Flavonoid genes of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>)

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.     

<Emphasis Type="Italic">S</Emphasis>-Allele characterization in self-incompatible pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.)

Gametophytic self-incompatibility, a natural mechanism occurring in pear and other fruit-tree species, is usually controlled by the S-locus with allelic variants ( S1, S2, Sn). Recently, biochemical and molecular tools have determined the S-genotype of cultivars in various species. The present study determined the S-locus composition of ten European pear cultivars via S-PCR molecular assay, thereby obviating time-consuming fieldwork whose results are often ambiguous because of environmental effects. To verify the S-PCR assay, two putative S-allele DNA fragments of Japanese pear were isolated; their sequences proved to be identical to those reported in the databank. Six S-allele fragments of European pear were then sequenced. While field data confirmed the molecular results, fully and half-compatible field crosses were not distinguishable.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Differential expression of ferritin genes in response to abiotic stresses and hormones in pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>)

In this study, the expression patterns of four ferritin genes (PpFer1, PpFer2, PpFer3, and PpFer4) in pear were investigated using quantitative real-time PCR. Analysis of tissue-specific expression revealed higher expression level of these genes in leaves than in other tested tissues. These ferritin genes were differentially expressed in response to various abiotic stresses and hormones treatments. The expression of ferritin wasn’t affected by Fe(III)-citrate treatment. Abscisic acid significantly enhanced the expression of all four ferritin genes, especially PpFer2, followed by N-benzylyminopurine, gibberellic acid, and indole-3-acetic acid. The expression peaks of PpFer1 and PpFer3 in leaves appeared at 6, 6, and 12 h, respectively, after pear plant was exposed to oxidative stress (5 mM H₂O₂), salt stress (200 mM NaCl), and heat stress (40°C). A significant increase in PpFer4 expression was detected at 6 h after salt stress or heat stress. The expression of ferritin genes was not altered by cold stress. These results suggested that ferritin genes might be functionally important in acclimation of pear to salt and oxidative stresses. Hormone treatments had no significant effect on expression of ferritin genes compared to abiotic stresses. This showed accumulation of ferritin genes could be operated by different transduction pathways under abiotic stresses and hormones treatments.     

Genome-wide analysis and characterization of molecular evolution of the <Emphasis Type="Italic">HCT</Emphasis> gene family in pear (<Emphasis Type="Italic">Pyrus bretschneideri</Emphasis>)

Lignin is a major component of stone cells in pear fruit, which significantly affects fruit quality. Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), a recently discovered enzyme in plants, is an important gene that participates in the formation of lignin. Although HCT gene cloning and expression patterns have been studied in several species, including pear, there is still no extensive genome-wide bioinformatics analysis on the whole gene family, and the evolutionary history of HCT gene family is still unknown. A total of 82 HCT genes were identified in pear, most of which have one or two exons, and all with the conserved HXXXD motif and transferase domains. Based on the structural characteristics and phylogenetic analysis of these sequences, the HCT gene family genes could be classified into four main groups. Structural analysis also revealed that 25 % of HCT genes share a MYB binding site. Expansion of the HCT gene family mostly occurred before the divergence between Arabidopsis and Rosaceae, with whole-genome duplication or segmental duplication events playing the most important role in the expansion of the HCT gene family in pear. At the same time, purifying selection also played a critical role in the evolution of HCT genes. Five of the 82 HCT genes were verified by qRT-PCR to correspond to the pattern of stone cell formation during pear fruit development. The genome-wide identification, chromosome localization, gene structures, synteny, and expression analyses of pear HCT genes provide an overall insight into HCT gene family and their potential involvement in growth and development of stone cells.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.