Leaf Cutin Monomers, Cuticular Waxes, and Blackspot Resistance in Rose

The fungal pathogen Diplocarpon rosae causes rose blackspot disease, a serious problem for roses (Rosa) in the managed landscape. To prevent this disease, homeowners and professional growers often apply chemical fungicide. However, increased use of fungicides poses an environmental hazard and an economic burden to the user. New landscape rose cultivars like ‘Knockout’ possess increased disease resistance, but the biological basis for this resistance is still unknown. To investigate the potential role of leaf cuticle in blackspot resistance in rose, five rose cultivars known to vary greatly in blackspot resistance were examined for variation in the major lipids of the leaf cuticle, specifically the monomers of the cutin polyester and the free cuticular waxes. This is the first report of cutin monomers in the Rosa genera. The rose cultivars selected for this study were ‘Knockout’, ‘Mister Lincoln’, ‘Garden Party’, ‘Purple Passion’, and ‘Bicolor’. ‘Knockout’ and ‘Garden Party’ had significantly lower total cutin monomer amount per leaf area than the other cultivars, whereas the most cutin monomers were observed on ‘Purple Passion’, ‘Bicolor’, and ‘Mister Lincoln’. Five major cutin monomers (mostly hydroxylated 16 carbon fatty acids) dominated the cutin profiles of both adaxial and adaxial surfaces of all cultivars, with the 10,16-dihydroxy hexadecanoic acids being most abundant. The proportion of 10,16-dihydroxy hexadecanoic acids was slightly higher in the adaxial than abaxial leaf cuticles of all cultivars. Correspondingly, other cutin monomers were relatively lower in the adaxial cuticle, except 16-hydroxy hexadecanoic acid that differed little. Uniquely, this is the first report of cutin monomer composition of isolated abaxial and adaxial leaf cuticles of any plant. Total leaf cuticular wax amounts were lowest on ‘Purple Passion’ and ‘Knockout’, intermediate in ‘Mister Lincoln’ and ‘Garden Party’, and highest on ‘Bicolor’, with alkanes as the most abundant wax class. Consistent with previously published disease susceptibility ratings, our visual scores showed that ‘Knockout’ was most resistant to blackspot pathogen infection with a visual disease rating score of 1.0, followed by ‘Mister Lincoln’ at 1.8, ‘Garden Party’ at 5.4, ‘Bicolor’ at 7.5, and ‘Purple Passion’ with the most visible disease damage at 8.8. Regression analysis revealed that the alkane and ester proportions were most closely associated with blackspot disease susceptibility ratings, being inversely (R2 = 0.63, P = 0.05) and directly (R2 = 0.81, P = 0.05) correlated, respectively. More studies on the role of cuticle in rose susceptibility to blackspot are now clearly warranted.

Characterization of Physiological and Biochemical Factors Associated with Postharvest Water Loss in Ripe Pepper Fruit during Storage

Fruit of pepper (Capsicum annuum L.) is hollow by nature, which limits its water reservoir capacity, and as such, small amounts of water loss result in loss of freshness and firmness, which reduce fruit quality, shelf life, and market value. In order to understand the basis for water loss from fruit, 10 pepper accessions with wide variation in water loss rate were used to study physiological and biochemical factors associated with postharvest water loss in ripe pepper fruit during storage. Postharvest water loss rate in ripe pepper fruit stored at 20 °C, and 85% relative humidity, was found to be associated with cell membrane ion leakage, lipoxygenase activity, and total cuticular wax amount. Total cuticular wax amounts were highest in the high-water-loss pepper fruit, and lowest in the low-water-loss fruit. However, total cuticle amount (isolated enzymatically and quantified gravimetrically), total cutin monomer amount, and the amount of individual cutin monomer and wax constituents (determined using gas chromatography mass spectrometry) indicated no direct association with postharvest water loss rates. Fruit fresh weight, pericarp weight, pericarp surface area, pericarp thickness, initial water content, and dry matter were highly associated with each other, but less so with water loss rate. Fruit of accessions displaying high fruit water loss rate matured and ripened earlier than fruit of accessions displaying low-water-loss rate. Cell membrane ion leakage and lipoxygenase activity were higher after storage than immediately after harvest. Pepper fruit total cuticle wax amount, lipoxygenase activity, and cell membrane ion leakage were directly related to postharvest water loss rate in pepper fruit during storage.

Cloning and Expression of the Cutinase A Gene of Botrytis cinerea

Cutinase of Botrytis cinerea has been suggested to play an important role in penetration of host tissues. A protein fraction with cutin hydrolyzing activity was purified from culture filtrates of B. cinerea induced for cutinase activity. An 18-kDa protein in this fraction was identified as cutinase and the corresponding gene cutA was cloned. The gene is present in a single copy in the genome of B. cinerea strain SAS56 and its predicted amino acid sequence shows significant homology (31 to 35% identity) to other fungal cutinases. RNA blot analysis showed that cutA mRNA is induced in vitro by the cutin monomer 16-hydroxy-hexadecanoic acid and repressed by glucose. The expression of cutA during infection of tomato leaves is low during early phases of infection, but high when the fungus has colonized the leaf and starts to sporulate.

Molecular basis of the early events in plant–fungus interaction

Fungal spores that land on aerial surfaces of plants first come into contact with plant cuticle. The cuticle is composed of an insoluble polyester called cutin, which is composed of hydroxy and hydroxyepoxy fatty acids and associated soluble waxes. The wax components can trigger differentiation of germinating fungal spores into infection structures. The penetration of the fungus into the plant requires enzymatic degradation of the polyester and the underlying carbohydrate barriers. The polyesterase, called cutinase, is induced by the contact with the plant surface. The small amount of cutinase carried by the spore generates a small amount of cutin monomers upon contact with the host, and the unique monomers trigger expression of the cutinase gene. This transcriptional level control can be demonstrated with isolated nuclei. Upon incubation of nuclei with cutin monomer and a soluble protein factor from the fungus, cutinase transcription is selectively activated. Structure activity relationships showed that the cutinase transcription activation required all of the structural elements of the cutin monomer. The cutinase transcript generated by the isolated nuclei was identical in size to the cutinase mRNA induced in the fungal cultures, which indicated usual initiation and termination. Fungal infection triggers defense reaction in plants. Plant peroxidases were implicated in the defense response of plants to fungal attack and stress. A highly anionic peroxidase involved in suberization of cell walls in tomato plants in response to fungal attack was cloned and sequenced. It was observed that in resistant lines of tomato the expression of this anionic peroxidase was induced 1 day earlier than in susceptible lines.Key words: cutinase, cutin monomers, pectate lyase, Fusarium solani pisi, anionic peroxidase.