A candidate for Lr19, an exotic gene conditioning leaf rust resistance in wheat

Lr19, one of the few widely effective genes conferring resistance to leaf rust in wheat, was transferred from the wild relative Thinopyrum ponticum to durum wheat. Since Lr19 confers a hypersensitive response to the pathogen, it was considered likely that the gene would be a member of the major nucleotide-binding site (NBS)-leucine-rich repeat (LRR) plant R gene family. NBS profiling, based on PCR amplification of conserved NBS motifs, was applied to durum wheat–Th. ponticum recombinant lines involving different segments of the alien 7AgL chromosome arm, carrying or lacking Lr19. Differential PCR products were isolated and sequenced. From one such sequence (AG15), tightly linked to Lr19, a 4,121-bp full-length cDNA was obtained. Its deduced 1,258 amino acid sequence has the characteristic NBS-LRR domains of plant R gene products and includes a coiled-coil (CC) region typical of monocots. The genomic DNA sequence showed the presence of two exons and a short intron upstream of the predicted stop codon. Homology searches revealed considerable identity of AG15 with the cloned wheat resistance gene Pm3a and a lower similarity with wheat Lr1, Lr21, and Lr10. Quantitative PCR on leaf-rust-infected and non-infected Lr19 carriers proved AG15 to be constitutively expressed, as is common for R genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and formation of extracellular matrix

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of α-,ω-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of α-,ω-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.     

Production of Perennial Vegetation in an Oasis-desert Transition Zone in NW China - Allometric Estimation,and Assessment of Flooding and Use Effects

River oases at the southern fringe of the Taklamakan desert in NW China are surrounded by belts of spontaneous vegetation that protect the oases from sand drift. As an important source of forage, fuel and construction wood, this foreland vegetation is also a component part of the agricultural system of the oases but has been, and still is, destroyed through overuse. Within a broader study that aimed to provide a basis for a sustainable management of this foreland vegetation, biomass and production were studied in four vegetation types dominated either by Alhagi sparsifolia, Calligonum caput-medusae, Populus euphratica, or Tamarix ramosissima that were thought to occur under different regimes of natural flooding in the foreland of Qira (Cele) oasis, Xinjiang, NW China. Shoot biomass components were closely correlated to basal area (Calligonum, Populus, Tamarix) or shrub volume and projection area (Alhagi), enabling non-destructive estimation of stand biomass from shoot diameters or shrub dimensions with sufficient precision using allometric regression equations. Relationships between shoot basal area and biomass of the woody species (Calligonum, Populus and Tamarix) agreed with predictions by a theoretical model of plant vascular systems, suggesting that they are determined by hydraulic and mechanical requirements for shoot architecture. Average aboveground biomass densities of typical stands in late summer were 2.97 Mg/ha in Alhagi, 3.6 Mg/ha in a row plantation and 10.9 Mg/ha in homogenous stands of Calligonum, 22–29 Mg/ha in 22 year-old Populus forests and 1.9–3.1 Mg/ha in Tamarix-dominated vegetation. Annual aboveground production including wood and assimilation organs ranged from 2.11 to 11.3 Mg/ha in plantations of Calligonum, 3.17 to 6.12 Mg/ha in Populus, and 1.55 to 1.74 Mg/ha (based on total ground area) or 3.10 to 7.15 Mg/ha (in homogenous stands) in Tamarix. Production of Alhagi is equal to peak biomass. A thinning treatment simulating use by the local population enhanced productivity of Calligonum, Populus and Tamarix. A complete harvest of Alhagi in late August decreased production in the following year. An artificial flood irrigation treatment did not sufficiently increase soil water content except in the uppermost layer and had no clear beneficial effect on growth of the four species and even a negative effect on Alhagi, which was due to increased competition from annual species. As biomass and production with or without artificial irrigation were much higher than values expected for rain-fed desert vegetation at a mean annual precipitation of 35 mm, it is concluded that the existence of all vegetation types studied is probably based on permanent access to groundwater and that natural floods or precipitation do not contribute to their water supply. The effects of agricultural groundwater use in the oasis on groundwater in the foreland of the oasis need further study. Sustainable use of this productive vegetation is possible but requires proper management.     

UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Geographic variations in underwater male Weddell seal Trills suggest breeding area fidelity

Adult Weddell seals (Leptonychotes weddellii) exhibit site fidelity to where they first breed but juveniles, and perhaps transient adult males, may disperse from their natal location. If there is mixing between adjacent breeding groups, we would expect that common vocalizations would exhibit clinal patterns. Underwater Trill vocalizations of male Weddell seals at Mawson, Davis, Casey, McMurdo Sound, Neumayer and Drescher Inlet separated by ca. 500 to >9,000 km, were examined for evidence of clinal variation. Trills are only emitted by males and have a known territorial defense function. Trills from Davis and Mawson, ca. 630 km apart, were distinct from each other and exhibited the greatest number of unique frequency contour patterns. The acoustic features (duration, waveform, frequency contour) of Trills from Neumayer and Drescher Inlet, ca. 500 km apart, were more distinct from each other than they were from the other four locations. General Discriminant Analysis and Classification Tree Analysis correctly classified 65.8 and 76.9% of the Trills to the correct location. The classification errors assigned more locations to sites >630 km away than to nearest neighbours. Weddell seal Trills exhibit geographic variation but there is no evidence of a clinal pattern. This suggests that males remain close to single breeding areas throughout their lifetime.     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Ornamental traits modification by Rol genes in Osteospermum ecklonis transformed with Agrobacterium tumefaciens

Summary Transgenic plants of Osteospermum ecklonis were produced by cocultivation of leaf fragments with Agrobacterium tumefaciens harboring rol genes from A. rhizogenes. The phenotypic alterations caused by the different transgenes were evaluated in field trials. The genetic manipulation produced transgenic plants characterized by the following features: 1) increased number of flowers (e.g., 35SrolC and rolABC); 2) early flowering (e.g., 35SrolC); 3) change of plant growth habit: erect (rolAB, rolABC and 35SrolC) with an increased number of branches (e.g., rolABC). The color of leaves was pale green in 35SrolC and dark green in rolAB transgenic plants. In conclusion this work reports: 1) genetic engineering of the ornamental species O. ecklonis, 2) modification of the main ornamental traits of this species by rol genes, and 3) segregation of the transgenes in the progeny.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.