[Essential oils from the rind of the fruit of Citrus trifoliata L].
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Citrus↗
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Furocoumarins from the seeds of Citrus trifoliata L.
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Chemical Phenomena↗
Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.
Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.
Acclimatization↗
Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress.
The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.
Antioxidants↗
[PONCIRUS TRIFOLIATA AND CITRUS MEYERI--NEW SOURCES OF LIMONIN].
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Chemistry, Pharmaceutical↗
Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.
Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.
Flavanones↗
Sequence analysis of a 282-kilobase region surrounding the citrus Tristeza virus resistance gene (Ctv) locus in Poncirus trifoliata L. Raf.
Citrus tristeza virus (CTV) is the major virus pathogen causing significant economic damage to citrus worldwide, and a single dominant gene, Ctv, provides broad spectrum resistance to CTV in Poncirus trifoliata L. Raf. Ctv was physically mapped to a 282-kb region using a P. trifoliata bacterial artificial chromosome library. This region was completely sequenced to about 8x coverage using a shotgun sequencing strategy and primer walking for gap closure. Sequence analysis predicts 22 putative genes, two mutator-like transposons and eight retrotransposons. This sequence analysis also revealed some interesting features of this region of the P. trifoliata genome: a disease resistance gene cluster with seven members and eight retrotransposons clustered in a 125-kb gene-poor region. Comparative sequence analysis suggests that six genes in the Ctv region have significant sequence similarity with their orthologs in bacterial artificial chromosome clones F7H2 and F21T11 from Arabidopsis chromosome I. However, the analysis of gene colinearity between P. trifoliata and Arabidopsis indicates that Arabidopsis genome sequence information may be of limited use for positional gene cloning in P. trifoliata and citrus. Analysis of candidate genes for Ctv is also discussed.
Amino Acid Sequence↗
Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).
Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.
Acclimatization↗
Refinement of the Citrus tristeza virus resistance gene (Ctv) positional map in Poncirus trifoliata and generation of transgenic grapefruit (Citrus paradisi) plant lines with candidate resistance genes in this region.
Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it's slow spread in R-2 plant lines or an initial appearance of infection and it's subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it's 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed.
Blotting, Northern↗
Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.).
Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F(1) progeny obtained by crossing 'Kiyomi' tangor (C. unshiu x sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F(1) progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.
Agrobacterium tumefaciens↗
Passive cutaneous anaphylaxis-inhibitory activity of flavanones from Citrus unshiu and Poncirus trifoliata.
This study examined the passive cutaneous anaphylaxis-inhibitory activity of the flavanones isolated from the pericarp of Citrus unshiu (Family Rutaceae) and the fruit of Poncirus trifoliata (Family Rutaceae). Naringenin, hesperetin and ponciretin potently inhibited IgE-induced beta-hexosaminidase release from RBL-2H3 cells and the PCA reaction. Among the flavanones examined, naringenin was the most potent with an IC50 value for beta-hexosaminidase release from RBL-2H3 cells of 0.029 mM. Intraperitoneally administered naringenin (5 mg/kg) inhibited PCA by 70 +/- 1.7 %, compared with the control group. The inhibitory activity of naringenin was found to be comparable to that of azelastine, which is a commercially available antiallergic drug. However, their glycosides, hesperidin, naringin and poncirin, did not inhibit the in vitro release of beta-hexosaminidase from the RBL-2H3 cells. On the other hand, these flavanones did not improve the oxazolone-induced dermatitis in the mouse ears. When the flavanone glycosides were administered to rats, the aglycones, but not the flavanone glycosides, were excreted in urine. This suggests that the flavanone glycosides can be activated by intestinal bacteria, and may be effective toward IgE-induced atopic allergies.
Animals↗
Fatty Acid methyl ester profiles for characterization of glomalean fungi and their endomycorrhizae.
Arbuscule-forming fungi in the order Glomales form obligate endomycorrhizal associations with plants that make them difficult to quantify, and taxonomy of the group is only beginning to be objectively understood. Fatty acid methyl ester (FAME) profiles were analyzed to assess the diversity and quantity of fatty acids in 53 isolates of 24 glomalean species. Spores and endomycorrhizal roots of sudan grass (Sorghum sudanense) and the citrus rootstock Carrizo citrange (Poncirus trifoliata x Citrus sinensis) were examined. Spores yielded reproducible FAME profiles from replicate spore collections extracted from soil pot cultures despite being grown in association with a host plant and with contaminating microorganisms present. Unweighted pair group analysis revealed relatively tight clusters of groups at the intraspecific, specific, and generic levels; however, lipid profiles at the family level were convergent. Thus, FAME profile comparisons provided a robust measure of similarity below the family level. FAME profiles in sudan grass roots containing vesicles and/or spores of Glomus intraradices were more similar to spore profiles than to profiles from nonmycorrhizal roots. The FAME profiles for Gigaspora species, which do not form vesicles or spores in roots, were less distinct from nonmycorrhizal roots. G. intraradices and G. rosea produced fatty acids in roots that were distinguishable from each other as well as from the host root. Production in citrus roots of the fatty acid 16:1(inf(omega)5) cis by two Glomus species was correlated with the development of mycorrhizal colonization as measured by clearing and staining procedures and by estimates of total incidence and vesicle intensity. FAME analysis of roots not only provided a measure of colonization development but also served as an index of carbon allocated to intraradical fungal growth and lipid storage.
Journal Article↗
A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.
Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.
Chromosome Mapping↗
Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.
The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.
Amino Acid Sequence↗
Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions.
Citrus fruits are sensitive to low temperatures and this often results in the development of chilling injuries during postharvest storage. In order to gain more insight into the molecular mechanisms involved in the acquisition of fruit chilling tolerance, we initiated a grapefruit (Citrus paradisi, cv. Marsh Seedless) flavedo cDNA sequencing project and used it to identify a cDNA similar to other Poncirus trifoliata and Citrus unshiu dehydrin genes reported to be responsive to low temperatures. The grapefruit dehydrin cDNA, designated cor15, encodes a predicted polypeptide of 15.1 kDa, that is almost completely identical with other reported citrus dehydrin proteins, except that it contains two large amino acid repeats, whereas P. trifoliata COR11 has only one such repeat and P. trifoliata COR19 and C. unshiu COR19 have three repeats. Together, the various grapefruit, P. trifoliata and C. unshiu dehydrins form a closely related and unique dehydrin gene family that differs from most other plant dehydrins in having an unusual K-segment similar to that of gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus. The grapefruit cor15 gene is consistently expressed in the fruit peel tissue at harvest, but its message levels dramatically decrease during storage at 2 degrees C. However, a pre-storage hot water treatment, which enhances fruit chilling tolerance, elicited retention of the constant level of cor15 gene expression during cold storage and eliminated its decline. The hot water treatment had no inductive effect on cor15 gene expression when the fruit were held at non-chilling temperatures. The effects of other stresses, such as exposure to ethylene, UV irradiation and wounding, on cor15 gene expression, were temporary and persisted for 1-2 days after the treatments.
Journal Article↗
Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.).
A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.
Blotting, Southern↗
[Inheritance of organelle genomes of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and flying dragon (Poncirus trifoliata)].
Cleaved Amplified Polymorphic Sequence (CAPS) was successfully applied to analyze the organelle composition of three eight-year-old trees of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and Flying Dragon (Poncirus trifoliata). Five chloroplast and five mitochondrial universal primer pairs were used. All chloroplast primer pairs (rbcL-rbcL, rbcL-PSA I, TrnH-Trnk, TrnD-TrnT, TrnK-TrnK) and three (nad 1 exon B-nad 1 exon C, 18S rRNA-5S rRNA, nad 4 exon 1-nad 4 exon 2) of the five mitochondrial primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eleven restriction endonucleases, including, Hin6 I, Bus RI, Taq I, Msp I, HinfI, AluI, Dra I, EcoR I, Hind III, BamH I and Pst I respectively, three polymorphic cpDNA-CAPS markers (rbcL-rbcL/Hin 6 I, TrnD-TrnT/BusR I, TrnD-TrnT/Taq I) and one mtDNA-CAPS marker (nad 1-nad1/Msp I) were found. The results showed that cpDNA in the somatic hybrid plants came from Flying Dragon, the mesophyll parent, and mtDNA from Cleopatra mandarin, the embryogenic suspension parent uniformly. In order to prove the reliability of CAPS results, and to get more detailed information about the mtDNA inheritance, RFLP analyses was conducted. Genomic DNA of the somatic hybrids and their corresponding parents were digested by five restriction endonucleases (Dra I, EcoR I, Hind III, BamH I and Pst I), and hybridized with five mitochondrial probes (Cob, Pro 2, Pro I, atp 6, 26S rRNA) as well as one chloroplast probe, i.e. the PCR product of Flying Dragon with the primer pair of trnd 1-trnt 1. The results were in line with those of CAPS, and no novel bands were detected, which indicated that no organelle DNA recombination or rearrangement have been detected in the hybrid plants. The research showed that novel pattern of nuclear-mitochondria-chloroplast interaction could be reached via protoplast fusion.
Cell Fusion↗
Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation.
The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.
Citrus sinensis↗