[Show abstract][Hide abstract] ABSTRACT: The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either α-amyrin and/or β-amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the β-amyrin synthases.
[Show abstract][Hide abstract] ABSTRACT: The genomic sequence encoding alpha-farnesene synthase-1 (AFS-1) was amplified from genomic DNA isolated from ‘Royal Gala’ apple (Malus×domestica Borkh.). The genomic sequence consists of six introns and seven exons, which is consistent with Class III terpene synthases.
Four variants of the genomic sequence were amplified. The four variants are based on the presence or absence of a repeat of
two sequences, one found in intron 4 (CAGTTATTTAATT) and the other in intron 5 (TA). Although there were small nucleotide
differences among the three apple cultivars ‘Royal Gala’, ‘Idared’, and ‘Ralls’, these resulted in only two amino acid changes
in the protein sequence, which are unlikely to explain the resistance or susceptibility of an apple cultivar to superficial
scald. Given that AFS-1 transcript levels are high in all cultivars, it appears that it is either the reactions downstream
of alpha-farnesene production that control the accumulation of oxidation products related to superficial scald or that the variation
in the level of its substrate, farnesyl diphosphate, may cause differences in the amount of alpha-farnesene produced.
Frontiers of Agriculture in China 03/2010; 4(1):74-78. DOI:10.1007/s11703-009-0091-1
[Show abstract][Hide abstract] ABSTRACT: Kiwifruit (Actinidia spp. Lindl.) flowers and fruit contain many compounds of interest to the flavour and fragrance industries. In particular, Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. flowers produce -linalool and important derivatives thereof, including linalool oxides, lilac aldehydes, alcohols and alcohol epoxides. Dynamic headspace sampling of whole A. arguta flowers showed that the peak emission rate of linalool, lilac alcohols and lilac aldehydes occurred around 0800hours. After solvent extraction, linalool levels remained constant throughout the day and night, but lilac alcohol levels peaked at noon. In whole flowers, linalool was found predominantly in pistils and petals, and the lilac compounds were found mainly in petals. Two highly homologous (96.6% nucleotide identity) terpene synthase cDNA sequences, AaLS1 and ApLS1, were isolated from A. arguta and Actinidia polygama (Sieb. et Zucc.) Maxim flower EST libraries respectively. Real-time PCR analysis revealed that AaLS1 was expressed constitutively throughout the day and night, and primarily in petal tissue. Functional analysis in Escherichia coli showed that AaLS1 and ApLS1 each encoded a linalool synthase which was confirmed by transient expression in planta. Enantioselective gas chromatography revealed that both terpene synthases produced only (S)-(+)-linalool. AaLS1, therefore, is likely to be the key enzyme producing the (S)-linalool precursor of the lilac alcohols and aldehydes in A. arguta flowers.
[Show abstract][Hide abstract] ABSTRACT: Kiwifruit vines rely on bees for pollen transfer between spatially separated male and female individuals and require synchronized flowering to ensure pollination. Volatile terpene compounds, which are important cues for insect pollinator attraction, were studied by dynamic headspace sampling in the major green-fleshed kiwifruit (Actinidia deliciosa) cultivar 'Hayward' and its male pollinator 'Chieftain'. Terpene volatile levels showed a profile dominated by the sesquiterpenes alpha-farnesene and germacrene D. These two compounds were emitted by all floral tissues and could be observed throughout the day, with lower levels at night. The monoterpene (E)-beta-ocimene was also detected in flowers but was emitted predominantly during the day and only from petal tissue. Using a functional genomics approach, two terpene synthase (TPS) genes were isolated from a 'Hayward' petal EST library. Bacterial expression and transient in planta data combined with analysis by enantioselective gas chromatography revealed that one TPS produced primarily (E,E)-alpha-farnesene and small amounts of (E)-beta-ocimene, whereas the second TPS produced primarily (+)-germacrene D. Subcellular localization using GFP fusions showed that both enzymes were localized in the cytoplasm, the site for sesquiterpene production. Real-time PCR analysis revealed that both TPS genes were expressed in the same tissues and at the same times as the corresponding floral volatiles. The results indicate that two genes can account for the major floral sesquiterpene volatiles observed in both male and female A. deliciosa flowers.
[Show abstract][Hide abstract] ABSTRACT: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs).
The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified.
This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.
[Show abstract][Hide abstract] ABSTRACT: The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.
[Show abstract][Hide abstract] ABSTRACT: A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.
[Show abstract][Hide abstract] ABSTRACT: Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (∼24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.
[Show abstract][Hide abstract] ABSTRACT: Linalool is an important chiral compound in the fragrance industry and is present in many products. Although, linalool has also been found in the fruit of kiwifruit and apple it is more abundant in the flowers, where it plays a key role as an intermediate to a number of interesting fragrance compounds. Three genes found to catalyse the production of linalool from geranyl diphosphate, have been mined from the HortResearch Plant EST database. The function of these genes has been proven using heterologous over-expression technologies. The similarities and differences between our genes and those already published are highlighted. Finally, we show the diversity in the fate of linalool in species of kiwifruit and apple, with discussion of the genes involved.
Developments in Food Science 12/2006; 43:93-96. DOI:10.1016/S0167-4501(06)80022-1
[Show abstract][Hide abstract] ABSTRACT: The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5'-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus x domestica Borkh). We sequenced cDNA libraries of cultivar 'Royal Gala' and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis -a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in 'Royal Gala', particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.
[Show abstract][Hide abstract] ABSTRACT: Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.
Transgenic Research 01/2004; 12(6):671-81. DOI:10.1023/B:TRAG.0000005103.83019.51 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Minor modifications were made sequentially to the nucleotide sequence of truncated cry1Ac9 to produce cry1Ac9A (one nucleotide change) and then cry1Ac9B (seven nucleotide changes). The derivative genes under the control of the CaMV 35S promoter were transformed into Nicotiana tabacum in order to determine whether these modified genes conferred resistance on the resulting transgenic tobacco plants to larvae of the potato tuber moth (Phthorimaea operculella). Over two trials with PTM larvae on the transgenic plants expressing the cry1Ac9B gene, lower larval growth, development and survival was evident for most of the lines compared to the control plants. In the second trial, for four of these lines (7, 25, 26 and 28) larval growth rates were very low (0.28, 0.3, 0.42 and 0.28, respectively) compared to the control growth rate (4.18) and leaf damage was minimal. Northern analysis and RT-PCR analysis showed that higher levels of cry1Ac9 mRNA were present in the transgenic tobacco lines containing cry1Ac9B than in the tobacco lines containing cry1Ac9A. These results suggest that certain minor modifications to the nucleotide sequence of cry1Ac9 are sufficient to improve the stability of its mRNA when expressed in tobacco and that this increase in steady state mRNA is sufficient to confer significant resistance to PTM larvae.
[Show abstract][Hide abstract] ABSTRACT: The rDNA internal transcribed spacer (ITS) region of 4 mealybug species, Pseudococcus viburni (Signoret), P. longispinus (Targiono-Tozzetti), P. calceolariae (Maskell), and P. similans (Lidgett), was isolated by polymerase chain reaction (PCR) amplification, cloned, and sequenced. In this region of the genome there were numerous differences, including nucleotide substitutions, insertions, or deletions between P. viburni, P. longispinus, and P. calceolariae, whereas P. calceolariae and P. similans were very similar. Based on sequence differences between the ITS regions, we designed PCR primers that were able to differentiate the 4 mealybug species and that correlated with morphological differences found between adult females of these species. The PCR amplification by using the species-specific primers enabled the differentiation of not only adult females but also eggs, juveniles, and adult males, which was not previously possible by using conventional identification methods.
[Show abstract][Hide abstract] ABSTRACT: The Bacillus thuringiensis cry9Aa2 gene encodes a 129 kDa protein with insecticidal activity against Lepidoptera, including the larvae of potato tuber moth (Phthorimaea operculella). The insecticidal moiety of Cry9Aa2 resides within the N-terminal 665 amino acids. Site-directed mutagenesis was used to modify a truncated version of the gene (cry9Aa2(T) nucleotides 1-1995), removing motifs likely to be deleterious to full-length transcription and transcript stability in plants. The codon usage of the gene was also altered to that more similar to the codon bias of dicotyledonous plant genes. The native gene and three modified versions of cry9Aa2(T), with incremental modifications from the 5' end, were each transformed into Nicotiana tabacum, under the control of the CaMV 35S promoter. Plants transformed with the native gene did not show resistance to potato tuber moth larvae. In contrast, significant levels of larval mortality and reductions in larval growth and leaf damage were observed on many of the plants transformed with the modified genes. The cry9Aa2(T) mRNA was barely detectable in plants transformed with the native gene, whereas significant accumulation of full-length cry9Aa2(T) transcript was seen in plants transformed with modified genes. Modifications in the 5'-terminal 693 nucleotides of cry9Aa2(T) had the most significant effect on increasing the steady-state levels of cry mRNA. Transcription initiation rates of both the native and modified cry9Aa2(T) genes were similar, suggesting that the lack of native transcript accumulation was a consequence of transcript instability and that the sequence modifications had significantly improved the stability of the cry9Aa2(T) transcript. This improvement in steady-state full-length transcript levels resulted in expression of the insecticidal gene in N. tabacum to levels which conferred significant resistance to potato tuber moth larvae.
[Show abstract][Hide abstract] ABSTRACT: Anthocyanin levels and the expression of six genes involved in anthocyanin
biosynthesis (PAL, CHS,
DFRand ANS) were studied during
apple (Malus domestica Borkh.) flower development. In
the petal, maximal accumulation of the six mRNAs occurred at an early stage of
flower development and then declined rapidly following petal expansion. During
petal development, the highest levels of CHI enzymatic activity and
anthocyanin concentration appeared about one day after maximum mRNA levels of
the six genes. Blocking UV or natural light (dark treatment) before flower bud
break reduced the expression of the six genes and inhibited anthocyanin
biosynthesis, resulting in either pink (UV block treatment) or pure white
(dark treatment) apple flowers. Furthermore, the pure white flowers (dark
treatment) were unable to resynthesise anthocyanins, even if they were
re-exposed to light or placed under UV-B plus white light
in vitrofollowing stage I of flower development. These
results suggest that anthocyanin biosynthesis and the activities of these
genes in the developing apple flower are controlled by both development and
light and that the key stage for the photoregulation is during the early
stages of development.
[Show abstract][Hide abstract] ABSTRACT: A protein phylogenetic tree was constructed from 24 homologous proteinase inhibitor I sequences identified in the EMBL/Genbank and Swiss-Prot databases and from translated amino acid data from four constitutive cDNA clones of proteinase inhibitor I characterized from potato tuber mRNA. The tree suggests that divergence of at least four paralogous proteins with functional specialization occurred at different times during the evolutionary history of the proteinase inhibitor I family. Five distinct regions in the primary structure, earlier identified by structural studies, were used to analyze the inhibitor family for hypervariability (Creighton and Darby, Trends Biochem Sci 14:319-324, 1989). Mutations did not occur with higher-than-random frequency within the proteinase binding region. When isoinhibitor, orthologous, or paralogous data subsets were subsequently analyzed the same results were obtained. Comparison of the amino acid sequences for all the known potato proteinase isoinhibitor I proteins identified ten highly variable sites. These also were distributed randomly. Thus hypervariability, which has been observed in all other serine proteinase inhibitor families to date, appears to be lacking in the proteinase inhibitor I family.