Steve Picton

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product γ-aminobutyric acid (GABA) in fruit, are discussed.

MOLECULAR CHARACTERISATION OF CDNA CLONES REPRESENTING PECTIN ESTERASE ISOZYMES FROM TOMATO

Two pectin esterase cDNA clones representing different isozymes with ca. 95% homology were isolated from an early ripening tomato fruit cDNA library. Both clones were longer than previously published sequences, and the encoded proteins possessed extended (229–233 amino acid) putative N-terminal extensions. In addition, the mRNA species corresponding to the two clones showed differential levels of expression in fruit.

Characterization and accumulation pattern of an mRNA encoding an abscission-related β-1,4-glucanase from leaflets of Sambucus nigra

A cDNA library was produced using mRNA extracted from ethylene-treated leaflet abscission zones of common elder (Sambucus nigra). Screening of the library with the insert from pBAC10, which encodes an abscission β-1,4-glucanase (cellulase) from bean (Phaseolus vulgaris), resulted in the isolation of a near-full-length cDNA which was designated JET 1. Northern analysis, using JET 1 as a probe, detected a transcript of 1.9 kb that accumulated prior to the first visible signs of cell separation. Accumulation of the JET 1 transcript is promoted by ethylene and primarily restricted to the tissue comprising the abscission zone. Sequence analysis of JET 1 indicates it is 1768 bp in length and shares significant homology at the amino acid level with β-1,4-glucanases from the leaf abscission zone of P. vulgaris (67%) and ripening avocado fruit (48%). The predicted peptide sequence of the S. nigra enzyme contains two potential glycosylation sites. Genomic Southern analysis of S. nigra DNA reveals that JET 1 may belong to a multi-gene family.

Stimulation and Inhibition of Expression of Ripening-related mRNAs in Tomatoes as Influenced by Chilling Temperatures

Summary The effects of chilling temperatures on developmental gene expression in tomato fruit have been examined using eight ripening-related cDNA clones as hybridization probes. Fruit were harvested at the breaker stage of maturity and ripened at 24 °C, or held at 4 °C for 6, 12 or 18 days before being transferred to 24 °C. When fruit were held at 24 °C without chilling, they accumulated lycopene and produced ethylene in a typical pattern. At 4 °C both lycopene accumulation and ethylene production were inhibited. With increasing periods at chilling temperatures, subsequent ripening at 24 °C, as indicated by lycopene accumulation, was reduced. Ethylene production at 24 °C was relatively unaffected by previous chilling temperatures. The patterns of accumulation of mRNAs homologous to the cDNA clones at both 4 °C and after transfer to 24 °C were variable. At 4 °C, expression of most mRNAs declined steadily to low levels over 18 days. However, expression of mRNA homologous to pTOM 13, a clone thought to be associated with ethylene synthesis, was unique in that levels increased to a maximum after 9 days at 4 °C before declining. After transfer of fruit to 24 °C, accumulation of this mRNA declined rapidly and then increased, but only to levels of about 30 % of the maximum observed at 4 0C. Expression of the other mRNAs generally increased after transfer to 24 °C, but the timing of maximum expression was dependent on the chilling period and the clone concerned. The appearance of polygalacturonase mRNA at 24 °C was markedly reduced after 18 days at 4 °C.

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 α-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.

Ethylene Genes and Fruit Ripening

Ethylene was identified as affecting plant growth at the start of this century (42), and is produced in at least trace amounts by almost all higher plants. It is involved in the control and coordination of a diverse range of plant growth and developmental processes including seed germination, root growth and development, vegetative growth, flower development, the ripening of fruits and the senescence and abscission of flower, leaf and fruit organs (Chapter G2). The hormone thus exerts its influence throughout the entire developmental progression of plants. Ethylene has also been implicated in the modulation of responses of plants to a wide range of biotic and abiotic stresses. The role of ethylene in regulating the ripening of tomato fruits has received particular attention, partly because of its intrinsic scientific interest, but also for reasons of experimental convenience and the economic importance of the tomato fruit as a major food crop.

The manipulation and modification of tomato fruit ripening by expression of antisense RNA in transgenic plants

The common cultivated tomato (Lycopersicon esculentum Mill.) provides a major focus for improvement of crop quality through genetic engineering. Identification of ripening-related cDNAs has enabled the modification of specific aspects of ripening by manipulating gene expression in transgenic plants. By utilizing ‘antisense RNA’ to modify expression of ripening genes, we have inhibited the production of the cell wall - metabolising enzymes polygalacturonase and pectinesterase and created transgenic plants that contain, effectively, single, targeted mutations affecting these genes. Furthermore, this approach has been used with previously unidentified cDNA clones to enable both functional identification and manipulation of genes involved in ethylene production (ACC oxidase) and carotenoid biosynthesis (phytoene synthase). The use of antisense RNA targeted to specific genes to alter ripening phenotypes and improve commercial utility of fruit by affecting shelf-life, processing characteristics and nutritional content is discussed.

Protein profiles in root-tips of two wheat (Triticum aestivum L.) cultivars with differential tolerance to aluminium

The molecular basis for differential aluminium tolerance of two wheat cultivars was investigated using two-dimensional gel electrophoresis of root-tip proteins labelled in vivo. Five proteins were specific to the tolerant cultivar in the absence of Al. The same five were induced in response to Al in both cultivars.

Sequence of a Cloned Tomato Ubiquitin Conjugating Enzyme

Ubiquitin is a small, abundant protein that is seemingly present in a11 eukaryotic cells and that is covalently ligated to specific protein substrates via an ATP-dependent reaction. This ubiquitinous has been demonstrated to target proteins for subsequent cellular degradation. Briefly, the process involves activation of ubiquitin, catalyzed by ubiquitin-activating enzymes, transfer to a family of ubiquitin-camer/conjugating proteins with different substrate specificities (UBCs or E~s) , and, finally, ubiquitin-protein ligation by either direct transfer of ubiquitin from E2 or mediated transfer utilizing ubiquitin-protein ligases (Hershko and Ciechanover, 1992; Jentsch, 1992). In this paper we report the sequence of a full-length tomato cDNA clone (LeE2,6.5). The cloned mRNA encodes a derived 148-amino acid sequence of 16.5 kD with an isoelectric point of 7.95. The peptide has a conserved region containing a putative active Cys residue at position 85 that is observed in other E2 sequences (Sullivan and Vierstra, 1989; Jentsch et al., 1990) and is thought to be required for the thiol ester formation with ubiquitin (van Nocker and Vierstra, 1991). In addition, a further Cys residue is present at position 108. The presence of two Cys residues and two ubiquitin thiol ester species has been observed for both wheat and Arabidopsis E2s, suggesting that E2s may interact with more than a single ubiquitin simultaneously (Sullivan and Vierstra, 1989; Sullivan et al., 1990). The cDNA clone ERT 17 was isolated as part of a research program to identify genes expressed at the onset of tomato fruit ripening (Picton et al., 1993). ERT 17 mRNA is detected during fruit development and increases to a peak after the onset of ripening. It is present both in leaves and at an increased leve1 in senescing and mechanically wounded leaf tissue. Although ripening, wounding, and foliar senescence are a11 ethylene-mediated processes, ERT 17 mRNA accumulation in fruit is not increased by ethylene treatment, and