Molecular characterization of a tomato polygalacturonase gene abundantly expressed in the upper third of pistils from opened and unopened flowers

 A polygalacturonase (PG) gene, TPG7 (Lyces;Pga1;8), has been cloned from tomato (Lycopersicon esculentum Mill., cv. Rutgers). RNA blot analysis reveals that TPG7 is highly expressed in pistils (ovary removed) from unopened and fully open flowers. Dissection of mature pistils demonstrated that TPG7 expression is limited to the top third (stigmatic region) of the pistils. This is contrasted with another tomato PG, TAPG4, which is also expressed in the same region of the pistil but only in mature pistils from fully open flowers. Hybridization of the TPG7 probe to anther RNA was nil to none and was barely detectable in RNA from leaf and flower abscission zones. The TPG7 polypeptide shares 39% sequence identity with the tomato fruit PG and between 63% and 73% sequence identities with six other tomato PGs. Received: 15 March 1999 / Revision received: 6 October 1999 / Accepted: 7 Oktober 1999     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

Differential regulation of the tomato ETR gene family throughout plant development

Ethylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ～ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue-specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.     

Polygalacturonase beta-subunit antisense gene expression in tomato plants leads to a progressive enhanced wound response and necrosis in leaves and abscission of developing flowers

Tomato (Lycopersicon esculentum var. Better Boy) plants were transformed with a tomato leaf wound-inducible polygalacturonase (PG) beta-subunit gene in the antisense orientation (PGbetaS-AS) under the control of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants exhibited small localized lesions, which eventually enlarged and spread throughout the entire surfaces of the leaves, resulting in cell death. The same lesions were also observed in the peduncle of developing flowers, extending to the whole flower causing abscission, resulting in a sterile phenotype. Leaves of transgenic plants exhibited elevated levels of PG activity, hydrogen peroxide, and enhanced defense signaling in response to wounding and elicitor treatment. The defense signaling increased was accompanied by an increased resistance toward tobacco hornworm (Manduca sexta) larvae. The cumulative results suggest that in the absence of the beta-subunit protein in tomato leaves, an increase in PG activity occurred that led to an enhanced wound response, the formation of lesions leading to severe necrosis, and an abscission of developing flowers.     

The never ripe mutation blocks ethylene perception in tomato.

Seedlings of tomato fruit ripening mutants were screened for their ability to respond to ethylene. Ethylene induced the triple response in etiolated hypocotyls of all tomato ripening mutants tested except for one, Never ripe (Nr). Our results indicated that the lack of ripening in this mutant is caused by ethylene insensitivity. Segregation analysis indicated that Nr-associated ethylene insensitivity is a single codominant trait and is pleiotropic, blocking senescence and abscission of flowers and the epinastic response of petioles. In normal tomato flowers, petal abscission and senescence occur 4 to 5 days after the flower opens and precede fruit expansion. If fertilization does not occur, pedicel abscission occurs 5 to 8 days after petal senescence. If unfertilized, Nr flowers remained attached to the plant indefinitely, and petals remained viable and turgid more than four times longer than their normal counterparts. Fruit development in Nr plants was not preceded by petal senescence; petals and anthers remained attached until they were physically displaced by the expanding ovary. Analysis of engineered 1-aminocyclopropane-1-carboxylate (ACC) synthase-overexpressing plants indicated that they are phenotypic opposites of Nr plants. Constitutive expression of ACC synthase in tomato plants resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. There were no obvious effects on senescence in leaves of ACC synthase overexpressers, suggesting that although ethylene may be important, it is not sufficient to cause tomato leaf senescence; other signals are clearly involved.     

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.     

Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a Role for Polygalacturonase in Ripening-Associated Pectin Disassembly

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.Fruit ripening is a genetically programmed event that is characterized by a number of biochemical and physiological processes that alter fruit color, flavor, aroma, and texture (Brady, 1987). Extensive cell wall modifications occur during ripening and are thought to underlie processes such as fruit softening, tissue deterioration, and pathogen susceptibility. These modifications are regulated at least in part by the expression of genes that encode cell wall-modifying enzymes (Fischer and Bennett, 1991). Pectins are a major class of cell wall polysaccharides that are degraded during ripening, undergoing both solubilization and depolymerization. In tomato the majority of ripening-associated pectin degradation is attributable to the cell wall hydrolase PG. Transgenic tomato plants with altered PG gene expression indicated that PG-dependent pectin degradation is neither required nor sufficient for tomato fruit softening to occur (Sheehy et al., 1988; Smith et al., 1988; Giovannoni et al., 1989). However, data from experiments using fruit of the same transgenic lines strongly suggested that PG-mediated pectin degradation is important in the later, deteriorative stages of ripening and in pathogen susceptibility of tomato fruit (Schuch et al., 1991; Kramer et al., 1992).In melon (Cucumis melo) substantial amounts of pectin depolymerization and solubilization take place during ripening (McCollum et al., 1989; Ranwala et al., 1992; Rose et al., 1998), implicating a role for PG in ripening-associated cell wall disassembly in melons. However, melons have been reported to lack PG enzyme activity (Hobson, 1962; Lester and Dunlap, 1985; McCollum et al., 1989; Ranwala et al., 1992). The possibility exists that PG is present in melon but that it does not conform to the expected enzymic properties in terms of abundance and/or lability, a point illustrated by recent reports in apple and strawberry, which were previously reported to lack PG activity but that do in fact accumulate low amounts of protein and/or measurable activity (Nogata et al., 1993; Wu et al., 1993). In light of the unexplained discrepancy between ripening-associated pectin depolymerization and undetectable PG activity in melons, we have undertaken a study to reexamine the status of PG in melon using the rapidly ripening cv Charentais (C. melo cv Reticulatus F1 Alpha).As reported for other cultivars, Charentais melons exhibit substantial solubilization and a downshift in the molecular-mass profile of water-soluble pectins, but this is associated with the later stages of ripening, after softening is initiated (Rose et al., 1998). By utilizing a molecular approach to analyze PG in melon, we have attempted to overcome some of the potential limitations of biochemical methods, such as low abundance of protein, reliance on other cell wall components, and unknown cofactors for activity and/or lability during extraction. In doing so, we have identified and characterized a multigene family encoding putative PGs from Charentais melon, including three PG homologs that are expressed abundantly during fruit ripening. The pattern of PG gene expression correlates temporally with the depolymerization of water-soluble pectins and an increase in pectin-degrading enzyme activity. Three additional PG homologs were also identified and shown to be expressed in mature anthers and fruit-abscission zones, tissues that, similar to ripening fruit, are undergoing cell separation. The most abundant ripening-associated putative PG mRNA, MPG1, was expressed in the filamentous fungus Aspergillus oryzae. The culture filtrate from the transformed A. oryzae strain XMPG1 exhibited endo-PG activity, further supporting a role for endo-PG in ripening-associated pectin disassembly in Charentais melon fruit.     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.