Two dimensional gel electrophoresis patterns of total, in vivo labelled and in vitro translated polypeptides from orange flavedo during maturation and following ethylene treatment

Changes in the pattern of silver stained (SS), in vivo [³⁵S]-methionine labelled (IL), and in vitro poly(A⁺)RNA translated (IT) polypeptides from the flavedo of orange fruits [ Citrus sinensis (L). Osbeck cv. Washington Navel] picked at three stages of maturity (mature-green, turning and fully coloured) and treated with different doses of ethylene (0, 1 and 10 μl 1⁻¹) were studied by two dimensional gel electrophoresis (using isoelectric focusing and nonequilibrium pH gradient as a first dimension). More than 500 SS, 300 IL, and 250 IT spots were detected in the gels. During maturation 32 SS, 2 IL and 2 IT spots decreased, whereas 2 SS, 3 IL and 2 IT spots increased. These results indicate that the maturation process is associated with a decrease of many accumulated flavedo proteins and with an increase of a reduced number of specific polypeptides, and that some of them may be regulated at the level of gene expression. All the spots which increased with maturity also increased with ethylene treatment, suggesting a role for this hormone in the maturation process. Ten IT spots which were not affected by maturity increased following ethylene treatment, while only 2 SS and 2 IL spots underwent this pattern of variation. Three spots recognized specifically by tobacco chitinase polyclonal antibodies remained essentially unaltered, whereas one spot whose intensity decreased significantly during maturation and ethylene treatment was identified as the large subunit of ribulose-1,5-bisphosphate carboxylase.     

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     

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.     

Persistence of [14C] gibberellin A3 and [3H] gibberellin A1 in senescing,ethylene treated Citrus and tomato fruit

The fate of [¹⁴C] gibberellin A₃ and [³H] gibberellin A₁ was examined in senescing fruit of Shamouti orange (Citrus sinensis L. Osbeck) and tomato (Lycopersicon esculentum Mill.). Gibberellin A₃ was highly persistent in Citrus peel (t 1/2=18 days) and to a lesser degree in tomato (t 1/2=5.5 days). Ethylene and ethephon caused a slight enhancement of gibberellin A₃ metabolism in Citrus and tomato fruit, respectively. Gibberellin A₁ was metabolized by Citrus peel at a relatively high rate (t 1/2 < 24 h) and ethylene slightly reduced this rate. It is concluded that the ethylene-induced enhancement of senescence does not involve major effects on the deactivation of applied gibberellins.Abbreviations GA₃ gibberellin A₃ - GA₁ gibberellin A₁     

Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments

An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 M 6-benzylaminopurine and 0.7 M gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature.     

Effect of the presence of olive fruit on ovarian maturation in the olive fruit fly, Dacus oleae, under laboratory conditions

Ovarian maturation was prevented in 7-, 22-, 23- and 28-day-old females of Dacus oleae (Gmelin) (Diptera: Tephritidae), developed in the pre-imaginal stages at LD 12:12, 19±1°C and kept as adults at LD 16:8, 26±1°C without access to olive fruits. In females of the above ages having continuous access to olive fruits and held under the same photoperiod and temperature conditions, 54, 87 and 100%, respectively, had mature oocytes. When the females had access to domes of paraffin wax, the percentages of females with mature oocytes were intermediate between those with and those without access to olive fruits. Under the above photoperiod and temperature conditions unfavorable for maturation, 50% of 4-week-old females had mature oocytes if exposed for one week to olive fruits during their first week and 91% if exposed during their 4th week of adult life. The respective percentages with wax domes in the cages were again intermediate between those with olives and those without olives or wax domes. The presence of olive fruits had also a strong positive effect on the ovarian maturation of females which developed from egg through the adult stage at LD 16:8, 26±1°C, a condition favoring ovarian maturation. Access to wax domes had again an intermediate effect on maturation. It is concluded therefore that the lack of ovarian maturation of D. oleae females which is observed under a short photophase during the pre-imaginal stages if followed by a long photophase and an increase of temperature during the adult stage, continues at least till the end of the 4th week of adult life in the absence of olive fruits but is averted when such fruits are offered to the adult flies.

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Résumé La maturation ovarienne des femelles adultes agées de 7, 22–23 et 28 jours de D. oleae ayant accompli leur développement embryonnaire, larvaire et nymphal en LD 12:12, 19±1°C et maintenues en LD 16:8, 26±1°C, sans accès à des olives, est inhibée. Dans les mêmes conditions, mais avec accès permanent à des olives, 54% des femelles ont des ovocytes mûrs à 7 jours, 87% à 22–23 jours et 100% à 28 jours. Avec des femelles ayant accès à des domes de paraffine, les pourcentages d'individus ayant des ovocytes mûrs sont intermédiaires entre ceux des deux situations précédentes. Dans les conditions cidessus défavorables à la maturation ovarienne, 50% des femelles de 28 jours ont des ovocytes mûrs si elles sont en présence d'olives durant leur première semaine de vie imaginale et 91% si elles sont en présence la 4éme semaine. Les pourcentages respectifs concernant les femelles pourvues de dômes de paraffine sont encore intermédiaires entre ceux des femelles avec olives et ceux de femelles privées de tout substrat. La présence d'olives a aussi un effet fortement positif sur la maturation ovarienne de femelles ayant accompli leur développement pre-imaginal et maintenues aussi à l'etat adulte en conditions LD 16:8, 26±1°C, favorisant la maturation ovarienne. L'accès à des dômes de paraffine a, là encore, un effet intermédiaire. On conclut par conséquent que la non maturation ovarienne des femelles de D. oleae qui est causée par une photopériode à jour court pendant la vie pre-imaginale accompagné par une photopériode à jour long et une augmentation de la température pendant la vie imaginale persiste au moins jusqu'à la fin de la quatrième semaine de la vie imaginale en absence d'olives, mais ne se produit pas si on met les femelles adultes en présence d'olives.

     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Gene expression during fruit ripening in avocado

The poly(A) ⁺RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.     

Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and ethylene production

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the -glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.     

Evidence for programmed cell death and activation of specific caspase-like enzymes in the tomato fruit heat stress response

The tomato (Lycopersicon esculentum) fruit is the best available model to study the stress response of fleshy fruit. Programmed cell death (PCD) plays an important role in stress responses in mammals and plants. In this study, we provide evidence that PCD is triggered in the tomato fruit heat stress response by detection of the sequential diagnostic PCD events, including release of cytochrome c, activation of caspase-like proteases and the presence of TUNEL-positive nuclei. Investigating the time course of these events for 12 h after heat treatment indicated that cytochrome c release and caspase-like protease activation occurred rapidly and were consistent with the onset of DNA fragmentation. In addition, LEHDase and DEVDase enzymes were specifically activated in tomato fruit pericarp during the heat treatment and recovery time. There was no significant activation of YVADase or IETDase proteases. Preincubation of pericarp discs with the broad-spectrum, cell-permeable caspase inhibitor Z-VAD-FMK, suppressed heat-induced cell death measured by trypan blue, accompanied by a decrease in LEHDase and DEVDase activities. Gui-Qin Qu and Xiang Liu contributed equally to this work.     

Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck)

Two dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to study the somatic embryogenesis (SE) in Valencia sweet orange (Citrus sinensis Osbeck). Twenty-four differentially expressed proteins were identified at five time points of citrus SE (0, 1, 2, 3, 4 weeks after embryo initiation) covering globular, heart/torpedo and cotyledon-shaped embryo stages. The general expression patterns for these proteins were consistent with those appeared at 4 weeks of citrus SE. The most striking feature of our study was that five proteins were predicted to be involved in glutathione (GSH) metabolism and anti-oxidative stress, and they exhibited different expression patterns during SE. Based on that oxidative stress has been validated to enhance SE, the preferential representation for anti-oxidative proteins suggests that they could have a developmental role in citrus SE. Some proteins involved in cell division, photosynthesis and detoxification were also identified, and their possible roles in citrus SE were discussed.     

Uniformity of plants regenerated from orange (Citrus sinensis Osb.) protoplasts

Summary Using 25 plants (protoclones) regenerated from orange (Citrus sinensis Osb.) protoplasts, several characters, including leaf and flower morphology, leaf oil, isozyme patterns and chromosome number, were examined. No significant variations in each character were recorded among the protoclones. Uniformity observed among protoclones was identical to that of nucellar seedlings.     

Identification of an ETR1-homologue from mango fruit expressing during fruit ripening and wounding

We have isolated a mango (Mangifera indica L.) cDNA homologue of the ethylene receptor gene ETR-1, referred to as METR1, which codes for a polypeptide of 802 amino acids with a predicted molecular weight of 89 kDa. The amino acid sequence is highly homologous (over 80 percnt;) to ETRs from other fruits. Genomic Southern blot analysis indicates that two or more ETR homologues exist in mango. RNA blot analysis revealed that the level of METR1 mRNA in the mesocarp increased during fruit ripening. In addition, it was found that the METR1 mRNA increases transiently during wounding of the tissue. This is the first report of an ETR homologue showing an induction during fruit ripening and wounding.     

Small fruit problem in Citrus trees

Summary Physiological causes of the small fruit problem which occurs in certain trees of orange [Citrus sinensis (L.) Osbeck cv. Valencia] were investigated in terms of water relations and gas exchange of fruits during early fruit development as well as tree carbohydrate reserves. These data from cv. Valencia trees with and without a small fruit potential were compared with those of the large fruited cv. Navel. Neither fruit water potential nor fruit transpiration nor tree carbohydrate reserves appeared to be a cause of the small fruit. Yield records showed the small fruit to be assocaited with a large number of fruit per tree. However, fruits from cv. Valencia trees with a small fruit potential respired faster than either fruits of the same cultivar and size from trees without the physiological disorder or fruits of the same size of cv. Navel and also exceeded the dark respiration of the respective leaves. Hence, the small fruit problem in cv. Valencia was partly attributed to inefficient fruit photosynthesis, causing excessive respiration of each of a larger number of fruits compared to fruits of a tree of the same cultivar but without the physiological disorder. Fruits of cv. Valencia respired more in their 2 months longer lifetime on the tree relative to those of cv. Navel. It is concluded that orchard management methods will have to be investigated to balance the fruit load of the cv. Valencia tree utilizing the carbon available for fruit growth and to minimise stress during the early fruit development.