Dani Eshel

pH Regulates Endoglucanase Expression and Virulence of Alternaria alternata in Persimmon Fruit

The phytopathogenic fungus Alternaria alternata produces one endo-1,4-beta-glucanase, AaK1, which is an important factor in disease development in persimmon fruit. During growth of A. alternata in media containing acidified yeast extract or cell walls from persimmon fruit, the fungus secreted ammonia and raised the medium pH. A rise in media pH from 3.8 to 6.0 in the presence of cell walls induced the expression of AaK1, whereas a glucose-induced decline in pH to 2.5 repressed transcription and enzymatic production. Treatments with buffered solutions at pH 6.0 during growth of A. alternata in the presence of glucose derepressed AaK1 expression and endo-1,4-beta-glucanase production and enhanced decay development on the fruit. The results suggest that conditions affecting environmental pH modulate gene expression of AaK1 and virulence of A. alternata in persimmon fruit

Resistance of gibberellin-treated persimmon fruit to Alternaria alternata arises from the reduced ability of the fungus to produce endo-1,4-β-glucanase.

ABSTRACT Black-spot symptoms, caused by Alternaria alternata, developed in persimmon fruits during prolonged storage at -1 degrees C. A preharvest treatment with gibberellic acid (GA(3)) extended the storage life of the fruit by delaying both black-spot development and fruit softening. Conversely, treatment of persimmon fruits with paclobutrazol (PBZ), an inhibitor of gibberellin (GA) synthesis, enhanced black-spot development and fruit softening during storage. Production of endo-1,4-beta-glucanase (EC 3.2.1.4, EG) by A. alternata in culture and in the presence of cell walls from PBZ-treated fruits as the carbon source, was enhanced by 150% over production in the presence of cell walls from control fruits, whereas endoglucanase (EG) production in the presence of cell walls from GA(3)-treated fruits was reduced by 49% relative to controls. To determine the importance of EG in symptom development, A. alternata EG was purified from a culture-inducing medium. It had a molecular mass of 41 kDa, its optimal pH and temperature for activity were 5.5 and 47 degrees C, respectively, and the pI was 4.3. Its K(m) and V(max) were 0.43 mg ml(-1) and 18 mumol reducing groups minute per milligrams of protein, respectively. The internal sequence of a 21-mer amino acid peptide from the purified EG showed 62% similarity and 38% identity to the EG-1 of Trichoderma reesei and of T. longibrachiatum. Purified EG induced black-spot symptoms on the fruit, similar to those caused by A. alternata, whereas boiled enzyme caused only pricking signs. Our results suggest that the black-spot symptoms caused by A. alternata, in persimmon, are related to the ability of the fungus to produce EG in developing lesions.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Postharvest chlorine treatments for the control of the persimmon black spot disease caused by Alternaria alternata

Abstract Black-spot symptoms, caused by Alternaria alternata, develop in ‘Triumph’ persimmon fruit during prolonged storage at −1oC. Preharvest dip treatment in the organic chlorine compound Troclosene sodium extended the storage life of the fruit by delaying development of black-spot disease (BSD). Troclosene sodium was more stable and efficient for the control of A. alternata than calcium hypochlorite. At 500 μg ml−1 Troclosene sodium significantly reduced the development of BSD in persimmon fruit sampled in 15 orchards in different growing regions of Israel. Following commercial dipping and storage at −1oC, the percentage of marketable fruit after 4 months of storage was 15–40% higher than in untreated fruit. Present results suggest that the BSD can be controlled by a simple dip treatment with chlorine disinfectant.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

Can loss of apical dominance in potato tuber serve as a marker of physiological age

The potato tuber constitutes a model system for the study of dormancy release and sprouting, suggested to be regulated by endogenous plant hormones and their balance inside the tuber. During dormancy, potato tubers cannot be induced to sprout without some form of stress or exogenous hormone treatment. When dormancy is released, sprouting of the apical bud may be inhibited by sprout control agents or cold temperature. Dominance of the growing apical bud over other lateral buds decreases during storage and is one of the earliest morphophysiological indicators of the tubers physiological age. Three main types of loss of apical dominance (AD) affect sprouting shape. Hallmarks of programmed cell death (PCD) have been identified in the tuber apical bud meristem (TAB-meristem) during normal growth, and are more extensive when AD is lost following extended cold storage or chemical stress. Nevertheless, the role of hormonal regulation in TAB-meristem PCD remains unclear.

Vicilin and the basic subunit of legumin are putative chickpea allergens

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized. A serum pool from paediatric patients allergic to chickpeas was used to detect IgE-binding proteins from chickpea seeds by immunoassay and immunoblot inhibition assay. Protein samples enriched in chickpea legumin and vicilin were obtained by anion exchange chromatography, and were identified by mass spectrometric analysis. IgE-immunoassays of globulin fractions from chickpeas revealed that vicilin (50 kDa) and the basic subunit of legumin (20 kDa) were bound by IgE from patient sera. Pea and lentil protein extracts strongly inhibited the IgE binding to chickpea globulin. We speculate that vicilin and the basic subunit of legumin are major chickpea allergens. Also, the globulin fraction of chickpea likely cross-reacts with the allergenic proteins of pea and lentil.

Induction of Rhizopus oryzae germination under starvation using host metabolites increases spore susceptibility to heat stress.

Sweetpotato is a nutritional source worldwide. Soft rot caused by Rhizopus spp. is a major limiting factor in the storage of produce, rendering it potentially unsafe for human consumption. In this study, Rhizopus oryzae was used to develop a concept of postharvest disease control by weakening the pathogen through induction of spore germination under starvation conditions. We isolated the sweetpotato active fractions (SPAFs) that induce spore germination and used them at a low dose to enhance spore weakening caused by starvation. Germination in SPAF at 1 mg/ml weakened the pathogen spores by delaying their ability to form colonies on rich media and by increasing their sensitivity to heat stress. The weakening effect was also supported by reduced metabolic activity, as detected by Alarmar Blue fluorescent dye assays. Spores incubated with SPAF at 1 mg/ml showed DNA fragmentation in some of their nuclei, as observed by TUNEL assay. In addition, these spores exhibited changes in ultrastructural morphology (i.e., shrinkage of germ tubes, nucleus deformation, and vacuole formation) which are hallmarks of programmed cell death. We suggest that induction of spore germination under starvation conditions increases their susceptibility to stress and, therefore, might be considered a new strategy for pathogen control.

Etiolated Stem Branching Is a Result of Systemic Signaling Associated with Sucrose Level

Hot or cold storage of potato tuber, a swollen stem, induces sucrose accumulation in the parenchyma that enhances branching during sprouting under dark conditions. The potato (Solanum tuberosum) tuber is a swollen stem. Sprouts growing from the tuber nodes represent loss of apical dominance and branching. Long cold storage induces loss of tuber apical dominance and results in secondary branching. Here, we show that a similar branching pattern can be induced by short heat treatment of the tubers. Detached sprouts were induced to branch by the heat treatment only when attached to a parenchyma cylinder. Grafting experiments showed that the scion branches only when grafted onto heat- or cold-treated tuber parenchyma, suggesting that the branching signal is transmitted systemically from the bud-base parenchyma to the grafted stem. Exogenous supply of sucrose (Suc), glucose, or fructose solution to detached sprouts induced branching in a dose-responsive manner, and an increase in Suc level was observed in tuber parenchyma upon branching induction, suggesting a role for elevated parenchyma sugars in the regulation of branching. However, sugar analysis of the apex and node after grafting showed no distinct differences in sugar levels between branching and nonbranching stems. Vacuolar invertase is a key enzyme in determining the level of Suc and its cleavage products, glucose and fructose, in potato parenchyma. Silencing of the vacuolar invertase-encoding gene led to increased tuber branching in combination with branching-inducing treatments. These results suggest that Suc in the parenchyma induces branching through signaling and not by excess mobilization from the parenchyma to the stem.

Cellular and Molecular Changes Associated with Onion Skin Formation Suggest Involvement of Programmed Cell Death

Skin formation of onion (Allium cepa L.) bulb involves scale desiccation accompanied by scale senescence, resulting in cell death and tissue browning. Understanding the mechanism of skin formation is essential to improving onion skin and bulb qualities. Although onion skin plays a crucial role in postharvest onion storage and shelf life, its formation is poorly understood. We investigated the mode of cell death in the outermost scales that are destined to form the onion skin. Surprisingly, fluorescein diacetate staining and scanning electron microscopy indicated that the outer scale desiccates from the inside out. This striking observation suggests that cell death in the outer scales, during skin formation, is an internal and organized process that does not derive only from air desiccation. DNA fragmentation, a known hallmark of programmed cell death (PCD), was revealed in the outer scales and gradually decreased toward the inner scales of the bulb. Transmission electron microscopy further revealed PCD-related structural alterations in the outer scales which were absent from the inner scales. De novo transcriptome assembly for three different scales: 1st (outer), 5th (intermediate) and 8th (inner) fleshy scales identified 2,542 differentially expressed genes among them. GO enrichment for cluster analysis revealed increasing metabolic processes in the outer senescent scale related to defense response, PCD processes, carbohydrate metabolism and flavonoid biosynthesis, whereas increased metabolism and developmental growth processes were identified in the inner scales. High expression levels of PCD-related genes were identified in the outer scale compared to the inner ones, highlighting the involvement of PCD in outer-skin development. These findings suggest that a program to form the dry protective skin exists and functions only in the outer scales of onion.