Mystery of the forbidden fruit: Historical epilogue on the origin of the grapefruit,Citrus paradisi (Rutaceae)

Attempts by the early colonial settlers of Barbados to plant orchards of shaddock (pummelo,Citrus grandis) from seedlings gave rise to the grapefruit(C. paradisi), an apomictic hybrid. Early botanists misidentified the grapefruit as a variety of shaddock, confusing it with a second hybrid growing on Jamaica. The botanist who first named the species, James Macfadyen, is shown here to have described the wrong fruit as a result of such misidentifications. Citrus historians of the 20th century have been unable to confirm the existence of a legendary Captain Shaddock, said to have brought the first seeds of the shaddock to Barbados. The present authors have found a basis for the legend, identifying a Captain Chaddock who traded in the West Indies in the 17th century. In addition, they have rectified the misidentifications of the grapefruit by early botanists that have confused the literature up to the present.     

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)

 Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing β-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis. Received: 2 February 2000 / Revision received: 21 June 2000 / Accepted: 29 June 2000     

An ultrastructural study of two forms of chilling-induced injury to the rind of grapefruit (Citrus paradisi, Macfed).

The ultrastructural manifestations of storage chilling-injury and concentric ring stipple indicate that the two types of injury are similar in some respects and different in others.The low temperature storage-induced injury involves the epidermal cells and several layers of epicarpal cells below. It is manifested as an increase in lipid material in the cytoplasm and vacuole and in eventual degradation and collapse of the cytoplasm. The field injury, concentric ring stipple, apparently involves primarily the epidermal cells which become extremely electron dense. The epicarp cells of the injured region do not show extensive damage but do exhibit increased lipid accumulation. This accumulation of lipid is a phenomenon common to both types of injury and may be indicative of an altered metabolism due to the low temperatures. The apparent loss of organization and compartmentation in the chilling injury is also suggestive of membrane degradation.The storage chilling-injury occurs after exposure to low temperatures for several weeks, whereas the concentric ring stipple is evidently induced by an exposure in the field for only a few hours. The ultrastructural differences observed in these two injuries, therefore, may be due to the different time- and stress- factors involved.     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164     

Development of indel markers from Citrus clementina (Rutaceae) BAC-end sequences and interspecific transferability in Citrus

? Premise of the study: Indel markers were developed from BAC-end sequences of Citrus clementina cv. Nules. Transferability and polymorphism were tested in the Citrus genus to estimate the potential of indel markers mined from a single genotype for use in genetic studies. ? Methods and Results: Using polyacrylamide gel electrophoresis and DNA silver staining, 89 indel markers were tested for their transferability and polymorphism. Thirty-eight markers were selected. Heterozygosity in C. clementina cv. Nules was confirmed for 33 of these indel pairs. A preliminary diversity study using a capillary electrophoresis fragment analyzer was conducted with 21 indels using 45 accessions representing Citrus genus diversity. Intraspecific and interspecific polymorphisms were observed. ? Conclusions: These results indicate the utility of indel markers developed from sequence data of a single genotype of interspecific origin. In Citrus, these markers will be useful for genetic mapping, germplasm characterization, and phylogenetic assignment of DNA fragments.     

Synthetic regulators of carotenoid biosynthesis in Citrus paradisi

The ability of 16 amines to induce carotenoid biosynthesis in Marsh seedless grapefruit is correlated with the octanol-water partition coefficient and the Hammett constants. The compounds fall into three series: p-RC₆H₄COOCH₂CH₂NEt₂ (R = H, NH₂, CN, NO₂, MeO, Me, tert-Bu, F, Cl, Br), p-RC₆H₄CH₂NEt₂ (R = H, Me, NO₂), and RC₆H₄OCH₂CH₂NEt₂ (R = o-Me, m-Me, p-Me). Total carotene content increased up to 12-fold. Lycopene, not normally accumulated, became a major pigment. The benzoates caused up to a 24-fold increase in the β-carotene content. Except for the larger accumulation of cyclic carotenes, the mode of action of these amines appears to be similar to that of 2-(4-chlorophenylthio triethylamine hydrochloride.     

Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability in Citrus

? Premise of the study: Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. ? Methods and Results: Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata, C. maxima, C. medica, C. sinensis, C. aurantium, C. paradisi, C. lemon, C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra- and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. ? Conclusions: These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps.     

Accumulation of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus paradisi,Citrus limonia and Citrus aurantium

The production of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus species is described. The levels of these compounds were examined by gas chromatography-mass spectrometry and their yields were compared with the amounts found in mature fruits. A simultaneous increase and decrease in the levels of nootkatone and valencene, respectively, were observed with the aging of callus cultures of Citrus paradisi. These results suggest that valencene might be a possible precursor of nootkatone in this species. The high level of nootkatone detected in 9-month-old callus cultures of Citrus paradisi might be associated with the corresponding cell morphological changes observed.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FID flame-ionisation detector - FW fresh weight - GLC gas liquid chromatography - K Kinetin - NAA naphthalene acetic acid     

Citrus meletensis (Rutaceae), a new species from the Pliocene of Valdarno (Italy)

There are few fossil remains that are assigned or compared toCitrus. A new and characteristic leaf from the Pliocene of Italy is described asCitrus meletensis and its systematic position within theRutaceae is discussed. Together with other sparse remains that are reviewed here it confirms the existence ofCitrus in the European Tertiary.     

Secondary product glucosyltransferase and putative glucosyltransferase expression during Citrus paradisi (c.v. Duncan) growth and development

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.     

Uptake of 14C-gibberellic acid by mature grapefruit (Citrus paradisi Macf.) as affected by relative humidity and method of application

Effects of the method of application and relative humidity on the uptake of ¹⁴C-gibberellic acid (¹⁴C-GA₃) by mature grapefruit (Citrus paradisi Macf.) were examined. Uptake was higher when ¹⁴C-GA₃ was applied as a 'drying-out' solution than as a 'non-drying' solution. When ¹⁴C-GA, was applied by the 'drying-out' method, which closely imitates field conditions, rates of uptake were very high while the solution was drying out and during the first few hours after drying. Uptake from the dry residue continued in decreasing rates till the end of the experiment (72 h). Uptake from the dry residue was higher when fruits were incubated at 100% than at 50% relative humidity (RH). Transfer of fruits from 50% to 100% RH as late as 48 h after drying still increased the rate of uptake. Drying-out treatment solutions produced higher uptake rates with neutral (pH 7) as well as acid (pH 4) treatment solutions, and in the presence of triton B-1956, Triton X-100 or L-77 surfactants.     

Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.     

Thamnosma (Rutaceae) in Africa

The African members of the remarkably disjunct Afro-American genus Thamnosma are revised. Six species are recognized, T africana, T. rhodesica and T. crenata in southern Africa, and T. somalensis, T. socotrana and T. hirschii in the Horn of Africa region, including the southern part of the Arabian Peninsula and Socotra. T. somalensis , sp. nov., is described from north-eastern Somalia. T. crenata , comb, nov., is based on T. africana var. crenata . A key to the species is given and two lectotypes and one neotype are selected.     

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. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Flavanone-specific 7-O-glucosyltransferase activity in Citrus paradisi seedlings: purification and characterization

The isolation and characterization of a flavanone-specific 7-O-glucosyltransferase and its resolution from other glucosyltransferases in Citrus paradisi (grapefruit) seedlings is described. This new enzyme in the subclass 2.4.1 catalyzes the glucosylation of the 7-OH group of naringenin (4',5',7-trihydroxyflavanone) to prunin and has been purified (943-fold) by fractional precipitation with ammonium sulfate and successive chromatography on Sephadex G-100, hydroxyapatite, UDP-glucuronic acid agarose, Mono Q, and Mono P columns. It has a pH optimum of 7.5-8.0, an apparent pI of 4.3, and an apparent Mr of 54,900. This glucosyltransferase has an expressed specificity for the 7-position of the flavanones naringenin (Kmapp 62 microM; Kmapp UDPG 51 microM) and hesperetin (Kmapp 124 microM; Kmapp UDPG 243 microM) and did not accept other flavone or flavonol aglycones. Characteristics of other flavonoid glucosyltransferase activities found in grapefruit seedlings are also described.     

A Six Nuclear Gene Phylogeny of Citrus (Rutaceae) Taking into Account Hybridization and Lineage Sorting

Background

Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species.

Methodology and Principal Findings

(1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test.

Conclusions

We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.