Masayuki Kita

A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds

RNA isolation is a prerequisite to the study of gene expression at the molecular level and has an increasingly important role in physiological and genetic investigations of fruit trees. However, RNA isolation is difficult in fruit trees that contain large amounts of polysaccharides and polyphenol compounds. Until now, no commercial kit has been developed specifically for, the isolation of high-quality RNA from fruit trees. Furthermore, because of the large amounts of polysaccharides and polyphenol compounds, the common protocols for RNA isolation are tedious and usually result in poor yields when applied to kiwifruit, apples, and peaches. Here we describe a simple method for RNA islation from fruit trees. This procedure involves easy grinding by zirconia balls; washing the fruit samples before extraction; extraction of RNA; and removal of proteins, polyphenols, and polysaccharides by insoluble polyvinylpyrrolidone (PVP) and potassium acetate with ethanol. The protocol results in high-quality RNA, as evidenced by performing reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis.

Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus

We isolated a cDNA clone encoding limonoid UDP‐glucosyltransferase (limonoid GTase) from the albedo of Satsuma mandarin (Citrus unshiu Marc.) and investigated the contribution to limonoid glucoside accumulation in fruit. The isolated cDNA clone (CitLGT) was 1732 bp in length encoding 511 deduced amino acids with a predicted molecular mass of 57.5 kDa. The products of in vitro translation from an expression vector had the limonoid GTase activity. Southern blot analysis of genomic DNA indicated that CitLGT was present as a single copy gene in the Citrus genome. The amount of transcript corresponding to CitLGT mRNA changed the same way as the fluctuation of limonin glucoside content during fruit development of navel orange (Citrus sinensis Osb.). This indicates that the transcription of CitLGT regulates the conversion of limonoid aglycones to glucosides in citrus fruit.

Characterization of genomic sequence showing strong association with polyembryony among diverse Citrus species and cultivars, and its synteny with Vitis and Populus

Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony.

Expressed sequence tags of ovary tissue cDNA library in Citrus unshiu Marc

To characterize the gene expression and regulation associated with anthesis stage of fruit development a total of 824 clones were randomly selected and sequenced from a cDNA library prepared from ovary tissue of Satsuma mandarin (Citrus unshiu Marc) at the anthesis stage. Clones derived from rDNA, mitochondrial and chloroplastic DNAs were removed and the remaining 544 cDNA clones were analyzed for homology with known sequences. From this analysis 455 clones were identified as homologues of a known gene or an expressed sequence tag (EST). We found 103 cDNA clones that were not detected in our previous analyses of Citrus ESTs including gene homologues related to transcription factors, molecular chaperones, and plant hormone biosynthesis/regulation. In addition, gene homologues related to amino acid biosynthesis, secondary metabolism and stress response were frequently detected, indicating that the ovary tissue may be metabolically active and have the intrinsic ability to react to biotic and abiotic stresses.

Refinement of cDNA Clone Expression Analysis in Random Sequencing from the Rapid Cell Development Phase of Citrus Fruit

Summary To characterize citrus fruit development, we analyzed changes in the levels of mRNAs in nine clones that included two glycine-rich protein (GRP) genes, an expressed sequence tag (EST) clone (EST1), and six putative stress-responsive genes (pcMFR1616.41, pcMFR1804.77, pcMFR1727.28, pcMFRI729.82, pcMFRI729.148 and pcMFR1804.36) during citrus development. GRPs were of two types; one clone (CitGRPI) showed a high degree of identity to typical GRP genes, the other (CitRNA-GRP3) resembled the genes of glycine-rich RNA-binding proteins. The CitGRP 1 transcript level was high in fruitlets, flowers, and mature leaves, but decreased to low levels in the fruit during later development stages. The CitRNA-GRP3 transcript was present in all the plant organs, except mature leaves. The EST 1 transcript level was maximal in the early development stage. Expression of the six putative stress-responsive genes also was regulated distinctly, both temporally and spatially. Collectively, the findings suggest that these genes have important roles in the normal development and enlargement of citrus fruit and that they are regulated by a wide range of internal and external stimuli.

Changes in the levels of mRNAs for putative cell growth-related genes in the albedo and flavedo during citrus fruit development

Abstract Changes in mRNA levels for the seven gene homologues to endoxyloglucan transferase-related protein, expansin, extensin, β-1,3 glucanase, glycine-rich protein, pectinacetylesterase and pectinesterase, which were obtained by random sequencing studies, were investigated in relation to rind development in citrus (Citrus unshiu Marc.) fruit. Expression patterns in the albedo and flavedo were classified into four types: Type-I, transcript levels low in early fruit development but increased at the ripening stage; Type-II, transcript levels high until mid-development and then decreased towards ripening; Type-III, detectable transcript limited to fruitlets at 26 days after flowering (DAF); Type-IV, ubiquitous during the development. Based on the expression patterns, we discuss the possible roles of these genes in rind development.

Molecular cloning of a homologue of dad-1 gene in citrus: distinctive expression during fruit development.

A cDNA homologue to the human defender against apoptotic death gene (dad-1), which is involved in programmed cell death, was isolated from satsuma mandarin (Citrus unshiu Marc.) fruit. It (Citdad-1-1) was 345 bp long, with a deduced protein sequence of 115 amino acids. Southern hybridization suggests that dad-1-related sequences are present as a small gene family in the citrus genome. Expression of Citdad-1-1 was progressively down-regulated in leaves as they matured, but not in juice sac/segment epidermis (edible part) towards fruit ripening. The role of dad-1 during citrus development is also discussed.

Characterization of a cDNA homologous to carotenoid-associated protein in citrus fruits

A cDNA (CitPAP) homologous to a gene encoding for Cucumis sativus carotenoid-associated protein (CHRC) has been isolated from satsuma mandarin (Citrus unshiu Marc.). Unlike ChrC whose expression was limited only in mature fruits (containing chromoplasts), CitPAP transcripts were detected in all the tissues examined including fruits, flowers and leaves. In this respect, CitPAP was rather close to a gene encoding for pepper plastid-lipid-associated protein (PAP), which exhibits ubiquitous expression in bell pepper organs containing chloroplasts or chromoplasts. CitPAP, however, differed from PAP in the magnitude and pattern of RNA accumulation. These results might indicate a novel function of CitPAP.

Allelic structures of UDP-glucose:limonoid glucosyltransferase affect limonoid bitterness in Citrus unshiu and C. sinensis

To develop a molecular indicator for determining the level of limonoid bitterness in Satsuma mandarin (Citrus unshiu) and navel orange (C. sinensis), the genetic background of the transcription of the limonoid glucosyltransferase gene was characterized in relation to the accumulation of non-bitter limonoid glucosides. Two types of cDNA clones (CitLGT-1 and CitLGT-2) encoding limonoid glucosyltransferase were isolated and characterized. The relationships between the gene expression patterns of CitLGTs and the non-bitter limonoid glucoside accumulation as well as the allelic structures of CitLGTs were determined. Southern blot analysis and segregation analysis of the progenies between Satsuma mandarin and tang or (C. unshiu × C. sinensis) indicated that CitLGT-1 and CitLGT-2 are transcribed from the alleles in a single locus. The genotype of the CitLGT locus in navel orange is homozygous for CitLGT-1 (CitLGT-1/CitLGT-1), whereas that in Satsuma mandarin is heterozygous for CitLGT-1 and CitLGT-2 (CitLGT-1/CitLGT-2). The levels of non-bitter limonoid glucosides in navel orange fruit were quite low at the early- to mid-developmental stage due to a defective CitLGT-2, whereas both non-bitter limonoid glucoside content and CitLGT-2 expression in Satsuma mandarin fruit were high throughout fruit development. These results indicate that the expression of CitLGT-2 is a prerequisite for the accumulation of non-bitter limonoid glucosides at the early- to mid-developmental stage of fruit. The allelism of the two CitLGTs, that is, whether the CitLGT-2 is present or not, is a useful molecular indicator for predetermining the levels of accumulation of non-bitter limonoid glucosides at the early- to mid-developmental stages of Satsuma mandarin and navel orange fruits.

Effect of jasmonates on ethylene biosynthesis and aroma volatile emission in Japanese apricot infected by a pathogen (Colletotrichum gloeosporioides).

The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.