Lisbon Lemon Research Articles

Optimization of in planta methodology for genome editing and transformation in Citrus.

Genetic transformation of many plant species relies on in vitro tissue culture-based approaches. This can be a labor-intensive process, requiring aseptic conditions and regenerating often recalcitrant species from tissue culture. Here, we have optimized an in planta transformation protocol to rapidly transform commercial citrus cultivars, bypassing the need for tissue culture. As a proof of concept, we used in planta transformation to introduce CRISPR/Cas9 constructs into Limoneira 8A Lisbon lemon and Pineapple sweet orange, cultivars that are challenging to transform with conventional techniques. Using our optimized protocol, the regeneration rate was significantly increased from 4.8% to over 95%, resulting in multiple gene-edited lines in lemon. We also successfully recovered gene-edited Pineapple sweet orange lines using this protocol; the transformation efficiency for these cultivars ranged between 0.63% and 4.17%. Remarkably, these lines were obtained within three months, making this in planta protocol a rapid methodology to obtain transformed citrus plants. This approach can rapidly and effectively introduce key genetic changes into a wide variety of citrus cultivars.

In silico characterization of the Limonene synthase gene in citrus species under freezing tolerance

Bitterness in citrus fruit extract is caused by changes and transformations in limonoids and the production of limonene, a bitter-tasting substance. Limonene synthase enzyme is responsible for producing limonene. This study examined the effect of low-temperature stress on the expression of the limonene synthase gene in four different citrus species: Miyagawa mandarin (Citrus unshiu), Thomson Navel Orange (Citrus sinensis var. Thomson navel), Ruby Star grapefruit (Citrus paradisi var. Ruby star), and Lisbon lemon (Citrus limon var. Lisbon). These species were grafted on two-year-old sour orange rootstocks. The experiment was carried out as a factorial in the form of a completely random design. Three temperatures were used for the cold treatment (−8, −4, 4 °C). The expression sequence of the Limonene synthase gene was predicted using bioinformatic software such as PLANT Care and PlantPan. The results showed significant variation in the frequency of amino acids in the LS gene. Leucine (11.36) and glutamic acid (7.57) had the highest frequency. The analysis of the Limonene synthase gene promoter revealed different Cis-element regions such as response elements to light, hormones, and abiotic stresses on the promoter of these genes. The gene promoter had many regions related to binding sites of MYB transcription factors, response regions to hormones, cold (Dehydrin with 15 cis-regulatory elements), and drought, emphasizing the role of this gene in abiotic stress response. The gene expression patterns of the Limonene synthase showed that the highest and lowest expression occurred at − 4 °C in Lisbon lemon (−132.28), Ruby Star grapefruit (−70.06), Thomson Navel Orange (−2.88), and Miyagawa Mandarin (−3.88), confirming the role of this gene in abiotic and biotic stress response.

Stepwise Regression Analysis of Citrus Genotype Under Cold Stress

Background: Citrus is among the cold temperature (freezing) susceptible plants. The performance of storage and breeding of horticultural crops is mainly dependent on the correlation among the traits. Objectives: The present study was conducted to identify the physiological and biochemical traits of some citrus cultivars with the greatest influence on stress tolerance and determine the direct and indirect effects of these traits on the median Lethal Time (LT50) using integrated pathway analysis (i.e., stepwise regression and causality coefficient). Methods: Pathway analysis (i.e., stepwise regression and causality coefficient) based on additive models, which is among the robust statistical methods to describe the relationship among the traits, was used to investigate the physiological and biochemical characteristics of four citrus cultivars (i.e., Japanese mandarin, Thomson orange, Ruby Star grapefruit, and Lisbon lemon) at temperature levels (-8, -4, and +4°C) in a completely randomized design with three replications in 2021. Results: The results showed that Japanese mandarin and Thomson orange cultivars had the highest stress tolerance (LT50). The results of the correlation coefficient demonstrated that the highest positive and significant correlations belonged to the LT50 with total flavonoid (0.443**), chlorophyll a with chlorophyll b (0.613**), carotenoid with chlorophyll a (0.929**), chlorophyll b (0.573**), and total chlorophyll (0.849**), relative moisture content with malondialdehyde (0.559**), glycine-betaine with catalase (0.919**), hydrogen peroxide with total flavonoid (0.405**), and catalase (0.611**). Conclusions: The results of stepwise regression for all traits indicated the importance and critical role of total flavonoid, proline, and LT50 characteristics.

Multi-omics Comparison Reveals Landscape of Citrus limon and Citrus sinensis Response to ‘Candidatus Liberibacter asiaticus’

Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage.

Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes (CBF1, CBF2, and CBF3) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1–5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus, indicating that CBF1 participates in the natural tolerance to CI.

Variation in phenolic acids and antibacterial attributes of peel extracts from ripe and unripe [Citrus limon (L.) Osbeck] fruit

This study reveals variation in phenolic acids and antimicrobial attributes of peel extracts from ripe and unripe lisbon lemon [Citrus limon (L.) Osbeck] fruit. Total phenolic contents in ripened lemon peels were significantly (p < 0.05) higher (138.66–344.33 GAE mg/g DW) than the unripen lemon peel (122.33–277.10 GAE mg/g DW). The total amount of individual phenolic acids as analyzed by RP-HPLC in unripe and ripened peels ranged from 3.8 ± 2.2 to 1453.6 ± 66.4 and 3.4 ± 1.8 to 1772 ± 64 mg/kg, respectively with gallic, caffeic, cinnamic, syringic, vanilic, vanillin, p-coumaric, m-coumaric and o-coumaric acids as the main compounds detected. As per disk diffusion measurement, different solvent (methanol, ethyl acetate and chloroform) extracts from ripened and unripe lemon peel exhibited considerable antimicrobial activity against four bacterial strains including Salmonella typhimurium, Bacillus subtilus, Escherichia coli and Staphylococcus aureus with 50% chloroform extracts having the best potential. The composition of phenolics and antimicrobial activity of tested peels varied notably in relation to extraction solvents and fruit maturity stages. Overall, the present results support potential uses of limon peels as a natural source of high-value phenolics/antimicrobial agents for nutra-pharmaceutical developments.

Reaction of some commercial citrus species and Iranian lime biotypes to witches' broom disease of lime

Witches' broom disease of lime (WBDL), associated with Candidatus Phytoplasma aurantifolia, has devastated the majority of Mexican lime orchards in southern Iran. Recognition of resistant cultivars and rootstocks can play a major role in WBDL management. In this study, graft-inoculation was used to address the reaction of 12 citrus commercial species and 39 collected lime biotypes from five main lime production areas of Hormozgan province, Iran, against WBDL phytoplasma. The experimental plants were allowed to grow from seed, then five seedlings of each species and biotypes were grafted with Ca. Phytoplasma aurantifolia infected shoots. Inoculated plants were inspected for symptom expression for two years. Direct and nested-PCR using P1/P7 and R16F2n/R16R2 primer pairs, respectively, were used for confirmation of phytoplasmal infection in the suspected plants. All commercial citrus species and 39 of the lime biotypes showed the symptoms of little leaf, internode shortening and witches' broom after two to twenty months from graft-inoculation. Incubation period in plants varied from two months in Astaraei lemon and R11 biotype of Rudan region to twenty months in S4 and S6 biotypes of Senderk region. With these results, we can say all commercial citrus species were sensitive to WBDL. Among the commercial citrus species, Lisbon lemon, grapefruit and Cleopatra mandarin cultivars showed symptoms of WBDL after 460, 425 and 375 days, respectively. Disease symptoms developed rapidly on Astaraei lemon and Bakraei within 85–105 days, respectively. Among the collected biotypes, one, five and one biotypes from Minab, Rudan and Hashtbandi, respectively, were resistant to WBDL phytoplasma. These biotypes were symptomless; however, the PCR assay for detection of the phytoplasma was positive in all of them. These results are promising regarding the potential of using the suitable biotypes to reduce the development of WBDL.

Analysis the effects of pollen grain sources on the fruits set and their characteristics of ‘Clementine’ mandarin using microscopic and molecular approaches

‘Clementine’ is one of the most important cultivars of mandarin that always has problems with fertilization and fruit set because of its self-incompatibility. Therefore, for the commercial fruit production, suitable and compatible pollen is required. To determine the best pollen source, ‘Clementine’ flowers were subjected to the different pollen grains including sweet lemon, Lisbon lemon, grapefruit, sour orange, as well as self-pollination and open pollination. Compatibility and incompatibility tests were carried out using controlled pollination method and microscopic study of pollen tube growth. Molecular markers were used to study the genetic relationships of the species and assess the role of genetic similarity on the fruits set and their attributes. The results of the microscopic study indicated that ‘Clementine’ mandarin is a self-incompatible cultivar but it is compatible with all the cultivars used in the experiment. Clementine fruit characteristics significantly influenced by pollen grain sources. The results of controlled pollination showed that the highest percentage of fruit set was obtained from flowers pollinated with sweet lemon pollen (73.75%) followed by Lisbon lemon pollen (69.25%) while the lowest fruit set was obtained from the self-pollinated flowers (11.75%). The highest and the lowest hale seeds were obtained from the flowers pollinated by Lisbon lemon pollen (12.22%) and self-pollinated ones (no viable seed), respectively. High correlation was observed between some of the studied characters that would be useful for selection purposes in the breeding programs. Results of microscopic evaluation of pollen grain tubes revealed that among the studied pollen grains, sour orange pollens were the most successful for penetration to the basal portion of style and into ovary of ‘Clementine’ mandarin. Assessment of genetic relationship among different Citrus species revealed that sour orange was the most similar species with ‘Clementine’ mandarin. High positive correlation was observed between genetic similarities of pollen sources at DNA level with the pollen grain sprouting and penetration to the different parts of style and ovary as well as seed formation percentages. Based on the studied fruit characteristics, grapefruit and Lisbon lemon pollens are suggested as the suitable pollinators for improving the qualitative and quantitative characteristics of ‘Clementine’ mandarin fruit.

A novel citrus viroid found in Australia, tentatively named citrus viroid VII.

A novel citrus viroid was discovered in a non-symptomatic Lisbon lemon (Citrus x limon L. Burm.f.) tree in New South Wales, Australia. Bioindexing, molecular detection and characterization involving sequencing combined with in silico analysis for the identification of the viroid-RNA hallmark properties of transmissibility and autonomous replication as well as specific sequence and structural motifs suggest that this viroid is a member of a new species in the genus Apscaviroid, family Pospiviroidae, which we have tentatively named "citrus viroid VII" (CVd-VII).

Regulation of ascorbic acid metabolism in response to different temperatures in citrus juice sacs in vitro

To elucidate the regulation of ascorbic acid (AsA) metabolism in response to temperatures, three varieties of citrus juice sacs (Valencia orange, Lisbon lemon, and Satsuma mandarin) in in vitro culture system were subjected to different temperatures (10°C, 20°C, and 30°C). The AsA accumulation was induced at 10°C, whereas it was not significantly affected at 30°C as compared with the control at 20°C in juice sacs of the three citrus varieties. The enhancement of AsA accumulation at 10°C was regulated by the expression of genes in AsA biosynthetic, oxidation, and regeneration pathway. In Valencia orange and Satsuma mandarin, the higher expression of CitVTC4 gene in biosynthetic pathway, and the higher expression of CitMDAR1 and CitMDAR2 genes in regeneration pathway, together with the lower expression of CitAO gene in oxidation pathway contributed to the higher AsA level at 10°C. In Lisbon lemon, the higher expressions of CitVTC1, CitVTC2, and CitVTC4 genes in biosynthetic pathway and the lower expression of CitAO gene in oxidation pathway contributed to the higher AsA level at 10°C.

Traits Driving Tolerance to Atmospheric Fluoride Pollution in Tree Crops

Increased emissions of fluoride into the atmosphere contribute to reducing the sustainability of agricultural systems worldwide. In order to improve the understanding of the factors behind such phenomenon, varieties of citrus (Citrus spp.), Valencia sweet-orange, Ponkan mandarin, and Lisbon lemon and coffee (Coffea spp.), Obata, Catuai, and Apoata, were treated with fluoride nebulization. The trees were exposed to nebulization for 60 min inside a chamber by using medium (0.04 mol L−1) and high (0.16 mol L−1) doses of fluoridic acid (HF) during three nonconsecutive days in a single week, for a total of 26 days of exposure during the experiment. Sixty days after beginning nebulization, we evaluated leaf gas exchange, (ultra)structural organization, tree growth, and fluoride and nutrient concentrations in plant tissue. Photosynthesis and leaf dry mass of citrus and coffee varieties were affected differently by fluoride toxicity, and based on the tolerance index (relative leaf dry mass of control versus leaf dry mass of trees treated with 0.16 mol L−1 HF), the order of sensitivity for the varieties of each species was as follows: for citrus, lemon > mandarin > sweet-orange; and for coffee, Apoata > Catuai > Obata. The ability of the trees to control fluoride absorption most likely explained this contrast in sensitivity among varieties because both photosynthesis and leaf growth were negatively correlated with leaf fluoride concentration. Although disorganization of the thylakoids, degeneration of vascular cells, and disruption of the middle lamella occurred in leaves of all varieties exposed to fluoride, the more severe damage was observed in those with greater sensitivity to the pollutant (i.e., lemon and Apoata coffee). Taken together, these results provided insights into the factors that explain poor performance of citrus and coffee trees under fluoride pollution and also revealed the traits driving the tolerance of these crops such a limiting condition, which included a combination of the following: (i) reduced fluoride absorption, (ii) increased photosynthesis, and (iii) improved maintenance of the ultrastructural organization of leaves.

Coupling a branch enclosure with differential mobility spectrometry to isolate and measure plant volatiles in contained greenhouse settings

Volatile organic compounds (VOCs) are off-gassed from all living organisms and represent end products of metabolic pathways within the system. In agricultural systems, these VOCs can provide important information on plant health and can ordinarily be measured non-invasively without harvesting tissue from the plants. Previously we reported a portable gas chromatography/differential mobility spectrometry (GC/DMS) system that could distinguish VOC profiles of pathogen-infected citrus from healthy trees before visual symptoms of disease were present. These measurements were taken directly from canopies in the field, but the sampling and analysis protocol did not readily transfer to a controlled greenhouse study where the ambient background air was saturated with volatiles contained in the facility. In this study, we describe for the first time a branch enclosure uniquely coupled with GC/DMS to isolate and measure plant volatiles. To test our system, we sought to replicate our field experiment within a contained greenhouse and distinguish the VOC profiles of healthy versus citrus infected with Candidatus Liberibacter asiaticus. We indeed confirm the ability to track infection-related trace biogenic VOCs using our sampling system and method and we now show this difference in Lisbon lemons (Citrus×limon L. Burm. f.), a varietal not previously reported. Furthermore, the system differentiates the volatile profiles of Lisbon lemons from Washington navels [Citrus sinensis (L.) Osbeck] and also from Tango mandarins (Citrus reticulata Blanco). Based on this evidence, we believe this enclosure-GC/DMS system is adaptable to other volatile-based investigations of plant diseases in greenhouses or other contained settings, and this system may be helpful for basic science research studies of infection mechanisms.

Early Changes in Host Response to C. Liberibacter asiaticus Infection in Citrus

Candidatus Liberibacter asiaticus (CLas), a non-culturable, phloem-limited bacterium, is one of the presumed causative agents of the devastating citrus disease known as Huanglongbing (HLB). HLB was first discovered in Florida in 2005, and has since spread throughout the state. It is now threatening both the California and Texas citrus industries. Diseased trees suffer from discolored leaves and fruit, reduced fruit yield with poor quality, and premature tree death. Though HLB is present globally, there is no known cure, and no known resistant citrus varieties. To understand how different citrus varieties respond to CLas infection, leaves from Lisbon Lemon, Tango Mandarin, and Washington Navel trees graft-inoculated with CLas were studied longitudinally by qPCR and 1H NMR metabolomics. Changes in the overall metabolite profile of the infected plants occurred earlier than detection of CLas by qPCR. Characteristic changes in pathways related to plant defense were altered by infection. These results suggest that metabolomics may be suitable for improved early detection of CLas infection and also clarify CLas-induced changes to host metabolism which may be used to inform discovery of targets for treatment of HLB and for methods of controlling CLas spread.

Identification, synthesis, and characterization of novel sulfur-containing volatile compounds from the in-depth analysis of Lisbon lemon peels (Citrus limon L. Burm. f. cv. Lisbon).

Lemons (Citrus limon) are a desirable citrus fruit grown and used globally in a wide range of applications. The main constituents of this sour-tasting fruit have been well quantitated and characterized. However, additional research is still necessary to better understand the trace volatile compounds that may contribute to the overall aroma of the fruit. In this study, Lisbon lemons (C. limon L. Burm. f. cv. Lisbon) were purchased from a grove in California, USA, and extracted by liquid-liquid extraction. Fractionation and multidimensional gas chromatography-mass spectrometry were utilized to separate, focus, and enhance unidentified compounds. In addition, these methods were employed to more accurately assign flavor dilution factors by aroma extract dilution analysis. Numerous compounds were identified for the first time in lemons, including a series of branched aliphatic aldehydes and several novel sulfur-containing structures. Rarely reported in citrus peels, sulfur compounds are known to contribute significantly to the aroma profile of the fruit and were found to be aroma-active in this particular study on lemons. This paper discusses the identification, synthesis, and organoleptic properties of these novel volatile sulfur compounds.

Regulation of ascorbic acid metabolism by blue LED light irradiation in citrus juice sacs

In the present study, the effects of red and blue LED lights on the accumulation of ascorbic acid (AsA) were investigated in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon. The results showed that the blue LED light treatment effectively increased the AsA content in the juice sacs of the three citrus varieties, whereas the red LED light treatment did not. By increasing the blue LED light intensity, the juice sacs of the three citrus varieties accumulated more AsA. Moreover, continuous irradiation with blue LED light was more effective than pulsed irradiation for increasing the AsA content in the juice sacs of the three citrus varieties. Gene expression results showed that the modulation of AsA accumulation by blue LED light was highly regulated at the transcription level. The up-regulation of AsA biosynthetic genes (CitVTC1, CitVTC2, CitVTC4, and CitGLDH), AsA regeneration genes (CitMDAR1, CitMDAR2, and CitDHAR) and two GSH-producing genes (CitGR and CitchGR) contributed to these increases in the AsA content in the three citrus varieties.

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.

Mechanism of Carotenoid Accumulation in Citrus Fruit

Citrus is a complex source of carotenoids with the largest number of carotenoids found in any fruit. Carotenoid concentration and composition vary greatly among citrus varieties. Satsuma mandarin (Citrus unshiu Marc.) predominantly accumulates β-cryptoxanthin in the juice sacs. Valencia orange (Citrus sinensis Osbeck) predominantly accumulates violaxanthin isomers in the juice sacs. Lisbon lemon (Citrus limon Burm.f.) accumulates low level of carotenoids in the juice sacs. To elucidate the carotenoid accumulation in citrus fruit maturation, the expression of genes related to carotenoid biosynthesis and catabolism was investigated in the three citrus varieties exhibited different carotenoid profile. The results showed that the carotenoid accumulation during citrus fruit maturation is highly regulated by the coordination of the expression for the genes related to carotenoid biosynthesis and catabolism in both flavedo and juice sacs. ‘Tamami’ is a hybrid between ‘Kiyomi’ tangor (Citrus unshiu Marc. × Citrus sinensis Osbeck) and ‘Wilking’ mandarin (Citrus nobilis Lour. × Citrus deliciosa Ten.). To elucidate the mechanism of the accumulation of β-cryptoxanthin in ‘Tamami’, a variety accumulating higher β-cryptoxanthin than Satsuma mandarin, the expression of genes related to carotenoid biosynthesis and catabolism was investigated in the juice sacs of ‘Tamami’. The results showed that the mechanism of β-cryptoxanthin accumulation in ‘Tamami’ was similar to that in Satsuma mandarin. Furthermore, in the recent studies, possible factors, which regulate carotenoid concentration and composition in citrus juice sacs were investigated in vitro.

Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro

In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation.

カンキツ果実におけるカロテノイド集積メカニズム

Citrus is a complex source of carotenoids with the largest number of carotenoids found in any fruit. Carotenoid concentration and composition vary greatly among citrus varieties. Satsuma mandarin (Citrus unshiu Marc.) predominantly accumulates β-cryptoxanthin in the juice sacs. Valencia orange (Citrus sinensis Osbeck) predominantly accumulates violaxanthin isomers in the juice sacs. Lisbon lemon (Citrus limon Burm.f.) accumulates low level of carotenoids in the juice sacs. To elucidate the carotenoid accumulation in citrus fruit maturation, the expression of genes related to carotenoid biosynthesis and catabolism was investigated in the three citrus varieties exhibited different carotenoid profile. The results showed that the carotenoid accumulation during citrus fruit maturation is highly regulated by the coordination of the expression for the genes related to carotenoid biosynthesis and catabolism in both flavedo and juice sacs. ‘Tamami’ is a hybrid between ‘Kiyomi’ tangor (Citrus unshiu Marc. × Citrus sinensis Osbeck) and ‘Wilking’ mandarin (Citrus nobilis Lour. × Citrus deliciosa Ten.). To elucidate the mechanism of the accumulation of β-cryptoxanthin in ‘Tamami’, a variety accumulating higher β-cryptoxanthin than Satsuma mandarin, the expression of genes related to carotenoid biosynthesis and catabolism was investigated in the juice sacs of ‘Tamami’. The results showed that the mechanism of β-cryptoxanthin accumulation in ‘Tamami’ was similar to that in Satsuma mandarin. Furthermore, in the recent studies, possible factors, which regulate carotenoid concentration and composition in citrus juice sacs were investigated in vitro.

Temporal Patterns inHomalodiscaspp. (Hemiptera: Cicadellidae) Oviposition on Southern California Citrus and Jojoba

A detailed study of the distribution of egg masses of Homalodisca vitripennis (Germar) and H. liturata Ball was done across a 2-yr period (2001-2003) on six host plants in southern California (Marsh grapefruit, Lisbon lemon, Washington navel, Dancy tangerine, rough lemon, and jojoba in Riverside; jojoba in Desert Center). The majority of egg masses in Riverside belonged to H. vitripennis (84-100%), whereas in Desert Center, all Homalodisca egg masses were H. liturata. Oviposition in Riverside occurred in two discrete periods, a late winter and spring period (mid-February to late May), followed by a short interval of very low oviposition during most of June, and then a summer period (late June to late September) followed by a relatively long period of very low oviposition in fall and early winter (October to mid-February). Levels of oviposition during the late winter-spring period were similar to those during the summer despite an observed larger population of adults during the latter period. Moreover, egg clutch size for H. vitripennis was generally greater in spring than during summer and was generally higher than that for H. liturata, especially on Riverside jojoba. Larger egg clutch size was seen on grapefruit than on lemon, navel, and tangerine during summer. There appeared to be temporal host shifts in oviposition; most evident was the shift from relatively high rates of oviposition on lemon and tangerine in late winter-early spring to relatively higher rates of oviposition on grapefruit and navel during summer.