Alan B. Bennett

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Cooperative disassembly of the cellulose–xyloglucan network of plant cell walls: parallels between cell expansion and fruit ripening

Modification of the plant primary cell wall is required for both cell expansion and for developmental events, such as fruit softening, where cell size remains static but where wall loosening is an important feature. Recent studies suggest that the cellulose-xyloglucan network is targeted by similar enzymatic activities in both expanding cells and ripening fruit but that unique isoforms are expressed in each process. Disassembly of this structural network probably involves the concerted and synergistic action of suites of these enzyme families, where one family of cell wall modifying proteins might mediate the activity of another, providing the basis for orchestrating ordered cell wall restructuring and turnover.

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.

Transgenic expression of pear PGIP in tomato limits fungal colonization.

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

Two divergent endo-beta-1, 4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence.

Antisense Acid Invertase (TIV1) Gene Alters Soluble Sugar Composition and Size in Transgenic Tomato Fruit

Invertase ([beta]-fructosidase, EC 3.2.1.26) hydrolyzes sucrose to hexose sugars and thus plays a fundamental role in the energy requirements for plant growth and maintenance. Transgenic plants with altered extracellular acid invertase have highly disturbed growth habits. We investigated the role of intracellular soluble acid invertase in plant and fruit development. Transgenic tomato (Lycopersicon esculentum Mill.) plants expressing a constitutive antisense invertase transgene grew identically to wild-type plants. Several lines of transgenic fruit expressing a constitutive antisense invertase gene had increased sucrose and decreased hexose sugar concentrations. Each transgenic line with fruit that had increased sucrose concentrations also had greatly reduced levels of acid invertase in ripe fruit. Sucrose-accumulating fruit were approximately 30% smaller than control fruit, and this differential growth correlated with high rates of sugar accumulation during the last stage of development. These data suggest that soluble acid invertase controls sugar composition in tomato fruit and that this change in composition contributes to alterations in fruit size. In addition, sucrose-accumulating fruit have elevated rates of ethylene evolution relative to control fruit, perhaps as a result of the smaller fruit size of the sucrose-accumulating transgenic lines.

Uniform ripening Encodes a Golden 2-like Transcription Factor Regulating Tomato Fruit Chloroplast Development

Pretty or Sweet The grocery-store tomato that looks beautiful but tastes like tart cardboard arises from selection processes favoring phenotypes that make commercial production more reliable. Significant in that selection process was a mutation that reduced the mottled color variations of unripe green tomatoes, leaving them a uniform, pale, green. Powell et al. (p. 1711) analyzed the molecular biology of the mutation. The uniform ripening mutation turns out to disable a transcription factor called Golden 2-like (GLK2). GLK2 expression increases the fruits photosynthetic capacity, resulting in higher sugar content. Controlling when tomatoes turn from green to red requires knocking out the gene that adds flavor. Modern tomato (Solanum lycopersicum) varieties are bred for uniform ripening (u) light green fruit phenotypes to facilitate harvests of evenly ripened fruit. U encodes a Golden 2-like (GLK) transcription factor, SlGLK2, which determines chlorophyll accumulation and distribution in developing fruit. In tomato, two GLKs—SlGLK1 and SlGLK2—are expressed in leaves, but only SlGLK2 is expressed in fruit. Expressing GLKs increased the chlorophyll content of fruit, whereas SlGLK2 suppression recapitulated the u mutant phenotype. GLK overexpression enhanced fruit photosynthesis gene expression and chloroplast development, leading to elevated carbohydrates and carotenoids in ripe fruit. SlGLK2 influences photosynthesis in developing fruit, contributing to mature fruit characteristics and suggesting that selection of u inadvertently compromised ripe fruit quality in exchange for desirable production traits.

Pedicel breakstrength and cellulase gene expression during tomato flower abscission

Six cellulase genes were isolated from total RNA of the ethylene-treated tomato (Lycopersicon esculentum Mill.) flower abscission zone by reverse-transcription polymerase chain reaction using degenerate primers to conserved amino acid sequences from known plant cellulases. Four of the gene fragments are homologous to fruit pericarp cellulases. The other two are novel cellulase genes, referred to as Cel5 and Cel6. Breakstrength and cellulase gene expression were then analyzed in naturally abscising flowers and flower explants. In both naturally abscising flowers and flower explants induced to abscise in air or ethylene, both new cellulase mRNAs were correlated with flower shedding. Whereas the Cel5 mRNA increased in later stages of abscission, the Cel6 mRNA was present in nonabscising flowers and then decreased in the final stage of abscission. A third cellulase, Cel1, increased during the final stage of abscission in flower explants and yet did not increase during shedding in planta, although it was detectable at low levels in all abscission stages. Cel1 and Cel5 mRNA decreased 99% when indole-3-acetic acid was added during ethylene treatment, consistent with low levels of abscission (3%). In contrast, Cel6 mRNA increased slightly when indole-3-acetic acid was added. These results suggest that abscission is a multistep process involving both activated and repressed cellulase genes and that the relative importance of each cellulase in the process depends on the physiological conditions under which abscission takes place.

The public-private structure of intellectual property ownership in agricultural biotechnology.

New findings indicate that there may be benefits from more collaborative models of intellectual property management in the public sector.

QTL ANALYSIS OF FRUIT ANTIOXIDANTS IN TOMATO USING LYCOPERSICON PENNELLII INTROGRESSION LINES

Antioxidants present in fruits and vegetables may help prevent some chronic diseases such as cancer, arthritis, and heart disease. Tomatoes provide a major contribution to human dietary nutrition because of their widespread consumption in fresh and processed forms. A tomato introgression line population that combines single chromosomal segments introgressed from the wild, green fruited species Lycopersicon pennellii in the background of the domesticated tomato, Lycopersicon esculentum, was used to identify quantitative trait loci (QTL) for nutritional and antioxidant contents. The concentration of ascorbic acid, total phenolics, lycopene and β-carotene, and the total antioxidant capacity of the water-soluble fraction (TACW) were measured in the ripe fruits. A total of 20 QTL were identified, including five for TACW (ao), six for ascorbic acid (aa), and nine for total phenolics (phe). Some of these QTL (ao6-2, ao6-3, ao7-2, ao10-1, aa12-4, phe6-2, and phe7-4) increased levels as compared to the parental line L. esculentum. For lycopene content, we detected four QTL, but none increased levels relative to L. esculentum. The two QTL (bc6-2 and bc6-3) detected for β-carotene increased its levels. The traits studied displayed a strong environmental interaction as only 35% of the water-soluble antioxidant QTL (including TACW, ascorbic, and phenolic contents) were consistent over at least two seasons. Also, only two QTL for phenolics were observed when plants were grown in the greenhouse and none was detected for ascorbic or TACW. The analysis demonstrates that the introgression of wild germplasm may improve the nutritional quality of tomatoes; however regulation appears to be complex with strong environmental effects.