Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

Genetic and molecular analysis in Cristobalina sweet cherry,a spontaneous self-compatible mutant

Self-compatibility in a naturally self-incompatible species like sweet cherry is a highly interesting trait for breeding purposes and a powerful tool with which to investigate the basis of the self-incompatible reaction in gametophytic systems. However, natural self-compatibility in sweet cherry is a very rare phenomenon. Cristobalina is a local Spanish sweet cherry cultivar that has proven to be spontaneously self-compatible. In this work, the nature of the self-compatibility in Cristobalina has been studied using genetic and molecular approaches. Pollination studies and microscopic observations of pollen tube growth were carried out to confirm the self-compatible character and the results obtained indicate that self-compatibility is caused by a failure of the pollen and not the style factor. Polymerase chain reaction (PCR) analysis of progenies derived from Cristobalina revealed that self-compatibility in this genotype is not related uniquely to one of the two pollen S alleles, but that pollen grains carrying either of the two haplotypes can overcome the incompatibility barrier. Moreover, PCR analysis and microscopic observation of pollen tube growth in progeny derived from Cristobalina also confirmed that the self-compatible descendants can carry either of the two S haplotypes of their progenitor. Isolation and sequencing of the style S-RNases and pollen SFBs revealed that the DNA sequences of these factors are the same as those described in other self-incompatible sweet cherry cultivars with the same S alleles. Possible mechanisms to explain self-compatibility in Cristobalina are discussed.     

Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers

PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S₁ to S₆ S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S₁₃, these also amplified S₇ to S₁₄ and an allele previously referred to as S_x, which we now label S₁₆. The genomic PCR products were cloned and sequenced. The partial sequence of S₁₁ matched that of S₇ and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S₇ to S₁₆, so that allele-specific primers are now available for all 13 S alleles of cherry (S₈, S₁₁ and S₁₅ are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S₃S₁₆), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S₆S₁₂), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S₃S₃, S₄S₆, S₄S₉ and S₄S₁₃, were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.Communicated by H.F. Linskens     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

The regulation of sweet cherry fruit abscission by polar auxin transport

Inconsistency of cropping is an important problem for UK sweet cherry production. Premature fruit abscission in Prunus can reduce yields severely, however, the environmental cues and hormonal signals that trigger abscission have not been identified. Auxin (IAA) is known to delay abscission by reducing the sensitivity of cells in the abscission zone to ethylene, a promoter of abscission. Therefore, the capacity for polar auxin transport (PAT) through sweet cherry pedicels was examined in relation to fruit abscission. Cherry ‘spurs’ (short shoots) with similar leaf areas and different fruit numbers were phloem-girdled to restrict assimilate movement. Abscission from spurs with many fruit (eight or more) occurred within 14 days of girdling, whereas abscission from spurs with few (two) fruit was minimal. The pedicels’ capacity for PAT in spurs with different fruit numbers was determined 1, 3 and 9 days after girdling (DAG). Fruit were analysed for endogenous IAA concentration 3, 5, 7 and 9 DAG. PAT inhibitors 2,3,5-triiodobenzoic acid or 1-N-naphthylphtalamic acid were applied to pedicels of fruit not expected to abscise, i.e. on spurs with few fruit. The effect of these inhibitors on fruit abscission was determined 14 DAG. The proportion of the transported [³H]-IAA was lower from the outset in pedicels from spurs with many fruit. By 9 DAG, symptoms of fruit abscission were apparent and 40% less [³H] -IAA was transported through pedicels on spurs with many fruit. Fruit endogenous IAA concentrations were similar in the two groups of spurs. Application of PAT inhibitors shortly after girdling increased fruit abscission by 30%. The results suggest that although a decline in PAT is not the only cause of fruit abscission, the maintenance of PAT contributes to fruit retention.     

Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor

Caenorhabditis elegans represents an excellent model in which to dissect the biosynthesis and assembly of the nematode cuticle. A sequenced genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of cuticle components. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. For many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterised bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed.     

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus × P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164     

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Combination of antagonistic yeasts with two food additives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit

AIMS: To evaluate beneficial effect of two food additives, ammonium molybdate (NH4-Mo) and sodium bicarbonate (NaBi), on antagonistic yeasts for control of brown rot caused by Monilinia fructicola in sweet cherry fruit under various storage conditions. The mechanisms of action by which food additives enhance the efficacy of antagonistic yeasts were also evaluated. METHODS AND RESULTS: Biocontrol activity of Pichia membranefaciens and Cryptococcus laurentii against brown rot in sweet cherry fruit was improved by addition of 5 mmol l(-1) NH4-Mo or 2% NaBi when stored in air at 20 and 0 degrees C, and in controlled atmosphere (CA) storage with 10% O2 + 10% CO2 at 0 degrees C. Population dynamics of P. membranefaciens in the wounds of fruit were inhibited by NH4-Mo at 20 degrees C after 1 day of incubation and growth of C. laurentii was inhibited by NH4-Mo at 0 degrees C in CA storage after 60 days. In contrast, NaBi did not significantly influence growth of the two yeasts in fruit wounds under various storage conditions except that the growth of P. membranefaciens was stimulated after storage for 45 days at 0 degrees C in CA storage. When used alone, the two additives showed effective control of brown rot in sweet cherry fruit and the efficacy was closely correlated with the concentrations used. The result of in vitro indicated that growth of M. fructicola was significantly inhibited by NH4-Mo and NaBi. CONCLUSION: Application of additives improved biocontrol of brown rot on sweet cherry fruit under various storage conditions. It is postulated that the enhancement of disease control is directly because of the inhibitory effects of additives on pathogen growth, and indirectly because of the relatively little influence of additives on the growth of antagonistic yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this study suggest that an integration of NH4-Mo or NaBi with biocontrol agents has great potential in commercial management of postharvest diseases of fruit.     

Requirement of tyrosylprotein sulfotransferase-A for proper cuticle formation in the nematode C. elegans

Tyrosine O-sulfation is one of the post-translational modification processes that occur to membrane proteins and secreted proteins in eukaryotes. Tyrosylprotein sulfotransferase (TPST) is responsible for this modification, and in this report, we describe the expression pattern and the biological role of TPST-A in the nematode Caenorhabditis elegans. We found that TPST-A was mainly expressed in the hypodermis, especially in the seam cells. Reduction of TPST-A activity by RNAi caused severe defects in cuticle formation, indicating that TPST-A is involved in the cuticle formation in the nematode. We found that RNAi of TPST-A suppressed the roller phenotype caused by mutations in the rol-6 collagen gene, suggesting that sulfation of collagen proteins may be important for proper organization of the extracellular cuticle matrix. The TPST-A RNAi significantly decreased the dityrosine level in the worms, raising the possibility that the sulfation process may be a pre-requisite for the collagen tyrosine cross-linking.     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.