems biology approaches are discussed. The potential role of epigenetic changes in generating useful variation is highlighted along with opportunities for enhancing the level of metabolites that have a beneficial effect on human health.     

The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening

In the years since we last reviewed the use of mutants to study tomato fruit ripening ( Grierson et al. 1987 ), considerable information has been gained by the cloning, sequencing and identification of many mRNAs implicated in this developmental process. Genes involved in cell wall degradation, colour change and ethylene synthesis have been cloned, and antisense techniques have been developed and used to produce genetically engineered mutant fruit deficient in these aspects of ripening (see Gray et al. 1992 ). Recently, a previously cloned ripening gene has been used to complement a naturally occurring fruit colour mutant, yellow flesh ( Fray & Grierson 1993a ), and a ripening impaired mutant, ripening inhibitor, has been used to identify several new ripening-related mRNAs ( Picton et al. 1993b ). The chromosomal region bearing the ripening inhibitor mutation has been subjected to high-resolution mapping ( Churchill, Giovannoni & Tanksley 1993 ) and chromosome walking experiments are in progress to identify this gene.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato

The post-translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O-glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O-glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY-OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O-glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY-SlEIN2 module in tomato fruit ripening.     

Lipids of ripening tomato fruit and its mitochondrial fraction

The lipid composition of tomato fruit and its mitochondrial fraction were examined at various stages of fruit ripeness. Phosphatidyl choline, phosphatidyl ethanolamine, monogalactosyl diglyceride, digalactosyl diglyceride and phosphatidyl inositol were found to be the major lipids of tomato pericarp at all stages of ripeness. Mitochondrial lipids resembled those of the parent tissue except for the absence of monogalactosyl diglyceride and a greater percentage of diphosphatidyl glycerol and phosphatidic acid. Changes in the lipid-protein ratio of mitochondria were noted with ripening.     

Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism

Ethylene and polyamine metabolism, both sharing a common precursor, S-adenosylmethionine (SAM), were investigated during detached tomato (Lycopersicon esculentum Mill. nothovar F1 Lorena) fruit ripening. Putrescine (PUT) was found to be the major polyamine in the fruits, always over 100 nmols/g FW, while spermidine (SPD) was between 7% and 3% of the level of PUT. Spermine (SPM) was not detected at any stage of ripening. The level of PUT and SPD, did not change significantly during ripening in spite of the almost continuous synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and only at the last stage of ripening was a drastic decrease in SPD content observed. The results obtained show that the onset of ACC synthesis and its accumulation within the tissue is not a consequence of a decrease in SPD synthesis.     

Immunological properties of β-fructofuranosidase from ripening tomato fruit

The amount of tomato fruit β-fructofuranosidase extractable from the cell walls during ripening parallelled the changes in activity of the enzyme. Using the techniques of radioimmunoassay, double immunodiffusion analysis and immunotitration, no differences in immunological properties of β-fructofuranosidase between the various stages of fruit ripening were detected.     

Mutations in tomato 1-aminocyclopropane carboxylic acid synthase2 uncover its role in development beside fruit ripening

The role of ethylene in plant development is mostly inferred from its exogenous application. The usage of mutants affecting ethylene biosynthesis proffers a better alternative to decipher its role. In tomato (Solanum lycopersicum), 1-aminocyclopropane carboxylic acid synthase2 (ACS2) is a key enzyme regulating ripening-specific ethylene biosynthesis. We characterised two contrasting acs2 mutants; acs2-1 overproduces ethylene, has higher ACS activity, and has increased protein levels, while acs2-2 is an ethylene underproducer, displays lower ACS activity, and has lower protein levels than wild type. Consistent with high/low ethylene emission, the mutants show opposite phenotypes, physiological responses, and metabolomic profiles compared with the wild type. The acs2-1 mutant shows early seed germination, faster leaf senescence, and accelerated fruit ripening. Conversely, acs2-2 has delayed seed germination, slower leaf senescence, and prolonged fruit ripening. The phytohormone profiles of mutants were mostly opposite in the leaves and fruits. The faster/slower senescence of acs2-1/acs2-2 leaves correlated with the endogenous ethylene/zeatin ratio. The genetic analysis showed that the metabolite profiles of respective mutants co-segregated with the homozygous mutant progeny. Our results uncover that besides ripening, ACS2 participates in the vegetative and reproductive development of tomato. The distinct influence of ethylene on phytohormone profiles indicates the intertwining of ethylene action with other phytohormones in regulating plant development.     

Molecular biology of fruit ripening and its manipulation with antisense genes

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified.Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.