Expression and functional analysis of two lycopene β-cyclases from citrus fruits

In the present study, two LCYb genes (CitLCYb1 and CitLCYb2) were isolated from Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck) and Lisbon lemon (Citrus limon Burm.f.) and their functions were analyzed by the color complementation assay in lycopene-accumulating E. coli cells. The results showed that CitLCYb1 and CitLCYb2 shared high identity at the amino acid level among the three citrus varieties. The N-terminal region of the two proteins encoded by CitLCYb1 and CitLCYb2 was predicted to contain a 51-residue chloroplastic transit peptide, which shared low similarity. In Satsuma mandarin, the secondary structures of the CitLCYb1 and CitLCYb2 encoding proteins without the transit peptide were quite similar. Moreover, functional analysis showed that both enzymes of CitLCYb1 and CitLCYb2 participated in the formation of β-carotene, and when they were co-expressed with CitLCYe, α-carotene could be produced from lycopene in E. coli cells. However, although CitLCYb2 could convert lycopene to α-carotene in E. coli cells, its extremely low level of expression indicated that CitLCYb2 did not participate in the formation of α-carotene during the green stage in the flavedo. In addition, the high expression levels of CitLCYb1 and CitLCYb2 during the orange stage played an important role in the accumulation of β,β-xanthophylls in citrus fruits. The results presented in this study might contribute to elucidate the mechanism of carotenoid accumulation in citrus fruits.     

The role of carotenoid cleavage dioxygenases in the regulation of carotenoid profiles during maturation in citrus fruit

To investigate the relationship between a carotenoid profile and gene expression for carotenoid cleavage dioxygenases, three citrus varieties that exhibit different 9-cis-violaxanthin levels in their juice sacs, Satsuma mandarin (Citrus unshiu Marc.; a variety accumulating a low level of 9-cis-violaxanthin), Valencia orange (Citrus sinensis Osbeck; variety accumulating a high level of 9-cis-violaxanthin), and Lisbon lemon (Citrus limon Burm.f.; a variety accumulating an undetectable level of 9-cis-violaxanthin) were used. Three cDNAs (CitCCD1, CitNCED2, and CitNCED3) were cloned. The recombinant CitCCD1 protein cleaved beta-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9-10 and 9'-10' positions and 9-cis-violaxanthin at the 9'-10' position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11-12 position to form xanthoxin, a precursor of abscisic acid (ABA). The gene expression of CitCCD1 increased in the flavedos and juice sacs of the three varieties during maturation. In Satsuma mandarin, the gene expression of CitNCED2 and CitNCED3 increased noticeably, accompanying a massive accumulation of ABA in the flavedo and juice sacs. In Valencia orange, the gene expression of CitNCED3 increased with a slight elevation of the ABA level in the flavedo, whereas neither the gene expression of CitNCED2 nor the ABA level increased noticeably in the juice sacs. In Lisbon lemon, the gene expression of CitNCED2 increased remarkably, accompanying increases in the ABA level in the flavedo and juice sacs. These results suggest that, in the juice sacs, the efficient cleavage reaction for ABA synthesis reduces the 9-cis-violaxanthin level in Satsuma mandarin and Lisbon lemon, whereas the low cleavage reaction maintains the predominant 9-cis-violaxanthin accumulation in Valencia orange.     

Catalytic activities and chloroplast import of carotenogenic enzymes from citrus

Citrus fruits are a rich source of carotenoids. cDNAs for carotenoid biosynthetic enzymes have been identified from their flavedos and juice sacs, and were used to examine expression patterns of carotenogenic genes during fruit development by several groups. However, functions of most of the gene products have not been verified yet. In this report, we examined catalytic activities of two carotenogenic enzymes from navel orange ( Citrus sinensis Osbeck), phytoene desaturase (CitPds) and lycopene-β-cyclase (CitLcyb), and one enzyme from Satsuma mandarin ( Citrus unshiu Marcow), lycopene-ɛ-cyclase (CitLcye). We also conducted in vitro import assay of the three proteins along with two other carotenogenic enzyme from navel orange, phytoene synthase (CitPsy) and carotenoid β-ring hydroxylase (CitChyb), using isolated chloroplasts, and confirmed their plastid localization and the presence of transit peptides that were cleaved upon import. Furthermore, we examined their suborganellar localization. CitPsy was found to be peripherally associated with the membrane, while CitPds was mainly recovered in the soluble fraction. By contrast, CitLcyb and CitLcye were targeted both to the soluble and to the membrane compartments, although the latter showed a stronger association to the membrane than the former. Finally, CitChyb was exclusively inserted into the chloroplast internal membranes. These data should help us better understand the mechanism of carotenoid biosynthesis.     

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.     

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.     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Abscisic acid increases in non-growing and paclobutrazol-treated fruits of seedless mandarins

Clementine ( Citrus reticulata [Hort.] Ex. Tanaka cv. Oroval) is a self-incompatible mandarin with a slow rate of fruit growth and high percentage of fruit abscission. Seedless Satsuma ( Citrus unshiu [Mak] Marc. cv. Clausellina) shows natural parthenocarpy and higher fruit set. Application of 25 μg fruit⁻¹ of paclobutrazol (PP333), an inhibitor of gibberellin biosynthesis, reduced the rate of growth and accelerated fruit abscission in both varieties. In contrast, gibberellin A₃ (GA₃) stimulated fruit growth only in the self-incompatible mandarin. Clementine fruits, in the absence of pollination, showed an approximately 2-fold transient increase in the free abscisic acid (ABA) content shortly after petal fall. In Satsuma, a very small accumulation of ABA was detected. Paclobutrazol treatment induced a 3-fold increase in ABA in Satsuma fruits but did not substantially affect the pattern of ABA accumulation in Clementine. In this variety, GA₃ suppressed the ABA increase observed in untreated fruits. These effects were observed 24 h after treatment. However, in Satsuma fruits, the effect of GA₃ on the ABA content was negligible. In addition, a comparative analysis of growing and non-growing fruits of Clementine showed that ABA, on a per unit weight basis, was always higher in the non-growing fruits. Treatment with 85 μM fluridone, an inhibitor of carotenoid biosynthesis and thus indirectly of ABA, delayed fruit abscission in Clementine, but also decreased fruit growth. Collectively, these observations indicate a relationship between high ABA content and a reduced rate of fruit growth and an acceleration of fruit abscission.     

Comparative sensitivity of 'Prior Lisbon' lemon and 'Valencia' orange trees to foliar sodium and chloride concentrations

Abstract While citrus rootstocks differ in capacity for sodium and chloride ion exclusion, citrus scion species also vary in foliar sensitivity to NaCl salinisation. Of two common scions, ‘Lisbon’ lemon appears more sensitive, whereas ‘Valencia’ orange in less sensitive to leaf salt. In an attempt to explain this difference. ‘Valencia’ orange (Citrus sinensis [L.] Osbeck) and ‘Prior Lisbon’ lemon (Citrus limon [L.] Burm. F.) were budded to rootstocks known to differ in their ability to exclude sodium ions viz, the strong excluder Trifoliata (Poncirus trifoliata [L.] Raf.), and the weaker excluder Troyer citrange (C. sinensis×P. trifoliata); neither rootstock shows strong exclusion of chloride ions. Budded trees were held under a photosynthetic photon flux density of 450 μmol m ² S ¹ and watered with nutrient solution containing either 0 or 50 mol m ³ NaCl. Growth and photosynthetic responses were measured over 58 d following onset of salinization: salinity effects on leaf gas exchange were studied in relation to changes in leaf water status, compatible solutes and foliar content of sodium and chloride ions, over that same period. Once root-zone salinization began to influence leaf solutes (day 30 onwards), lemon showed a steeper increase in leaf chloride than occurred for orange. Although rootstock differences were without effect on this ingress of chloride ions for either scion, sodium ions were excluded from both scions to a larger extent by Trifoliata than by Troyer citrange. Carbon dioxide assimilation of scion foliage was reduced earlier and to a much larger extent by rootzone salinization in lemon than in orange. Furthermore, comparisons of CO₂ assimilation in relation to leaf tissue solutes between scions (on either rootstock) showed stronger responses for both sodium and chloride ions in lemon than in orange. Faster ingress of chloride into lemon leaves was identified as the crucial factor which predisposed towards expression of that contrast between scions. Although contrasts between scions in photosynthetic responses to salinization matched a faster ingress of chloride into lemon than into orange leaves, the sharper photosynthetic response of ‘Prior Lisbon’ lemon to salinity was not solely attributable to higher concentrations of chloride ions (cell sap basis). A difference between species in subcellular compartmentation of the chloride ion under saline conditions was invoked.     

Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between <Emphasis Type="Italic">C. reticulata</Emphasis> and <Emphasis Type="Italic">C. limon</Emphasis>: the case of pulp carotenoid biosynthesis pathway

Allopolyploidy is known to induce novel patterns of gene expression and often gives rise to new phenotypes. Here we report on the first attempt to relate phenotypic inheritance in an allotetraploid somatic hybrid with gene expression. Carotenoid compounds in the fruit pulp of the two parental species and the hybrid were evaluated quantitatively by HPLC. Only very low levels of β-carotene and β-cryptoxanthin were observed in Citrus limon, while β-cryptoxanthin was a major component of C. reticulata, which also displayed high levels of phytoene, phytofluene, β-carotene, lutein, zeaxantin and violaxanthin. Total carotenoid content in mandarin juice sacs was 60 times greater than that in lemon. The allotetraploid hybrid produced all the same compounds as mandarin but at very low levels. Transgressive concentration of abscisic acid (ABA) was observed in the somatic hybrid. Real-time RT-PCR of total RNA from juice sacs was used to study expression of seven genes (CitDxs, CitPsy, CitPds, CitZds, CitLcy-b, CitChx-b, and CitZep) of the carotenoid biosynthetic pathway and two genes (CitNced1 and CitNced2) involved in abscisic acid synthesis from carotenoid. Gene expression was significantly higher for mandarin than lemon for seven of the nine genes analyzed. Lemon under expression was partially dominant in the somatic hybrid for three upstream steps of the biosynthetic pathway, particularly for CitDxs. Transgressive over expression was observed for the two CitNced genes. A limitation of the upstream steps of the pathway and a downstream higher consumption of carotenoids may explain the phenotype of the somatic hybrid.     

Comparative Studies of Net Photosynthesis and Transpiration of Some Citrus Species and Relatives

Comparisqns were made between ‘Campbell Valencia’ orange (Citrus sinensis (L.) Osbeck), and ‘Gabon Cherry-orange’ (Citropsis gabunensis (Engl.) Swing.), ‘Frost Lisbon’ lemon (Citrus limon (L.) Burm. f.) and ‘Eremolemon’ (Eremocitrus glauca (Lindl.) Swing. ×Citrus limon (L.) Burm. f.) and between diploid and autotetraploid ‘Lisbon’ lemons with respect to the influences of temperature and humidity on net photosynthesis and transpiration. Net photosynthesis, leaf conductance to water vapor and water-use-efficiency of Citropsis gabunensis were lower than with Citrus sinensis.‘Eremolemon’ had higher net photosynthesis and higher water-use-efficiency than ‘Lisbon’ lemon, but only small differences were observed between the two species in leaf conductance to water vapor. Small, nonsignificant, differences were observed between diploid and tetraploid ‘Lisbon’ lemons in responses of net photosynthesis and leaf conductance to temperature and humidity. Temperatures above 30°C and increases in vapor pressure difference caused declines in net photosynthesis and increases in vapor pressure difference resulted in decreases in leaf conductance to water vapor by all of the species used in these studies.     

Chlorophyllase activity in chlorophyll-free citrus chromoplasts

Chromoplast fractions from mature, chlorophyll-less ‘Valencia’ orange (Citrus sinensis L. Osbeck) flavedo (= the outer coloured layer of citrus peel) showed considerable chlorophyllase activity. Acetone powders prepared from chromoplast fractions had 2.5× higher specific activity than those prepared from whole flavedo. Exposure of mature, chlorophyll-less fruit to ethylene caused a 2.5 to 4.0 fold increase in chlorophyllase activity. Juice chromoplasts showed negligible chlorophyllase activity. The results suggest that chlorophyllase activity as well as its induction by ethylene are not dependent upon the presence of chlorophyll in the tissue.     

Cellulase Activity, Endogenous Abscisic Acid, and Ethylene in Four Citrus Cultivars during Maturation

At maturity, the fruit of two early maturing orange cultivars, Hamlin and Pineapple (Citrus sinensis [L.] Osbeck), contained more ethylene and abscisic acid than the late maturing Valencia and Lamb Summer (C. sinensis [L.] Osbeck) cultivars. Ethylene (up to 95 nl/l in internal atmosphere) and abscisic (50 μg/kg dry weight flavedo) increased most rapidly in Pineapple, leading to increased cellulase activity and loosening of the fruit. Fruit of the two late maturing cultivars contained less than 25 nl/1 ethylene and 40 μg abscisic acid/kg dry weight of flavedo at peak maturity. Cellulase activity and loosening of the fruit of these late maturing cultivars was slight.     

In vivo sucrose stimulation of colour change in citrus fruit epicarps: Interactions between nutritional and hormonal signals

During ripening, citrus fruit-peel undergoes 'colour break', a process characterized by the conversion of chloroplast to chromoplast. The process involves the progressive loss of chlorophylls and the gain of carotenoids, changing peel colour from green to orange. In the present work, the in vivo and in vitro effects of supplemented nutrients (sucrose and nitrogen) and phytohormones (gibberellins [GA] and ethylene) on colour change in fruit epicarp of Satsuma mandarin ( Citrus unshiu (Mak.) Marc., cv. Okitsu), were studied. The rate of colour break was correlated positively with sucrose content and negatively with nitrogen content. The removal of leaves blocked natural sucrose build-up and nitrogen reduction in the peel. Defoliation also inhibited chlorophyll disappearance and carotenoid accumulation, thereby preventing colour break. In vivo sucrose supplementation promoted sucrose accumulation and advanced colour break. In both in vivo and in vitro experiments, colour change promoted by sucrose was unaffected by ethylene but delayed by GA_3. In non-supplemented plants, ethylene accelerated colour break while GA₃ had no detectable effects. Ethylene inhibitors effectively counteracted the sucrose effects on colour change. Collectively, these results suggest that the chloroplast to chromoplast conversion in citrus fruit epicarps is stimulated by sucrose accumulation. The sugar regulation appears to operate via ethylene, whereas GA may act as a repressor of the sucrose-ethylene stimulation.