Gavin S. Ross

Analyses of Expressed Sequence Tags from Apple

The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5′-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BackgroundKiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs).ResultsThe ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified.ConclusionThis large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A β-galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl-β-d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the β-configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na2CO3-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the β-galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A [beta]-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-[beta]-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing [beta]-(1–>4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61–71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple [beta]-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

An apple polyphenol oxidase cDNA is up-regulated in wounded tissues

A full-length cDNA clone encoding apple (Malus domesticus) polyphenol oxidase (PPO) was isolated from a fruit peel cDNA library. Southern analysis indicated that apple PPO is encoded by a divergent multigene family. By northern analysis, PPO mRNA was only detected in a fruit sample taken one week after full bloom. PPO mRNA accumulated in wounded tissues, and also in peel tissue showing the symptoms of superficial scald, a post-harvest disorder. The induction of PPO mRNA provides the first evidence for transcriptional control of PPO expression after wounding or the manifestation of a physiological disorder.

Gene encoding polygalacturonase inhibitor in apple fruit is developmentally regulated and activated by wounding and fungal infection

A cDNA encoding polygalacturonase-inhibiting protein (PGIP) from mature apple fruit has been cloned and characterized. The open reading frame encodes a polypeptide of 330 amino acids, in which 24 amino acids at the N-terminus comprise the signal peptide. Apple PGIP contains 10 imperfect leucine-rich repeat sequence motifs averaging 24 amino acids in length. In addition to the 1.3xa0kb PGIP transcript, the cloned cDNA also hybridized to RNA molecules with sizes of 3.2 and 5.0xa0kb. Genomic DNA analysis revealed that the apple PGIP probably belongs to a small family of genes. PGIP transcript levels varied in fruit collected at different maturities, suggesting the gene is developmentally regulated. Very high PGIP transcript levels were detected in decayed areas and the tissue adjacent to the inoculation sites of Penicillium expansum and Botrytis cinerea. However, no increase in the amount of PGIP transcript in tissue distant from the decayed region was observed. Wounding on fruit also induced PGIP gene expression but to a much lessser extent when compared with decayed areas. After storage at 0xa0°C for 1 month, the abundance of PGIP transcript in ripe fruit was substantially increased. The PGIP gene in immature and ripe fruit was rapidly up-regulated by fungal infections, while in stored fruit the induction was very limited and concurred with an increase of fruit susceptibility to fungal colonization. Since PGIP gene expression is regulated by fruit development and responds to wounding, fungal infection and cold storage, these observations suggest that apple PGIP may have multiple roles during fruit development and stress response.

Apple ACC-oxidase and polygalacturonase: ripening-specific gene expression and promoter analysis in transgenic tomato

Levels of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and polygalacturonase (PG) mRNAs were characterized during ripening of Royal Gala, Braeburn and Granny Smith apples. Both ACC-oxidase and PG mRNAs were up-regulated in ripening fruit of all three cultivars. Expression in Royal Gala was detected earlier than in Braeburn and Granny Smith, relative to internal ethylene concentration. Genomic clones corresponding to the ACC-oxidase and PG mRNAs expressed in ripe apple fruit were isolated and ca. 2 kb of each promoter was sequenced. The start point of transcription in each gene was mapped by primer extension, and sequences homologous to elements in other ethylene-responsive or PG promoters were identified. The fruit specificity of the apple ACC-oxidase and PG promoters was investigated in transgenic tomato plants using a nested set of promoter fragments fused to the β-glucuronidase (gusA) reporter gene. For the ACC-oxidase gene, 450 bp of 5′ promoter sequence was sufficient to drive GUS expression, although this expression was not specific to ripening fruit. Larger fragments of 1966 and 1159 bp showed both fruit and ripening specificity. For the PG gene, promoter fragments of 1460 and 532 bp conferred ripening-specific expression in transgenic tomato fruit. However GUS expression was down-regulated by 2356 bp of promoter, suggesting the presence of a negative regulatory element between positions -1460 and -2356.

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)+ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.

A stress‐, pathogenesis‐, and allergen‐related cDNA in apple fruit is also ripening‐related

Abstract The screening of a ripe apple (Malus domestica ’Golden Delicious) cDNA expression library with polyclonal antibodies to spinach sucrose‐phosphate synthase resulted in the isolation of a cDNA clone pAP15. This clone encodes a homologue of a class of stress and pathogenesis‐related clones, and is also highly homologous to a class of allergenic proteins from apple without known biological function. The pAP15 cDNA insert is 817 base pairs in length and codes for a protein of 17.5 kD. Genomic Southern analysis suggests that the gene belongs to a family of at least 15 members. Northern analysis indicates that pAP 15 is ripening‐related, with an accumulation ofhomologous mRNA coincident with ethylene production during apple fruit ripening.