Gregory A. Tucker

Biochemistry of fruit ripening

Introduction - G A Tucker Avocado - G B Seymour and G A Tucker, Banana - G B Seymour Citrus fruit - E A Baldwin Exotics - J E Taylor Grape - A K Kanellis and K A Roubelakis-Angelakis Kiwifruit - N K Given (deceased) Mango - C Lizada Melon - G B Seymour and W B McGlasson Pineapple and papaya - R E Paull Pome Fruit - M Knee Soft fruit - K Manning Stone fruit - C J Brady Tomato - G Hobson and D Grierson. Index.

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in “Never ripe” mutants and the lack of activity in “ripening inhibitor” mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.

Effects of dietary polyphenols on gene expression in human vascular endothelial cells.

Previous studies have shown that consumption of fruit and vegetables plays a role in preventing the onset of CVD. These beneficial effects have been linked to the presence of polyphenolic compounds in plant-derived foods and their antioxidant capacity. It has been hypothesised that polyphenols may also have a direct effect on vascular endothelial cell growth and the expression of genes involved in angiogenesis and other roles of the endothelium. Previous studies in this area have tended to use concentrations of polyphenols that are supraphysiological (1-100 microm). The effects of more physiological concentrations (0.1 microm) of various individual polyphenols on gene expression were therefore investigated in cultured human umbilical vein endothelial cells (HUVEC) using both microarray and quantitative RT-PCR methodologies. Treatment of HUVEC with ferulic acid, quercetin or resveratrol (0.1 microm) resulted in changes to gene expression that for the three treatments amounted to significant (>2-fold) down-regulation of the expression of 363 genes and significant (>2-fold) up-regulation of 233 genes of the 10 000 genes present on the microarray. The majority of these genes were affected by resveratrol. Quantitative RT-PCR studies indicated that resveratrol (0.1 microm) significantly increased the expression of the gene encoding endothelial NO synthase (eNOS), which synthesises the vasodilator molecule NO, and both resveratrol and quercetin decreased expression of the potent vasoconstrictor, endothelin-1 (ET-1), while ferulic acid had no effect. The effects of resveratrol (0.1 microm) were also investigated when HUVEC were under oxidative stress following treatment with H2O2 (0-50 microm), which dose-dependently increased expression of eNOS and ET-1. Resveratrol stimulated eNOS mRNA in the absence of H2O2 and still allowed the increase with H2O2, but the effects were not additive. In contrast, resveratrol blocked the stimulatory effect of H2O2 on ET-1 expression. Hence, resveratrol has potent effects at a physiological concentration (0.1 microm) that would be expected to result in vasodilation and therefore help reduce blood pressure and the risk of CVD.

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening.

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.

Effect of the colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening

The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383–390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.

Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing.

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall-degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.

Polyuronide solubilization during ripening of normal and mutant tomato fruit

Abstract The amount and molecular size of soluble polyuronide extractable from ripening tomatoes is markedly affected by residual enzyme activity. The efficacy of phenol-acetic acid-water treatment to remove this residual activity is demonstrated. Data obtained using treated wall preparations confirms that there is an increase in soluble polyuronide during normal ripening and that this also occurs in the ‘Never-ripe’ mutant, and to a lesser degree in the ‘ripening-inhibitor’ mutant. However, changes in the molecular size of this polyuronide during normal ripening were not as extensive as previously reported and few changes were apparent in either of the mutants. Measurements were also made of polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) activity during ripening. The level of polygalacturonase activity does not appear to correlate with the amount of soluble polyuronide released, but may be related to the extent of depolymerisation. No relationship was apparent between the level of pectinesterase and either soluble polyuronide released or depolymerization.

Developmental Abnormalities and Reduced Fruit Softening in Tomato Plants Expressing an Antisense Rab11 GTPase Gene

A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall–modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a “mutant phenotype.” Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.

Flower abscission in mutant tomato plants

The effect of two mutations of the tomato known as Never ripe (Nr) and ripening inhibitor (rin) on abscission of the flowers was investigated. In the presence of ethylene the rate of abscission of normal and rin explants was similar, while that of Nr explants was delayed. The appearance and subsequent increases in both polygalacturonase (EC 3.2.1.15) and β-1-4-glucanase (EC 3.2.1.4) enzyme activities were similar in normal and rin explants, but retarded in Nr explants. Of these two, only polygalacturonase activity was exclusively associated with abscission-zone tissue.