<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.     

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.     

High-efficiency and stable genetic transformation of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) leaf segments and regeneration of transgenic plants

Pear (Pyrus communis L.) is a nutrient-dense fruit with strong consumer demand and high commercial value. However, most cultivated pear varieties are often susceptible to diseases caused by fungi, bacteria, and viruses. The purpose of the present study was to establish an efficient genetic transformation and regeneration protocol, paving the way for genetic engineering of pear cultivars with enhanced disease resistance. Major factors that influence transformation and regeneration were examined and optimal conditions were established for efficient transformation from leaf explants of ‘Old Home’, a valuable pear interstem and rootstock. High transformation efficiency was achieved largely due to an improved infection/transformation induction strategy. Co-cultivation of Agrobacterium cells and leaf segments on a liquid induction medium yielded a fivefold increase in transformation frequency. Southern hybridization analysis revealed presence of reporter gene uidA in the genomic DNA samples from independent transgenic plants, confirming the integration of the transgene in recipient pear genomes. The stability of T-DNA integration was evaluated by the consistent presence of the Km selectable marker and the expression pattern of the introduced reporter gene uidA was analyzed by GUS histochemical assay.     

Quantitative trait loci controlling vegetative propagation traits mapped in European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.)

The ease of vegetative propagation by hardwood cuttings is a critical trait for consideration by breeders of woody perennial rootstocks. This is especially so for Pyrus, because most Pyrus rootstock are known to be difficult to propagate. This report presents progress on the identification of loci controlling rooting of hardwood cuttings in European pear (Pyrus communis L.). Quantitative trait loci (QTLs) controlling the development of adventitious roots on hardwood cuttings were identified in both parents of a mapping population developed by crossing “Old Home” and “Louise Bonne de Jersey,” with the goal of investigating the genetic control of several rootstock related traits, which would be useful for rootstock breeding. A QTL for root development was identified on chromosome 7, co-located in both parents and exhibiting male and female additive and dominance effects. These results will assist in developing genetic markers that can be utilized by rootstock breeders for marker-assisted selection for this complex trait.     

Identifying QTLs for fire-blight resistance via a European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) genetic linkage map

The existence of different levels of susceptibility to fire blight (Erwinia amylovora) in European pear (Pyrus communis L.) cultivars suggests that it is possible to identify QTLs related to resistance in pear germplasm. Given the polygenic nature of this trait, we designed two genetic maps of the parental lines 'Passe Crassane' (susceptible) and 'Harrow Sweet' (resistant) using SSRs, MFLPs, AFLPs, RGAs and AFLP-RGAs markers. RGA-related markers should theoretically map in chromosome regions coding for resistance genes. The 'Passe Crassane' map includes 155 loci, for a total length of 912 cM organised in 18 linkage groups, and the 'Harrow Sweet' map 156 loci, for a total length of 930 cM divided in 19 linkage groups; both maps have a good genome coverage when compared to the more detailed apple maps. Four putative QTLs related to fire blight resistance were identified in the map. A suite of molecular markers, including two AFLP-RGAs, capable of defining resistant and susceptible haplotypes in the analysed population was developed.     

<Emphasis Type="Italic">GhDRIN</Emphasis>1, a novel drought-induced gene of upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) confers abiotic and biotic stress tolerance in transgenic tobacco

A novel stress tolerance cDNA fragment encoding GhDRIN1 protein was identified and its regulation was studied in cotton boll tissues and seedlings subjected to various biotic and abiotic stresses. Phylogenetic and conserved domain prediction indicated that GhDRIN1 was annotated with a hypothetical protein of unknown function. Subcellular localization showed that GhDRIN1 is localized in the chloroplasts. The promoter sequence was isolated and subjected to in silico study. Various cis-acting elements responsive to biotic and abiotic stresses and hormones were found. Transgenic tobacco seedlings exhibited better growth on amended MS medium and showed minimal leaf damage in insect bioassays carried out with Helicoverpa armigera larvae. Transgenic tobacco showed better tolerance to water-deficit and fast recovered upon rewatering. Present work demonstrated that GhDRIN1, a novel stress tolerance gene of cotton, positively regulates the response to biotic and abiotic stresses in transgenic tobacco.     

Endophytic yeasts in <Emphasis Type="Italic">Malus domestica</Emphasis> and <Emphasis Type="Italic">Pyrus communis</Emphasis> fruits under anthropogenic impact

Yeast abundance and species diversity on the surface and in inner tissues of Malus domestica and Pyrus communis fruits under high anthropogenic impact in the city of Moscow (Russia) were studied. Results demonstrated that abundance of epiphytic yeasts on the fruits increased gradually, reaching the maximum of 3.2 × 10⁴ CFU/g on mature fruits. During summer, abundance of endophytes did not change significantly (variation near 2.5 × 10³ CFU/g) until complete maturation, while in September their numbers increased to 10⁴ CFU/g. Basidiomycetous yeasts (Filobasidium wieringae, F. magnum, Rhodotorula glutinis, and Rhodosporidiobolus colostri) predominated on the fruit surface. Ascomycetous species were the most diverse group inside the fruits, which quantitatively increased through maturation. It was found that the share of opportunistic species Candida parapsilosis in internal tissues was significant during the entire period of fruit formation and development under anthropogenic impact in the city. Specific properties of epiphytic and endophytic yeast communities developing in natural ecological niches under synanthropic conditions and anthropogenic impact are discussed.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Effective biotic elicitation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. shoot cultures by lysates from <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 μg g⁻¹ dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 μg g⁻¹ dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 μg g⁻¹ dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of γ-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 μg g⁻¹ dry wt, respectively.     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

<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.     

Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Construction and functional analysis of pathogen-inducible synthetic promoters in <Emphasis Type="Italic">Brassica napus</Emphasis>

In this study, we selected two known pathogen-inducible cis-acting elements, F and E17, to construct synthetic pathogen-inducible promoters for analysis in transformed canola (Brassica napus L.). The synthetic promoter approach was used, which involved the insertion of dimers and combining two cis-acting elements (E17 and F) upstream of the minimal CaMV 35S promoter. Canola plants were transformed by three constructs, pGEE, pGFF, pGFFEE containing synthetic promoters (SP), SP-EE, SP-FF and SP-FFEE, respectively. Analyses of histochemical and fluorometric GUS expression indicated that synthetic promoters responded to fungal elicitors and phytohormone treatments. The SP-FF promoter showed high responses against methyl jasmonate and Sclerotinia sclerotiorum, while SP-EE demonstrated inducibility only in response to salicylic acid and Rhizoctonia solani. The SP-EE promoter similar to SP-FFEE, did not respond to S. sclerotiorum and methyl jasmonate. However, SP-FFEE was highly induced by R. solani elicitors and showed that the level of GUS expression was greater than that by either of E17 or F elements alone. These three synthetic promoters did not activate the expression of the reporter gene in response to cold, heat, UV and wounding.