Characterization of Pinalate,a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content

The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and zeta- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% zeta-carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3-6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in zeta-carotene desaturase or in zeta-carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.     

Hormonal and Nutritional Changes in the Flavedo Regulating Rind Color Development in Sweet Orange [Citrus sinensis (L.) Osb.]

The objective of this research was to determine the changes in the levels of endogenous gibberellins GA₁ and GA₄, abscisic acid (ABA), and ethylene during fruit coloring of on-tree fruits of sweet orange. The time course of carbohydrates and nitrogen content in the flavedo prior to fruit color break and during peel ripening were also studied. To identify nutritional and hormonal changes in the fruit, 45?days before fruit color break the peduncles of 15?C30 fruits per tree of ??Washington?? navel, ??Navelate,?? and ??Valencia Delta Seedless?? sweet orange, located in single-fruited shoots, were girdled to intercept phloem transport. A set of 15?C30 fruits per tree remained intact on the peduncle for control. Girdling significantly delayed fruit coloration for more than 2?months; the delay paralleled higher GA₁ and GA₄ concentrations in the flavedo and retarded the rise of ABA concentration prior to color break. Girdling also reduced carbohydrate concentrations and increased N concentrations in the flavedo compared to control fruits; no ethylene production was detected. Therefore, in sweet orange, fruit changes color by reducing active gibberellin concentrations in the flavedo, which are involved in regulating sugars and ABA accumulation and in reducing N fraction concentration as rind color develops. This was demonstrated in vivo without removing the fruit from the tree. Comparable results were obtained with experiments carried out over four consecutive years in two countries (Spain and Uruguay).     

Altered sensitivity to ethylene in ‘Tardivo’, a late‐ripening mutant of Clementine mandarin

‘Tardivo’ mandarin is a mutant of ‘Comune’ Clementine with a delay in peel degreening and coloration, allowing late harvesting. In this work, we have explored if the late‐harvesting phenotype of ‘Tardivo’ mandarin is related to altered perception and sensitivity to ethylene. The peel degreening rate was examined after a single ethephon treatment or during a continuous ethylene application in fruits at two maturation stages. In general, ethylene‐induced peel degreening was considerably delayed and reduced in fruits of ‘Tardivo’, as well as the concomitant reduction of chlorophyll (Chl) and chloroplastic carotenoids, and the accumulation of chromoplastic carotenoids. Analysis of the expression of genes involved in Chl degradation, carotenoids, ABA, phenylpropanoids and ethylene biosynthesis revealed an impairment in the stimulation of most genes by ethylene in the peel of ‘Tardivo’ fruits with respect to ‘Comune’, especially after 5 days of ethylene application. Moreover, ethylene‐induced expression of two ethylene receptor genes, ETR1 and ETR2, was also reduced in mutant fruits. Expression levels of two ethylene‐responsive factors, ERF1 and ERF2, which were repressed by ethylene, were also impaired to a different extent, in fruits of both genotypes. Collectively, results suggested an altered sensitivity of the peel of ‘Tardivo’ to ethylene‐induced physiological and molecular responses, including fruit degreening and coloration processes, which may be time‐dependent since an early moderated reduction in the responses was followed by the latter inability to sustain ethylene action. These results support the involvement of ethylene in the regulation of at least some aspects of peel maturation in the non‐climacteric citrus fruit.     

Terpene down-regulation in orange reveals the role of fruit aromas in mediating interactions with insect herbivores and pathogens

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.     

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.     

Cell number, cell size and hormone levels in semi-isogenic mutants of Lycopersicon pimpinellifolium differing in fruit size

Tomato fruit growth parameters, cell number and cell size, and hormone levels [IAA, abscisic acid (ABA), zeatin (Z)/zeatin riboside (ZR), isopentenyladenosine (i-Ado)/isopentenlyadenine (i-Ade)], in the wild-type ( Lycopersicon pimpinellifolium Mill.) and a semi-isogenic mutant (mutant III) differing in fruit size were investigated during fruit development. An image-processing system was used for the determination of cell number and single cell size per fruit and hormone levels were measured by radioimmuno-assay (RIA). The bigger fruits of mutant III showed higher cell numbers throughout fruit development and cells enlarged faster than in wild-type fruits. During the first 10 days of fruit growth, the main cell division period after fertilization, high concentrations of cytokinins were found, these being correlated with high cell division activity. There were only slight differences in IAA and ABA levels in the different sized fruits. The results emphasized the importance of the cell number per fruit at anthesis as a determining factor of final fruit size in tomatoes. A possible relationship between cytokinins and subsequent fruit development is discussed.     

Variations of Flavonoid Composition and Antioxidant Properties among Different Cultivars,Fruit Tissues and Developmental Stages of Citrus Fruits

A large number of biologically active compounds are present in ripe citrus fruits. However, few studies have been focused on the changes in flavonoids and the evolution of antioxidant activity during citrus fruit growth. In this study, fruits of five citrus cultivars cultivated in China were sampled at 60–210 days post‐anthesis (DPA) at intervals of 30 days. The amounts of main flavonoids in the peel and pulp were analyzed by HPLC and their activities were studied by DPPH, ABTS and FRAP. The results showed that the contents of hesperidin, diosmin, eriodictyol, rutin and nobiletin increased before 90 DPA and then decreased with the growth and development of fruits, but an opposite tendency was observed for naringin and narirutin. The antioxidant activities in citrus peel and pulp were found to be significantly correlated with some flavonoids. The results may be of guiding values in citrus production and utilization of citrus fruit by‐products.     

Are citrus species favorable hosts for the Mediterranean fruit fly? A demographic perspective

We studied, under laboratory conditions, demographic parameters of adult Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (medfly), obtained from three sweet orange varieties, lemon, and bitter oranges. These data were combined with immature developmental rates and survival on the same hosts to estimate host‐specific population parameters. Pairs of newly emerged adults from each citrus variety were held individually in transparent plastic cages, and females were allowed to oviposit in either red domes (artificial, pre‐punctured plastic oviposition devices), or intact, whole citrus fruits. We found strong effects of larval host (citrus fruits) on adult longevity and fecundity. In all five citrus varieties, females did not manage to deposit eggs into fruit pulp. The proportion of eggs laid in either the flavedo or albedo area of the fruit peel differed depending on the citrus variety. In all cases except bitter oranges, females oviposited fewer eggs in citrus fruits than in the artificial oviposition substrates, suggesting that most citrus fruits cause a significant reduction in the reproductive potential of medflies. Negative correlations were found between fecundity and (a) the density of oil glands, and (b) the amount of essential oils in the flavedo area of citrus fruits. There was no correlation between fecundity and other fruit physical characteristics, such as resistance of fruit peel to pressure and thickness of the flavedo. Apparently, resistance of citrus fruits to medfly infestation is directly related to citrus essential oils. The intrinsic rate of increase (r) was higher in bitter oranges than in the three sweet orange varieties tested. A negative r was estimated for flies that developed and oviposited in lemons, indicating a tendency for population decrease in this host. The suitability of citrus fruits for medfly development and the practical implications of our findings for management of medflies in citrus orchards are discussed.     

Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck)

There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. The peel of Citrus fruit accumulates large amounts of ABA during maturation. To understand the regulation of ABA biosynthesis in Citrus, two full-length cDNAs (CsNCED1 and CsNCED2) encoding NCEDs were isolated and characterized from the epicarp of orange fruits (Citrus sinensis L. Osbeck). Expression of the CsNCED1 gene increased in the epicarp during natural and ethylene-induced fruit maturation, and in water-stressed leaves, in a pattern consistent with the accumulation of ABA. The second gene, CsNCED2, was not detected in dehydrated leaves and, in fruits, exhibited a differential expression to that of CsNCED1. Taken together, these results suggests that CsNCED1 is likely to play a primary role in the biosynthesis of ABA in both leaves and fruits, while CsNCED2 appears to play a subsidiary role restricted to chromoplast-containing tissue. Furthermore, analysis of 9-cis-violaxanthin and 9'-cis-neoxanthin, as the two possible substrates for NCEDs, revealed that the former was the main carotenoid in the outer coloured part of the fruit peel as the fruit ripened or after ethylene treatment, whereas 9'-cis-neoxanthin was not detected or was in trace amounts. By contrast, turgid and dehydrated leaves contained 9'-cis-neoxanthin but 9-cis-violaxanthin was absent. Based on these results, it is suggested that 9-cis-violaxanthin may be the predominant substrate for NCED in the peel of Citrus fruits, whereas 9'-cis-neoxanthin would be the precursor of ABA in photosynthetic tissues.     

The Abscisic Acid Content of Citrus with Special Reference to Lemon

Free and conjugated abscisic acid (ABA) in the fruit peel offour species of citrus and their distribution in the variousreproductive organs of Citrus limon L. cv. Eureka during ontogenywere determined by an indirect enzyme-linked immunosorbent assay(ELISA). The different citrus species varied in free ABA (3-8g g dry wt.) and conjugated ABA (10-39 /xg gl dry wt.). In general,the amount of conjugated ABA was 4-fold that of free ABA. Inthe lemon flower, the combined style and stigma tissues containedthe predominant (>65%) amount of both free and conjugatedABA. During fruit development, free and conjugated ABA of thedifferent tissues showed dynamic changes. In the vesicles, freeABA showed progressive increases with development and reacheda high level at maturity whereas the conjugated ABA showed acorresponding decrease with fruit development. Free ABA in theseed increased with fruit development. The content of ABA incitrus fruit was significantly correlated ( = 8.06 + 2.81 Inx; /-2 = 0.84^**) with the weight of the fruit Key words: Abscisic acid, citrus species, reproductive organs, development     

Mineral Content of the Pulp and Peel of Various Citrus Fruit Cultivars

Biological Trace Element Research - The aim of the study was to compare the mineral content between the peel and the pulp of citrus fruits and to determine which citrus fruit, among orange (Citrus...     

ABA-deficiency results in reduced plant and fruit size in tomato

Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.     

Overexpression of the persimmon abscisic acid β‐glucosidase gene (DkBG1) alters fruit ripening in transgenic tomato

β‐Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) β‐glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA‐BG in fruits is still unclear. In this study, through RNA‐seq analysis of persimmon fruit, 10 full‐length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95 days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA‐glucose ester (ABA‐GE) to release free ABA. Compared with wild‐type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1‐OE) accelerated fruit ripening onset by 3–4 days by increasing ABA levels at the pre‐breaker stage and induced early ethylene release compared with wild‐type fruits. DkBG1‐OE altered the expression of ripening regulator NON‐RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA‐GE.     

影响柑桔树开花结果性能的生态生理因素

本文报道1988年和1989年春夏之际异常和正常的桔园小气候环境对柑桔树开花结果物候期、抽梢生长、幼果发育、生理落果、当年果实产量和品质等性能的影响。结果表明,柑桔树的座果率受许多生态生理因素制约,但第一次生理落果主要与开花期桔园的小气候环境有关,第二次生理落果则与树体营养生理代谢密切相关。据此提出了预测预报柑桔树第二次生理落果的指标及其防御措施。