Gene expression during fruit ripening in avocado

The poly(A) ⁺RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

The biosynthetic pathway for indole-3-acetic acid changes during tomato fruit development

Phytohormone metabolism during fruit ripening is critical to the controlof this developmental process, yet we know little about pathways for theproduction of many of these signaling compounds. Using stable isotope labelingin both an in vitro aseptic tomato fruit culture systemanddetached greenhouse-grown tomato fruit, we have shown by mass spectral analysisthat tomato uses the tryptophan-independent pathway to produce IAA fromanthranilate or indole. We also show that there is a developmental switch fromtryptophan utilization to tryptophan-independent production that occurs betweenmature green and red-ripe stages of fruit development. Moreover, this pathwayswitch does not appear to be associated with ripening per se in that fruit fromneverripe tomato plants also utilize the tryptophanindependent pathway.     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Fine mapping of QTLs of chromosome 2 affecting the fruit architecture and composition of tomato

Negative correlations between quality traits and fruit size may hamper the breeding of fresh market tomato varieties for better organoleptic qualities. In a recent QTL analysis, QTLs with large effects on fruit weight, locule number and several quality traits were detected in the distal 50 cM of chromosome 2, but favorable alleles for fruit weight and locule number were unfavorable to quality traits. Substitution mapping was undertaken to determine whether the effects were due to a single QTL or to several tightly linked QTLs. Several chromosomal segments were characterized using near-isogenic lines. Five of them appeared to be involved in one or several traits. Considering the five segments from the top to the bottom of the region, the QTLs detected in each segment controlled the variation of: (1) fruit weight, (2) soluble solids content and dry matter weight, (3) fruit weight, (4) locule number and (5) fruit weight, dry matter weight, total sugars, titratable acidity and soluble solids content. This last cluster illustrates an antagonism between fruit weight and four quality traits, as favorable alleles are not conferred by the same parent in both cases. Nevertheless, several antagonistic QTLs were separated from each other in the first four segments, holding the promise for marker-assisted improvement of fruit quality traits without compromising the fruit size.     

Genes from Lycopersicon chmielewskii affecting tomato quality during fruit ripening

Three chromosomal segments from the wild tomato, L. chmielewskii, introgressed into the L. esculentum genome have been previously mapped to the middle and terminal regions of chromosome 7 (7M, 7T respectively), and to the terminal region of chromosome 10 (10T). The present study was designed to investigate the physiological mechanisms controlled by the 7M and 7T segments on tomato soluble solids (SS) and pH, and their genetic regulation during fruit development. The effects of 7M and 7T were studied in 64 BC₂F₅ backcross inbred lines (BILs) developed from a cross between LA 1501 (an L. esculentum line containing the 7M and 7T fragments from L. chmielewskii), and VF145B-7879 (a processing cultivar). BILs were classified into four homozygous genotypes with respect to the introgressed segments based on RFLP analysis, and evaluated for fruit chemical characteristics at different harvest stages. Gene(s) in the 7M fragment reduce fruit water uptake during ripening increasing pH, sugars, and SS concentration. Gene(s) in the 7T fragment were found to be associated with higher mature green fruit starch concentration and red ripe fruit weight. Comparisons between tomatoes ripened on or off the vine suggest that the physiological mechanisms influenced by the L. chmielewskii alleles are dependent on the translocation of photosynthates and water during fruit ripening.