Citrus Ichangensis Research Articles

Genome-wide identification, expression analysis of WRKY transcription factors in Citrus ichangensis and functional validation of CiWRKY31 in response to cold stress

BackgroundIchang papeda (Citrus ichangensis), a wild perennial plant of the Rutaceae family, is a cold-hardy plant. WRKY transcription factors are crucial regulators of plant growth and development as well as abiotic stress responses. However, the WRKY genes in C. ichangensis (CiWRKY) and their expression patterns under cold stress have not been thoroughly investigated, hindering our understanding of their role in cold tolerance.ResultsIn this study, a total of 52 CiWRKY genes identified in the genome of C. ichangensis were classified into three main groups and five subgroups based on phylogenetic analysis. Comprehensive analyses of motif features, conserved domains, and gene structures were performed. Segmental duplication plays a significant role in the CiWRKY gene family expansion. Cis-acting element analysis revealed the presence of various stress-responsive elements in the promoters of the majority of CiWRKYs. Gene ontology (GO) analysis and protein-protein interaction predictions indicate that the CiWRKYs exhibit crucial roles in regulation of both development and stress response. Expression profiling analysis demonstrates that 14 CiWRKYs were substantially induced under cold stress. Virus-induced gene silencing (VIGS) assay confirmed that CiWRKY31, one of the cold-induced WRKYs, functions positively in regulation of cold tolerance.ConclusionSequence and protein properties of CiWRKYs were systematically analyzed. Among the 52 CiWRKY genes 14 members exhibited cold-responsive expression patterns, and CiWRKY31 was verified to be a positive regulator of cold tolerance. These findings pave way for future investigations to understand the molecular functions of CiWRKYs in cold tolerance and contribute to unravelling WRKYs that may be used for engineering cold tolerance in citrus.

Transcriptome analysis of the pulp of citrus fruitlets suggests that domestication enhanced growth processes and reduced chemical defenses increasing palatability

To identify key traits brought about by citrus domestication, we have analyzed the transcriptomes of the pulp of developing fruitlets of inedible wild Ichang papeda (Citrus ichangensis), acidic Sun Chu Sha Kat mandarin (C. reticulata) and three palatable segregants of a cross between commercial Clementine (C. x clementina) and W. Murcott (C. x reticulata) mandarins, two pummelo/mandarin admixtures of worldwide distribution. RNA-seq comparison between the wild citrus and the ancestral sour mandarin identified 7267 differentially expressed genes, out of which 2342 were mapped to 117 KEGG pathways. From the remaining genes, a set of 2832 genes was functionally annotated and grouped into 45 user-defined categories. The data suggest that domestication promoted fundamental growth processes to the detriment of the production of chemical defenses, namely, alkaloids, terpenoids, phenylpropanoids, flavonoids, glucosinolates and cyanogenic glucosides. In the papeda, the generation of energy to support a more active secondary metabolism appears to be dependent upon upregulation of glycolysis, fatty acid degradation, Calvin cycle, oxidative phosphorylation, and ATP-citrate lyase and GABA pathways. In the acidic mandarin, downregulation of cytosolic citrate degradation was concomitant with vacuolar citrate accumulation. These changes affected nitrogen and carbon allocation in both species leading to major differences in organoleptic properties since the reduction of unpleasant secondary metabolites increases palatability while acidity reduces acceptability. The comparison between the segregants and the acidic mandarin identified 357 transcripts characterized by the occurrence in the three segregants of additional downregulation of secondary metabolites and basic structural cell wall components. The segregants also showed upregulation of genes involved in the synthesis of methyl anthranilate and furaneol, key substances of pleasant fruity aroma and flavor, and of sugar transporters relevant for sugar accumulation. Transcriptome and qPCR analysis in developing and ripe fruit of a set of genes previously associated with citric acid accumulation, demonstrated that lower acidity is linked to downregulation of these regulatory genes in the segregants. The results suggest that the transition of inedible papeda to sour mandarin implicated drastic gene expression reprograming of pivotal pathways of the primary and secondary metabolism, while palatable mandarins evolved through progressive refining of palatability properties, especially acidity.

Comprehensive analysis of KCS gene family in Citrinae reveals the involvement of CsKCS2 and CsKCS11 in fruit cuticular wax synthesis at ripening

Cuticular wax covers the surface of fleshy fruit and plays a protective role in fruit development and postharvest storage, including reducing fruit water loss, resisting biotic and abiotic stress and affecting fruit glossiness. The β-ketoacyl-CoA synthase (KCS) is the rate-limiting enzyme of very long chain fatty acids (VLCFAs) synthesis, which provides precursors for the synthesis of cuticular wax. In this study, a total of 96 KCS genes were identified in six Citrinae species, including 13, 16, 21, 14, 16 and 16 KCS genes in the primitive species (Atalantia buxifolia), the wild species (Citrus ichangensis), and four cultivated species (Citrus medica, Citrus grandis, Citrus sinensis and Citrus clementina), respectively. Compared with primitive species, wild and cultivated species showed expansion of KCS gene family. Evolutionary analysis of KCS gene family indicated that uneven gain and loss of genes resulted in variable numbers of KCS genes in Citrinae, and KCS genes have undergone purifying selection. Expression profiles in C. sinensis revealed that the KCS genes had diverse expression patterns among various tissues. Furthermore, CsKCS2 and CsKCS11 were predominantly expressed in the flavedo and their expression increased sharply with ripening. Subcellular localization analysis indicated that CsKCS2 and CsKCS11 were located in the endoplasmic reticulum. Further, heterologous expression of CsKCS2 and CsKCS11 in Arabidopsis significantly increased the content of cuticular wax in leaves. Thus, CsKCS2 and CsKCS11 are involved in the accumulation of fruit cuticular wax at ripening. This work will facilitate further functional verification and understanding of the evolution of KCS genes in Citrinae.

Characterization of Odor-Active Compounds of Ichang Lemon (Citrus wilsonii Tan.) and Identification of Its Genetic Interspecific Origin by DNA Genotyping.

Ichang lemon is a citrus fruit whose rind gives off a delicious and much appreciated fragrance and flavor. The volatile components of the fruit peel of Ichang lemon were investigated by GC-MS and GC-O (AEDA method). Simultaneously, its genetic origin was identified by using diagnostic SNP markers specific to ancestral species and multiallelic SSR and InDel markers. Ichang lemon combines three ancestral genomes (Citrus maxima, Citrus ichangensis, and Citrus reticulata) and may be a pummelo × Yuzu hybrid. Although the major compounds of the Ichang lemon aromatic profile were present in Citrus junos, a few pummelo-specific compounds were also detected, such as indole and nootkatone, in agreement with its maternal lineage. 3-Methyl-3-sulfanylbutyl acetate, reported to occur in passion fruit and brewed coffee, was identified by GC-MS, GC-QTOF-MS, and GC-FTIR for the first time in citrus. This odor-active compound has a sulfurous, tropical fruity, green note.

Comparative Genetic Analysis between the Jeju ‘Inchangkyool’ and Chinese ‘Ichangensis’ (Citrus ichangensis) using Internal Chloroplast trnL-trnF Intergenic Spacers and Transcribed Spacer Sequence Regions

Comparative Genetic Analysis between the Jeju ‘Inchangkyool’ and Chinese ‘Ichangensis’ (<i>Citrus ichangensis</i>) using Internal Chloroplast trnL-trnF Intergenic Spacers and Transcribed Spacer Sequence Regions

Coordination of multiple regulation pathways contributes to the tolerance of a wild citrus species (Citrus ichangensis ‘2586’) against Huanglongbing

Huanglongbing (HLB), a highly destructive disease of citrus production worldwide, is presumably caused by ‘Candidatus Liberibacter asiaticus’ (CLas) because Koch's postulates have not been fulfilled. No HLB resistant genotypes have been so far identified in commercial citrus species, hindering the development of resistance breeding. China is an important center of citrus germplasm resources with abundant wild citrus species. However, studies on the susceptibility or resistance of these wild citrus and their response mechanisms to CLas infection have been limited. In this study, we evaluated the symptom development and transcriptional differences among two wild citrus species, Citrus ichangensis ‘2586’ and C. daoxianensis, and the highly susceptible C. sinensis. No typical symptoms was observed and much less CLas accumulated in C. ichangensis, compared with C. daoxianensis and C. sinensis. Much greater number of differentially expressed genes were identified and pathways were perturbed in diseased C. sinensis, followed by diseased C. daoxianensis then C. sinensis. Many pathways including cell wall, secondary metabolism, stress responses, RNA transcriptional regulation and protein modification were differentially regulated in three diseased citrus species but with different preferences. Particularly, genes associated with carbohydrate metabolism, callose deposition and photosynthesis process were significantly regulated in CLas-infected C. daoxianensis and C. sinensis but not in CLas-infected C. ichangensis. Taken together, it seem to indicate that the relatively successful defenses of C. ichangensis against multiplication of CLas and its symptom development may involve a novel mechanism in which the carbohydrate metabolism and photosynthesis process was balanced with the normal physiology of the plant, and coordinated with other regulation pathways. Evaluation of wild citrus resources to CLas infection is promising for citrus HLB resistance breeding in the future.

Analysis of Flavonoid Metabolites in Citrus Peels (Citrus reticulata "Dahongpao") Using UPLC-ESI-MS/MS.

Flavonoids are a kind of essential substance for the human body because of their antioxidant properties and extremely high medicinal value. Citrus reticulata “Dahongpao” (DHP) is a special citrus variety that is rich in flavonoids, however little is known about its systematic flavonoids profile. In the present study, the presence of flavonoids in five important citrus varieties, including DHP, Citrus grandis Tomentosa (HZY), Citrus ichangensis Swingle (YCC), Citrus sinensis (L.) Osbeck (TC), and Citrus reticulata ‘Buzhihuo’ (BZH), was determined using a UPLC-ESI-MS/MS-based, widely targeted metabolome. Results showed that a total of 254 flavonoid metabolites (including 147 flavone, 39 flavonol, 21 flavanone, 24 anthocyanins, 8 isoflavone, and 15 polyphenol) were identified. The total flavonoid content of peels from DHP was the highest. DHP could be clearly separated from other samples through clustering analysis and principal component analysis (PCA). Further, 169 different flavonoid metabolites were observed between DHP peels and the other four citrus peels, and 26 down-regulated differential metabolites displayed important biological activities in DHP. At the same time, a unique flavonoid component, tricin 4′-O-syringyl alcohol, was only found in DHP, which could be used as a marker to distinguish between other varieties. This work might facilitate a better understanding of flavonoid metabolites between DHP peels and the other four citrus peels and provide a reference for its sufficient utilization in the future.

Rapid screening and quantification of major organic acids in citrus fruits and their bioactivity studies.

Organic acids (OAs) are small non-volatile molecules with widespread usage in processed foods, feeds and instant beverages. The prime aim of this study was to explore major OAs in local citrus fruits (Citrus limetta, Citrus aurantifolia, Citrus nobilis, Citrus karna, Citrus medica, Citrus ichangensis and Citrus aurantium) and assessment of their bioactivities. A RP-HPLC-DAD method was developed using buffer free solvent system for rapid detection and quantification of major OAs from citrus fruits and derived products. Method validation studies showed good linear calibration curve (0.985-0.998) for all OAs. The values of %RSD ranged between 0.0001-1.129 and 0.142-1.941 for interday and intraday variability respectively. The limit of detection and limit of quantification values for different OAs were ranged between 1.5-12 and 5-40µgmL-1. The juice of above mentioned citrus fruit cultivars were assessed for OAs, total phenolics, free radical scavenging antioxidants and their antimicrobial potential against selected bacterial and fungal strains. The results showed variable contents of phenolics [0.28±0.001-1.17±0.014mg (GAE) mL-1] and antioxidant compounds (1.26±0.009-2.84±0.006mg of trolox equivalents mL-1) in all juice samples besides significant antifungal activity against C. albicans and A. niger strains. However, in case of antibacterial activity, only C. aurantifolia showed inhibitory effects against selected strains. It was found that citrus fruits have immense potential for their utilization as economic source of natural OAs and development of value added products, beverages and bio-preservatives.

A method to evaluate the bioactive function of fruit extracts of Chinese wild Citrus with microtubular activity

China is one of the most important centers of origin for Citrus genetic resources. Due to the high content of secondary metabolites, mining wild Chinese Citrus for novel medical applications is promising. In this study, extracts of Chinese wild species from different taxonomical groups were screened for potential effects on microtubules (MTs) in vitro. MT density as a readout for nucleation, and frequency distribution over MT lengths as a readout for elongation and decay were determined by quantitative image analysis via a standardized coverslip assay using fluorescently labelled neurotubulin. Extract from peels of Citrus ichangensis Swing. strongly increased the density of MTs; whereas, extract from peels of Citurs limon (L.) Burm.f. exerted the opposite effect. Extract from pulp of Citrus limonia Osbeck promoted MT elongation, and in addition induced a small population of very long MTs. These data suggest that wild Chinese Citrus harbour compounds that act specifically on different aspects of MT nucleation, elongation, and decay.

Molecular phylogeography and population evolution analysis of Citrus ichangensis (Rutaceae)

Ichang papeda (Citrus ichangensis), a wild and endemic perennial plant in Rutaceae, is characterized by the existence of wild and natural populations in southwestern and middle-west China. We analyzed a total of 231 individuals across 16 natural populations using chloroplast SSR markers, nuclear SSR markers, and single-copy nuclear genes. Standard population genetic analyses as well as Bayesian and maximum likelihood models were used to clarify the genetic diversity, population differentiation, barriers to gene flow, bottleneck events, isolation by distance, history migration, demographic history among populations, and phylogeny evolution. The chloroplast and nuclear genome analyses revealed a low level of genetic diversity in C. ichangensis. Clear signals of recent bottlenecks and strong patterns of isolation by distance were detected among different subpopulations, indicating a low extent of historical gene flow for this species and that genetic drift would occur after population differentiation. Bayesian clustering analyses revealed a clear pattern of genetic structure, with one cluster spanning the potential refugia in Wuling Mountains and Ta-pa Mountains, and other two clusters covering a more limited distribution range. The demographic history also supported the scenario that two isolated clusters originated in parallel from the genetic diversity center. Taxonomically, Ichang papeda may be a member of subgenus Citrus. Owing to the complicated topography, the mountainous regions and the Yangtze River have provided long-term stable habitats for C. ichangensis and acted as main barriers for its expansion, which might facilitate the process of speciation. Statistical population models and genetic data indicated strong genetic structure in C. ichangensis, which might result from the restricted gene flow, genetic drift, and population bottlenecks.

Anaphylaxis to the Citrus Fruit Yuzu

Introduction: We report a selective anaphylaxis to yuzu (Citrus junos). Case Presentation: A 42-year-old woman, with no atopic history, has experienced two anaphylactic reactions after consuming yuzu. Skin prick tests (SPTs) were performed with juice, peel and pulp of commercially available lemon, orange, grapefruit, tangerine and yuzu, 2 cultivars of Citrus junos, 2 cultivars of Citrus reticulata, one Citrus ichangensis and food consumed during the culprit meal.

Effectiveness of aromatherapy in decreasing maternal anxiety for a sick child undergoing infusion in a paediatric clinic

We examined whether aromatherapy involving inhalation of yuzu (Citrus ichangensis×Citrus reticulata) oil was effective in decreasing mothers' anxiety for her sick child receiving an infusion at a paediatric clinic. Controlled clinical trial. Mothers of sick children who arrived at the hospital were asked to complete an anonymous questionnaire. After a doctor examined the child and confirmed the necessity for infusion, the mothers who agreed to participate in our study were allocated to an aromatherapy or a control group. A diffuser was filled with yuzu oil before the subjects entered the aromatherapy room. The mother was shown how to use the aromatherapy diffuser while the child was receiving an infusion in the same room. Fifteen minutes after entering the room, the mothers were asked to complete an another questionnaire. We measured the mother's anxiety with the state anxiety score from the State-Trait Anxiety Inventory. There were 60 subjects in the aromatherapy group and 61 in the control group. Both groups were well balanced in terms of demographic characteristics. Using analysis of variance, we demonstrated a significant difference in two-factor interactions between the control and aromatherapy groups. Maternal state anxiety was significantly lower in the aromatherapy than in the control group. Inhalation of yuzu oil was shown to decrease maternal anxiety for a sick child. A multicentre randomized controlled trial or double-blind study is necessary to obtain objective evidence of this benefit of aromatherapy.

Botanicals in Dietary Supplements

Botanicals are accepted worldwide as medicinal agents and nutraceuticals. Extensive scientific investigations have been performed over the past 200 years which have resulted in the evolution of botanical utilization. This special issue highlights some of this research in 7 review articles and in 24 original research articles. Review Articles. W. Jiratchariyakul and G. B. Mahady wrote about the botanical status and popular herbs in EU, US, and Thailand. M. Miroddi et al. updated the market and the regulatory of botanical products in EU and US. Traditional Chinese and Indian Medicines (TCM and TIM) play an important role in Asian countries. Z. Wang et al. presented and discussed the role of TCM in the treatment of epidemic type II diabetes mellitus. M. M. Pandey et al. reported the use of TIM as a nutritional supplement in malnutrition. Y. Kamisah et al. presented the chemoprevention and antioxidation of Parkia speciosa. A. P. Bartolome et al. reviewed laboratory evidence of Bidens pilosa. T.-P. Huynh et al. discussed the promising botanical compounds for prevention and treatment of eye diseases. Research Articles. New biological and pharmacological activities of botanicals are reported. They included the enhancement of learning, memory and antistress of Acanthopanax trifoliatus (P. Sithisarn et al.), the sedative effect of Ziziphus mauritiana (A. M. M. San et al.), the cardioprotective effect of Phyllanthus emblica (L. Chularojmontri et al.), the cytoprotection and antioxidative stress of Citrus maxima (L. Chularojmontri et al.), anti-influenza viral activity of Momordica charantia (V. Pongthanapisith et al.), antioxidation from Nypa fruticans (N. Prasad et al.), Nigella glandulifera (J. Zhao et al.), TIM (M. M. Pandey et al.), Herba Cynomorii (J. Chen et al.). The alleviation of metabolic disorder of Citrus ichangensis (X. Ding et al.), and anticancer activity of Vitex agnus-castus (S. Li et al.). The mechanisms of anticancer action are deeply investigated with the botanical compounds, zerumbone from Zingiber zerumbet (N. M. Nadzri et al.), phenyl butenoid dimmer from Zingiber cassumunar (T. Anasamy et al.), and girinimbine from Murraya koenigii (S. Mohan et al.). Biochanin A, the major isoflavone from Trifolium pratense, prevented the bone loss in the ovariectomized rat (S.-J. Su et al.). Quercetin isolated from Caesalpinia mimosoides had neuroprotective effect (N. Tangsaengvit et al.) and inhibited eosinophile (M. K. Asano Sakai-Kashiwahara). C. Li et al. reported new anti-inflammatory triterpenoids from Illicium difengpi. Besides the herbal activities, the cohort study of TCM, Si-Wu-Tang, in postpartum women was performed to evaluate the health benefits. The effectiveness of the modern herbal drug was carried out using the double-blind randomized controlled clinical trial, as shown under the title “Antiherpetic effects of Gynura procumbens” (S. Jarikasem et al.). The research on the quality assessment of the botanicals was also presented in this issue. It included the HPLC analysis of Moringa oleifera (B. Vongsak et al.) and Pueraria tuberosa (S. Rastogi et al.). In addition, the arsenic accumulation in Zingiberaceous rhizomes was reported (C. Ubonnuch et al.). Weena Jiratchariyakul Ludger Beerhues Gail B. Mahady Tanawan Kummalue Molvibha Vongsakul

Phylogenetic relationships of Citrus and its relatives based on rbcL gene sequences

We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed.

Segregation for Double Spine Trait in Hybrids of Microcitrus inodora

Several Microcitrus species are valuable as parents in breeding citrus rootstocks and scions because of useful characteristics, such as monoembryony, very short juvenility, short fruit maturation time, and resistance to Phytophthora and nematodes. The large-leaf Australian wild lime, Microcitrus inodora (F. M. Bail.) Swing., also possesses a trait that is generally undesireable in new hybrids, double axillary spines. Most F1 hybrids between M. inodora and single-spine Microcitrus and Citrus species were observed to possess the double spine trait. In crosses of M. australasica (F. Muell.) Swing. × M. inodora, M. papuana H. F. Winters × M. inodora, and M. warburgiana (F. M. Bail.) Tan. × M. inodora, double spines were present in 80%, 81%, and 87% of the progeny, respectively. In hybrids of Citrus species with M. inodora, percentage of double spines varied from 43% in progeny from M. inodora × Citrus aurantium L., to 100% in progeny from M. inodora × Citrus ichangensis Swing. When double-spined hybrids of M. inodora were used as parents in crosses with other single-spined Citrus species or hybrids, progenies varied from 0 to 86% with double spines. The patterns of inheritance suggest that the double spine trait is controlled by an allele of a nuclear gene that is dominant and homozygous in M. inodora, but that one or more other independently segregating alleles are necessary for expression of the double spine trait in advanced generation hybrids.

Limonoids in Miyamashikimi (Skimmia japonica Thunb.).

Miyamashikimi (Skimmia japonica Thunb.) seeds contain the following limonoids that are found in seeds of Citrus species: deacetylnomilin, nomilin, limonin, obacunone, deacetylnomilinic acid, nomilinic acid, isoobacunoic acid; and those found in seeds of Fortunella species: retrocalamin, calamin, cyclocalamin, methyl isoobacunoate diosphenol and isoobacunoate diosphenolic acid; and those found in seeds of Citrus ichangensis: ichangensin. They also contain 17-β-D-glucopyranoside of limonin, nomilin, obacunone, deacetylnomilin, calamin, retrocalamin, cyclocalamin and ichangensin. Skimmia japonica is the only plant which contains all of these limonoids. These data reveal unusual and unexpectedly high levels of citrus-like limonoids which suggest that Skimmia japonica is taxonomically closer to Citrus than previously believed.

Healthy, Precocious, and Fertile Hybrids Obtained from Microcitrus spp. (Citrus ichangensis)

Microcitrus is one of five genera that are partially sexually cross-compatible with the genus Citrus. The genus Microcitrus contains seven species with characteristics that may be valuable for breeding citrus scions and rootstocks, including zygotic embryony, short juvenile period, short fruit maturation time, and resistance to nematodes and Phytophthora. However, relatively few F1 hybrids between Microcitrus and Citrus have been reported, and most of these have been pollen- and ovule-sterile. Some of these intergeneric hybrids have also been highly susceptible to cold damage. To create a genetic bridge for recombination of useful traits from Microcitrus into Citrus, two selections of Citrus ichangensis (an exceptionally cold hardy species with zygotic embryony and short juvenile period) were hybridized with Microcitrus warburgiana and two selections of Microcitrus inodora. Seed were collected from these crosses and germinated in a warm greenhouse. A total of 94 M. inodora xC. ichangensis hybrids and 34 M. warburgiana xC. ichangensis hybrids) were obtained and transplanted to 4-gallon pots in a screenhouse. At 33 months after planting the seed, 42% of the M. inodora × C. ichangensis hybrids and 67% of the M. warburgiana × C. ichangensis hybrids had flowered. Pollen germination tests on agar plates indicated that several hybrids produced large quantities of viable pollen. Numerous crosses were completed using some of these F1 hybrids as pollen and seed parents. Several F1 hybrids were confirmed to be highly fertile by recovery of healthy F2 and backcross hybrids with Microcitrus sp., Citrus sp., Poncirus trifoliata, and other Microcitrus (C. ichangensis) selections.

Limonoid and flavonoid composition in varieties of Papeda and Papedocitrus

The limonoids of the seeds and the flavonoids of the leaves of a number of species of the Citrus sub-genus Papeda were examined. The seeds contained only the normal citrus limonoids and did not contain ichangensin, the unique limonoid found in C. ichangensis. A flavonoid analysis showed that a number of papeda varieties including C. latipes and C. hystrix may be a papeda hybrid species. C. junos had flavanone neoheperidosides, which are also found in cultivars related to the pummelo. All papedas examined had rutin present, which is characteristic of cultivars related to the citron. Citrus ichangensis had the most complex flavone/flavonol pattern of all the papedas examined. This suggests that Citrus ichangensis may truly be a unique papeda species unlike that of the other papedas.

Cold-induced changes in ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase in citrus

The effects of cold acclimation on ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase) were examined in leaves of moderately cold-hardy ‘Valencia’ orange ( Citrus sinensis L. Osbeck) and in two cold-hardy genotypes, ‘Trifoliata’ orange ( Poncirus trifoliata L. Raf.) and ‘FF-6-7-2’ citrus hybrid ( Citrus ichangensis Swing. X Fortunella margarita Lour. Swing.). Soluble protein extracted from leaves and analyzed by electrophoresis showed significant differences in composition among the three citrus genotypes. A 12 kD polypeptide with unknown function was present in large quantity in ‘Valencia’, but not in the other two genotypes. Probing with antibodies specific for Rubisco and PEPCase indicated that ‘Trifoliata’ had two polypeptides of Rubisco small subunit (13 and 14 kD) whereas ‘Valencia’ and ‘FF-6-7-2’ had only one (14 kD). Cold acclimation differentially altered the expression of the two carboxylases. In all three genotypes, cold acclimation resulted in increased amount of Rubisco large subunit, but decreased amount of the small subunit. However, the amount of PEPCase was substantially enhanced by cold acclimation in the cold-hardy genotypes (‘FF-6-7-2’ and ‘Trifoliata’), but was largely reduced by the cold treatment in the moderately cold-hardy genotype (‘Valencia’). The enhanced expression of PEPCase during cold acclimation may be important for the induction of freezing tolerance in citrus.

Limonoids in seeds of three citrus hybrids related to citrus ichangensis

The identity and concentrations of the limonoids present in the seeds of three Citrus ichangensis derived hybrid cultivars, Mochiyu, Yuko and Ichang lemon, were determined. The seeds from Mochiyu and Yuko contained ichangensin, which is unique to the C. ichangensis group. Ichangensin was not contained in the seeds from Ichang lemon; however these seeds contained relatively high levels of deacetylnomilin and deacetylnomilinic acid, which are the putative intermediate precursors of ichangensin. These results support the hypothesis that these three citrus cultivars are derived from C. ichangensis. The seeds of all three cultivars also contained limonoids common to all species of Citrus and its hybrids such as limonin, nomilin, nominilic acid, ichangin, and obacunone.