Expression of expansin genes in strawberry varieties with contrasting fruit firmness.

Fruit softening is associated with cell wall disassembly mediated by the action of a complex set of enzymes and proteins. Expansins, a group of proteins with unknown enzymatic activity, are proposed to be involved in this process. In order to study the involvement of expansins in strawberry fruit softening we have analyzed the expression level of five expansin mRNAs (FaEXP1, FaEXP2, FaEXP4, FaEXP5 and FaEXP6) in the cultivars "Selva", "Camarosa" and "Toyonaka", which differ in fruit firmness during ripening. We have found a correlation between mRNA expression levels and fruit firmness for FaEXP1, FaEXP2 and FaEXP5. For these three mRNAs we have observed higher expression levels in the softest cultivar (Toyonaka) than in the other two firmer cultivars (Selva and Camarosa) at the beginning of ripening. This correlation was not found in the case of FaEXP4 and FaEXP6, although both genes displayed a different expression pattern in the three cultivars analyzed. Western-blot analysis revealed that the accumulation of expansin proteins begins earlier in the softest cultivar during ripening.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Detachment-accelerated ripening and senescence of strawberry (Fragaria × ananassa Duch. cv. Akihime) fruit and the regulation role of multiple phytohormones

To study the detachment stress on the ripeness of strawberry fruit, physiological characteristics of strawberry fruit on and off plant during ripeness and senescence processes were investigated. The results indicated that the ripeness of strawberry fruit upon detachment was accelerated, in terms of firmness, soluble solid content and especially color development. The color of fruit off plant changed rapidly from white to full red in 1–2 days. The respiratory rate in fruit off plant was strengthened, higher than that on plant. Abscisic acid level and ethylene production in fruit off plant were also higher than those on plant and auxin degradation was exacerbated by detachment. Expression levels of FaMYB1, FabHLH3 and FaTTG1 were generally reduced with phenotypes of redder color and more anthocyanin accumulation in fruit off plant. Results also suggested that the detachment initially stimulated ethylene and abscisic acid production and auxin degradation, which modulated ripening-related gene expression and at last enhanced fruit pigmentation.     

High-Temperature Perturbation of Starch Synthesis Is Attributable to Inhibition of ADP-Glucose Pyrophosphorylase by Decreased Levels of Glycerate-3-Phosphate in Growing Potato Tubers

A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.     

Effect of exogenous application of gibberellic acid on color change and phenylalanine ammonia-lyase, chlorophyllase, and peroxidase activities during ripening of strawberry fruit (Fragaria x ananassa Duch.)

The effect of exogenously applied gibberellic acid (GA₃) on the postharvest color change of strawberry fruit was evaluated through their external color and surface color parameters. A significant delay on color evolution was observed in fruits treated with GA₃. The evolution of activities of phenylalanine ammonia-lyase (PAL), chlorophyllase, and peroxidase was also analyzed. PAL activity increased during strawberry ripening, but in fruits treated with GA₃ the increase in such activity was slower, and, probably as consequence, the development of red color was delayed. Moreover, the activity of chlorophyllase and peroxidase, enzymes possibly involved in chlorophyll metabolism, decreased during strawberry ripening. However, a delay was observed in the decrease of such activities in GA₃-treated fruits.Abbreviations PAL phenylalanine ammonia-lyase - GA₃ gibberellic acid₃ - PVPP polyvinylpolyprrolidone - CEAU chlorophyllase enzymic activity unit - PEAU peroxidase enzymic activity unit - LSD least significant difference. Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina. Author for correspondence.Members of the Research Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid