William S. Conway

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.

Chemical changes in the cortical tissue and cell walls of calcium-infiltrated ‘Golden Delicious’ apples during storage

Abstract ‘Golden Delicious’ apples (Malus×domestica Borkh.) were either untreated or pressure-infiltrated after harvest with 0, 1, 2, 3 or 4% CaCl2 solutions (w/v) and stored at 0xa0°C for up to 6 months. The chemical composition of the fruit cortical tissue and cell walls of the 2–4 mm layer under the epidermis was studied. Storage of untreated fruit resulted in a decrease in K, P, Mg, S and Suc content of the tissue, while Fru and Glc increased. In the cell wall, Ca, Mg and total polysaccharide content increased while S, P, total neutral sugar and protein content decreased. During storage, fruit infiltrated with 0% CaCl2 showed a decrease in total polysaccharide and uronic acid content while neutral sugar content increased. After 6 months, the 0% treated fruit had higher levels of total P, Na and S compared to the untreated fruit. These changes in the cell wall of both untreated and 0% treated fruit resulted in an overall decrease in cell wall content of the apple tissue during storage. Analyses of Ca-infiltrated tissue and cell wall characteristics showed an interaction between CaCl2 treatment and time in storage for total and cell wall-bound minerals, total neutral sugar (Glc, Ara, Gal and Rha), protein and cell wall content. CaCl2 infiltration resulted in an increase in both total and cell wall-bound Ca of the apple tissue during storage, with a maximum reached at 2% CaCl2 for fruit stored 4 or 6 months. Ca-infiltrated fruit had higher levels of total K and Na, cell wall-bound Mg, and reduced loss of Ara and Gal after 6 months storage compared to fruit treated with 0% CaCl2, resulting in reduced cell wall degradation of 2% CaCl2 treated fruit during storage. The major changes in the tissue and cell walls occurred after 6 months storage, indicating that this stage was critical for quality maintenance.

Cell wall composition of calcium-treated apples inoculated with Botrytis cinerea

Abstract The role of calcium in maintaining cell wall integrity was investigated in cortical tissue of apple fruit. Carbohydrate, phenolic, protein and mineral element composition were determined in cell walls of high- and low-calcium content fruit inoculated with Botrytis cinerea . Cell walls in fungal-infected tissue of low-calcium fruit showed a decrease in non-cellulosic polysaccharides containing galacturonosyl, rhamnosyl, arabinosyl, xylosyl and galactosyl moieties and increases in cellulose, wall-bound phenolics, protein and mineral elements. In infected tissue of high-calcium fruit, the compositional changes in the cell wall were generally smaller compared with those observed in the low-calcium treatment. The results of this investigation indicate that the effect of calcium in reducing decay is associated with maintaining cell wall structure by delaying and/or modifying chemical changes in cell wall composition.

Characterization of a calcium/calmodulin- regulated SR/CAMTA gene family during tomato fruit development and ripening

BackgroundFruit ripening is a complicated development process affected by a variety of external and internal cues. It is well established that calcium treatment delays fruit ripening and senescence. However, the underlying molecular mechanisms remain unclear.ResultsPrevious studies have shown that calcium/calmodulin-regulated SR/CAMTAs are important for modulation of disease resistance, cold sensitivity and wounding response in vegetative tissues. To study the possible roles of this gene family in fruit development and ripening, we cloned seven SR/CAMTAs, designated as SlSRs, from tomato, a model fruit-bearing crop. All seven genes encode polypeptides with a conserved DNA-binding domain and a calmodulin-binding site. Calmodulin specifically binds to the putative targeting site in a calcium-dependent manner. All SlSRs were highly yet differentially expressed during fruit development and ripening. Most notably, the expression of SlSR2 was scarcely detected at the mature green and breaker stages, two critical stages of fruit development and ripening; and SlSR3L and SlSR4 were expressed exclusively in fruit tissues. During the developmental span from 10 to 50 days post anthesis, the expression profiles of all seven SlSRs were dramatically altered in ripening mutant rin compared with wildtype fruit. By contrast, only minor alterations were noted for ripening mutant nor and Nr fruit. In addition, ethylene treatment of mature green wildtype fruit transiently stimulated expression of all SlSRs within one to two hours.ConclusionsThis study indicates that SlSR expression is influenced by both the Rin-mediated developmental network and ethylene signaling. The results suggest that calcium signaling is involved in the regulation of fruit development and ripening through calcium/calmodulin/SlSR interactions.

Calcium effect on the mycelial cell walls of Botrytis cinerea

Abstract The goal of this study was to determine whether calcium ion, (one of the electrolytes released after plant cell attack), may have a direct effect on fungal growth and chemistry of the fungal cell wall. B. cinerea was grown on Richards solution containing different amounts of CaCl 2 , and the cell walls were extracted from the mycelium after 7 days of growth. Mineral, neutral and aminosugar, protein and uronic acid contents were determined. At 1 g l −1 CaCl 2 , only the aminosugar content increased. At 2 g l −1 CaCl 2 , neutral sugar synthesis was reduced, whereas the uronic acid content increased. For higher CaCl 2 concentrations, the calcium ion content of the cell wall increased, resulting in reduced protein and neutral sugar contents. Meanwhile, the cell wall proportion of the mycelia increased on a dry weight basis due to an increase in uronic acid, Ca, P, Na and neutral sugar contents of the cell wall with increasing CaCl 2 in the media. The resulting thickening of the fungal cell wall caused by calcium ion may be an important factor in the host-pathogen relationship.

Influence of prestorage heat and calcium treatments on lipid metabolism in ‘Golden Delicious’ apples

Abstract Heating ‘Golden Delicious’ apples for 4 days at 38° and/or pressure infiltrating the fruit with a CaCl2 solution after harvest, maintains firmness and reduces decay during storage. The possibility that these beneficial effects involve changes in membrane lipid metabolism was investigated. Lipids of hypodermal cortical tissue were analyzed after 0, 1, 2 or 4 days at 38° and after storage (15 weeks at O° plus 1 week at 20°) of fruit that were untreated (Ctl), heated 4 days at 38° (HT), infiltrated with 2% CaCl2 (Ca) or heated then infiltrated (HT + Ca) before storage. Overall, effects of HT were much more pronounced than those of Ca and effects of HT + Ca were intermediate between those of HT or Ca alone. An initial phase of membrane damage induced by heating, indicated by glycerolipid loss over the first 1–2 days, could explain why HT for less than 3–4 days has an adverse effect on post-storage quality. HT effects on plastids, including accelerated chlorophyll and monogalactolipid loss, as well as carotenoid accumulation, are likely to cause the distinct yellowing of the fruit. HT-induced reductions in steryl glycosides and cerebrosides prior to storage similar to those that occurred in Ctl and Ca fruit during storage, and the phospholipid (PL) content of HT fruit after storage was close to that of Ctl fruit at harvest. Also, the ratio of linoleate to oleate in PL was much higher in HT and HT + Ca than in Ctl fruit at the end of storage. One or more of these effects of HT on membrane lipids could be involved in the ultimate benefits to fruit quality.

Polygalacturonases of a latent and wound postharvest fungal pathogen of muskmelon fruit

Abstract Partially purified endo- and exo-polygalacturonases (PG) from two fungal pathogens (Phomopsis cucurbitae and Rhizopus stolonifer) were compared in relation to their ability to macerate netted muskmelon tissue at different stages of fruit development. PG extracts from P. cucurbitae, a latent infection pathogen, produced little maceration until fruit were 50 days post-anthesis (10 days postharvest). In contrast, PGs from R. stolonifer, a wound pathogen, produced high levels of maceration at all stages of fruit development from 20 to 50 days post-anthesis. Both pathogens demonstrated highest levels of total PG activity in mesocarp and lowest levels in exocarp (peel) tissues. Isoelectrofocusing–polyacrylamide gel electrophoresis indicated two prominent PG isozymes in R. stolonifer and nine isozymes in P. cucurbitae. Cell wall carbohydrate analysis showed an approximately 6-fold decrease in galactosyl residue content between 10 and 50 days post-anthesis in uninfected fruit. Infected fruit showed approximately 7- and 8-fold decreases in galacturonic acid content when infected with P. cucurbitae and R. stolonifer, respectively. Significant decreases in cell wall rhamnosyl and arabinosyl residues occurred during infection of fruit with both pathogens. These results support a role for cell wall pectin degradation during the decay process of muskmelon by these pathogens. The ability to macerate fruit tissue, as related to the latent infection phenomenon, may be due to substrate specificity or inhibitors present in muskmelon fruit tissue.

Biological characteristics of Monilinia fructicola isolates from stone fruits in eastern West Virginia

Abstract Monilinia fructicola, the causal agent of brown rot, was recovered from decayed stone fruits (peach, plum and nectarine) in 11 West Virginia orchards. There was significant variation among these isolates with respect to colony morphology, growth rate, sporulation level, sensitivity to fenbuconazole, vegetative compatibility and virulence. Species identification was confirmed using ITS sequences from the nuclear ribosomal RNA gene. The cultural phenotypes on potato dextrose agar (PDA) ranged from white to dark, melanized colonies. The growth rate of the isolates on PDA ranged from 0.3 to 3.2 mm day−1 at 4 °C, from 2.9 to 7.6 mm day−1 at 10 °C, and from 4.8 to 19 mm day−1 at 24 °C. There was a statistically significant relationship between the growth of the isolates on PDA and their aggressiveness on nectarines at 24 °C, especially at higher inoculum concentrations. Sporulation of 3-day-old cultures on peach agar at 24 °C varied from profuse to no sporulation, with some isolates sporulating only sparsely after 10 days. The EC50 for fenbuconazole ranged from 0.003 to 0.129 μg μL−1 and for two reference isolates was 0.020 and 0.016 μg μL−1. Only a few vegetative compatibility groups were identified among isolates within orchards, reflecting the lack of sexual recombination in this region.

Carbon, nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ∼7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5–6, with an optimum at pH 4.5. PG activity was detectable 0–70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca2+, Mg2+, Mn2+ and Fe2+ and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.