Grantley W. Lycett

Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins

The nucleotide sequence and derived amino acid sequence were determined for a full-length version of the tomato cDNA clone, pTOM75, the mRNA for which has previously been shown to accumulate in roots, ripening fruit and senescing leaves. Computer analysis of the predicted protein product, which we have named tomato ripening-associated membrane protein (TRAMP) indicates strong homology to known transmembrane channel proteins from other organisms. Northern analysis showed that this gene was induced by waterstress and that this induction was unaffected in an ABA-deficient genetic back-ground.

Increased 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Shoots of Flooded Tomato Plants Raises Ethylene Production to Physiologically Active Levels

Soil flooding increased 1-aminocyclopropane-1-carboxylic (ACC) acid oxidase activity in petioles of wild-type tomato (Lycopersicon esculentum L.) plants within 6 to 12 h in association with faster rates of ethylene production. Petioles of flooded plants transformed with an antisense construct to one isoform of an ACC oxidase gene (ACO1) produced less ethylene and had lower ACC oxidase activity than those of the wild type. Flooding promoted epinastic curvature but did so less strongly in plants transformed with the antisense construct than in the wild type. Exogenous ethylene, supplied to well-drained plants, also promoted epinastic curvature, but transformed and wild-type plants responded similarly. Flooding increased the specific delivery (flux) of ACC to the shoots (picomoles per second per square meter of leaf) in xylem sap flowing from the roots. The amounts were similar in both transformed and wild-type plants. These observations demonstrate that changes in ACC oxidase activity in shoot tissue resulting from either soil flooding or introducing ACC oxidase antisense constructs can influence rates of ethylene production to a physiologically significant extent. They also implicate systemic root to shoot signals in regulating the activity of ACC oxidase in the shoot.

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.

Silencing of the Major Salt-Dependent Isoform of Pectinesterase in Tomato Alters Fruit Softening

Pectinesterase (PE; E.C. 3.1.1.11) is an enzyme responsible for the demethylation of galacturonyl residues in high-molecular-weight pectin and is believed to play an important role in cell wall metabolism. In this study, Pmeu1, a ubiquitously expressed PE gene, has been characterized by antisense suppression in tomato (Solanum lycopersicum). Transgenic tomato plants showed reduced PE activity levels in both green fruit and leaf tissue to around 65% and 25% of that found in wild-type plants, respectively. Pmeu1 was observed to encode a salt-dependent PE isoform that correlated with PE1 as previously described in fruit tissue. Silencing of Pmeu1 did not result in any detectable phenotype within the leaf tissue despite the gene product representing the major isoform in this tissue. In comparison, silencing in fruit resulted in an enhancement to the rate of softening during ripening. The role of PMEU1 in fruit ripening is discussed.

Tomato Rab11a characterization evidenced a difference between SYP121 dependent and SYP122 dependent exocytosis

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat beta-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory events.

The role of Rab GTPases in cell wall metabolism

The synthesis and modification of the cell wall must involve the production of new cell wall polymers and enzymes. Their targeted secretion to the apoplast is one of many potential control points. Since Rab GTPases have been strongly implicated in the regulation of vesicle trafficking, a review of their involvement in cell wall metabolism should throw light on this possibility. Cell wall polymer biosynthesis occurs mainly in the Golgi apparatus, except for cellulose and callose, which are made at the plasma membrane by an enzyme complex that cycles through the endomembrane system and which may be regulated by this cycling. Several systems, including the growth of root hairs and pollen tubes, cell wall softening in fruit, and the development of root nodules, are now being dissected. In these systems, secretion of wall polymers and modifying enzymes has been documented, and Rab GTPases are highly expressed. Reverse genetic experiments have been used to interfere with these GTPases and this is revealing their importance in regulation of trafficking to the wall. The role of the RabA (or Rab11) GTPases is particularly exciting in this respect.

A rab11-like gene is developmentally regulated in ripening mango (Mangifera indica L.) fruit

A full-length cDNA clone from mango (Mangifera indica L.) fruit has homology to the rab11/YPT3 class of small GTPases. The corresponding mRNA is expressed in fruit, only during ripening. The likely involvement of this RabX protein in trafficking cell-wall modifying enzymes through the trans-Golgi network is discussed.

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the β-subunit and most of the α-subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the α polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.

Cell wall composition of tomato fruit changes during development and inhibition of vesicle trafficking is associated with reduced pectin levels and reduced softening.

Fruit development entails a multitude of biochemical changes leading up to the mature green stage. During this period the cell wall will undergo complex compositional and structural changes. Inhibition of genes encoding elements of the machinery involved in trafficking to the cell wall presents us with a useful tool to study these changes and their associated phenotypes. An antisense SlRab11a transgene has previously been shown to reduce ripening-associated fruit softening. SlRab11a is highly expressed during fruit development which is associated with a period of pectin influx into the wall. We have analysed the cell wall polysaccharides at different stages of growth and ripening of wild type and antisense SlRab11a transgenic tomato (Solanum lycopersicum cv, Ailsa Craig) fruit. Our results demonstrated intriguing changes in cell wall composition during the development and ripening of wild type Alisa Craig tomato fruit. Analysis of SlRab11a expression by TaqMan PCR showed it to be expressed most strongly during growth of the fruit, suggesting a possible role in cell wall deposition. The SlRab11a antisense fruit had a decreased proportion of pectin in the cell wall compared with the wild type. We suggest a new approach for modification of fruit shelf-life by changing cell wall deposition rather than cell wall hydrolytic enzymes.

The effect of chimeric transgene architecture on co-ordinated gene silencing

Abstract. Two gene constructs were made consisting of a 244-bp sense fragment from the 5′ end of a polygalacturonase cDNA, the 3′ end of which was ligated to a 414-bp fragment from the 5′ end of a phytoene synthase cDNA. In the first construct, the phytoene synthase fragment was in a sense orientation (sense/sense chimeric gene) and in the second construct the phytoene synthase fragment was in an antisense orientation (sense/antisense chimeric gene). Both chimeric genes were inserted between a cauliflower mosaic virus promoter and terminator. Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants transformed with each construct gave rise to transformants with three distinct phenotypes: plants with red fruit, plants with pure yellow fruit and plants with red and yellow sectored fruit. For both chimeric constructs, expression of the endogenous polygalacturonase and phytoene synthase genes were found to be co-ordinately suppressed in yellow tissue, but showed normal expression in red tissue. Data from microscopic analyses of fruit chromoplasts, from the three phenotypes, implied that phytoene synthase suppression from each construct predominantly had two states within a cell: on or off.