Pamela Dunsmuir

Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening

The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. Fruit in which Exp1 protein accumulation was suppressed to 3% that of wild-type levels were firmer than controls throughout ripening. Suppression of Exp1 protein also substantially inhibited polyuronide depolymerization late in ripening but did not prevent the breakdown of structurally important hemicelluloses, a major contributor to softening. In contrast, fruit overexpressing high levels of recombinant Exp1 protein were much softer than controls, even in mature green fruit before ripening commenced. This softening was correlated with the precocious and extensive depolymerization of structural hemicelluloses, whereas polyuronide depolymerization was not altered. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein.

Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity

The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3′ flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30-to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2-to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO2 concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide.

DIFFERENTIAL EXPRESSION OF EXPANSIN GENE FAMILY MEMBERS DURING GROWTH AND RIPENING OF TOMATO FRUIT

AbstractcDNA 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.

Homologous recombination in plant cells after Agrobacterium-mediated transformation.

A single amino-acid change in the acetolactate synthase (ALS) protein of tobacco confers resistance to the herbicide chlorsulfuron. A deleted, nonfunctional fragment from the acetolactate synthase gene, carrying the mutant site specifying chlorsulfuron resistance plus a closely linked novel restriction site marker, was cloned into a binary vector. Tobacco protoplasts transformed with Agrobacterium tumefaciens carrying this vector yielded chlorsulfuron-resistant colonies. DNA gel blot analysis of DNA from these colonies suggested that in three transformants homologous recombination had occurred between the endogenous ALS gene and the deleted ALS gene present in the incoming T-DNA. Plants were regenerated from these chlorsulfuron-resistant colonies, and in two of the transformants, genetic analysis of their progeny showed that the novel gene segregated as a single Mendelian locus. Possible models for the generation of these recombinant plants are discussed.

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.

Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis-related PR1b gene

Correlation of the temporal and spacial pattern of induction of the pathogenesis-related (PR) genes PR1a, PR1b, and PR1c with viral infections in certain tobacco cultivars has implicated PR proteins in viral disease resistance. To test whether the PR1 proteins of tobacco are involved in viral resistance, transgenic Nicotiana tabacum plants were constructed which constitutively express the PR1b gene. This protein was secreted from cells of transgenic plants and accumulated in the extracellular space at levels equivalent to those found in nontransgenic plants in association with disease resistance. Transgenic plants derived from the cultivar (cv.) Xanthi (susceptible to tobacco mosaic virus [TMV] infection) exhibited no delayed onset or reduction in the severity of systemic symptoms after TMV infection. In transgenic plants derived from cv. Xanthi-nc (TMV resistant), the time of appearance, the size and general morphology, and the number of viral lesions produced were similar to the parental control plants after TMV infection. These data indicate that the PR1b protein of tobacco is not sufficient for TMV resistance, and imply that the PR1 proteins may not function as unique antiviral factors.

The expression of cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringae pv tabaci

SummaryWe used in vitro growth inhibition assays to demonstrate that synthetic cecropin protein has potent activity against a range of plant pathogenic bacteria. We then prepared transgenic tobacco plants which express cecropin mRNA and protein. We have used Pseudomonas syringae pv tabaci infection of these transgenic tobacco as a model system to evaluate whether the plants which express cecropin protein also have increased tolerance to infection. We found no dramatic difference in disease response between plants which are expressing cecropin protein and control plants which were derived from the transformation with a binary vector which did not carry the gene encoding cecropin protein.

Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin

Abstract Expansins are proteins that cause cell wall loosening, and are involved in many aspects of cell wall modification during development. In tomato, the expansin gene LeExp1 shows ripening-related accumulation of mRNA and protein, and transgenic silencing of the expression of this gene results in tomato fruit that are significantly firmer than corresponding azygous controls throughout ripening. Examination of postharvest quality characteristics of fruit suppressed in accumulation of LeExp1 protein found that increased firmness resulted in significantly improved fruit integrity during storage at 13xa0°C. Based upon the first appearance of noticeable deterioration, fruit shelf life was extended by 5–10 days, depending upon the packaging. However, the increased firmness of LeExp1-suppressed fruit did not result in increased resistance to the necrophytic pathogens Botrytis cinerea and Alternaria alternata . Juice prepared from LeExp1-suppressed fruit following a microwave break had a soluble solids content (°Brix), insoluble solids content (precipitate weight ratio) and serum viscosity similar to controls. Resuspension of the insoluble pelleted particulate material in 15% of the serum produced a thick paste, which allowed estimation of gross viscosity in a Bostwick consistometer. The viscosity of paste from LeExp1-suppressed fruit was 19% greater than that from corresponding azygous controls, presumably due to changes in the insoluble particulate components affecting flow characteristics. No significant effects of the LeExp1 transgene on fruit size or fruit number per plant were noted. The data suggest that fruit suppressed in expression of LeExp1 have improved shelf life and processing properties.

Suppression of a ripening-related endo-1,4-β-glucanase in transgenic pepper fruit does not prevent depolymerization of cell wall polysaccharides during ripening

The function of the ripening-related endo-1,4-β-D-glucanase (EGase) CaCel1 in fruit softening was investigated by suppression of CaCel1 gene expression in transgenic pepper (Capsicum annuum L.) plants using constitutive expression of a truncated sense CaCel1 transgene. In suppressed lines, immunodetectable CaCel1 protein and extractable CMCase activity were reduced to at or below the limit of detection in ripe mature red fruit, suggesting that in pepper ripening-related CMCase activity is the product of a single gene. However, the abundances of two mRNAs derived from the CaCel1 gene by differential transcription initiation were affected differently in suppressed lines. Accumulation of a 1.7 kb CaCel1 transcript was strongly suppressed, whereas the abundance of a 2.1 kb CaCel1 transcript was only partially reduced. This implies that the 1.7 kb mRNA is responsible for producing CaCel1 protein, while the 2.1 kb mRNA is translationally inactive, and as such is recalcitrant to co-suppression. Chelator-soluble polyuronides exhibited little or no depolymerization during ripening, but matrix glycans including xyloglucan were extensively depolymerized. Depolymerization of non-xyloglucan matrix glycans was the prominant cell wall change observed during pepper ripening. However, the lack of CaCel1 activity in suppressed fruit had no detectable effect on ripening-related matrix glycan depolymerization, which occurred at wild-type levels. Recombinant CaCel1 protein purified from a transgenic pepper line over-expressing functional CaCel1 was active against pepper matrix glycansin vitro, and showed greater activity against non-xyloglucan polysaccharides than against xyloglucan. Transgenic suppression of CaCel1 EGase activity has not identified the natural cell wall substrate for this enzyme, and shows that activities other than CaCel1 are responsible for the depolymerization of matrix glycans occurring during ripening in pepper.

Constitutive overexpression of a ripening-related pepper endo-1,4-β-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening

The ripening-related pepper endo-1,4-β-D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71xa0kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.