Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Synthesis and biological evaluation of novel anticancer bivalent colchicine-tubulizine hybrids

A series of novel antimitotic hybrids were synthesized in good yields by linking of azide-containing colchicine congeners with acetylene-substituted tubulizine-type derivatives using copper-mediated 1,3-dipolar cycloaddition. Obtained compounds exhibit good cytotoxicity against HBL100 epithelial cell lines (IC(50)=0.599-2.93 μМ). Several newly synthesized compounds are the substoichiometric inhibitors of microtubule assembly (R=0.41-0.78). The results highlight the importance of the length of spacer linking the tubulin binding ligands in heterodimeric molecules.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

QTLs for Woolly Poplar Aphid (Phloeomyzus passerinii L.) Resistance Detected in an Inter-Specific Populus deltoides x P. nigra Mapping Population

The genus Populus represents one of the most economically important groups of forest trees. It is composed by approximately 30 species used for wood and non-wood products, phytoremediation and biomass. Poplar is subjected to several biological and environmental threats although, compared to annual crops, we know far less about the genetic bases of biotic stress resistance. Woolly poplar aphid (Phloeomyzus passerinii) is considered a main pest of cultivated poplars in European and American countries. In this work we present two high density linkage maps in poplar obtained by a genotyping by sequencing (GBS) approach and the identification of QTLs involved in Ph. passerinii resistance. A total of 5,667 polymorphic markers (5,606 SNPs and 61 SSRs) identified on expressed sequences have been used to genotype 131 plants of an F1 population P ×canadensis obtained by an interspecific mate between Populus deltoides (resistant to woolly poplar aphid) and Populus nigra (susceptible to woolly poplar aphid). The two linkage maps, obtained following the two-way pseudo-testcross mapping strategy, have been used to investigate the genetic bases of woolly poplar aphid resistance. One major QTL and two QTLs with minor effects (mapped on LGV, LGXVI and LG XIX) explaining the 65.8% of the genetic variance observed in the progeny in response to Ph. passerinii attack were found. The high density coverage of functional markers allowed the identification of three genes belonging to disease resistance pathway as putative candidates for P. deltoides resistance to woolly poplar aphid. This work is the first report on genetic of woolly poplar aphid genetic resistance and the resistant loci associated markers identified represent a valuable tool in resistance poplar breeding programs.     

Stathmin/Op18 is a novel mediator of vinblastine activity

Microtubule (MT) dynamic instability is tightly regulated by stabilizing and destabilizing proteins, the latter being exemplified by stathmin/Op18, a protein known to destabilize MTs. Studies in cells have indicated that the level of stathmin expression modifies the cytotoxicity of antimicrotubule drugs, such as vinblastine (VLB). Using isothermal titration calorimetry and analytical ultracentrifugation, we show that VLB increases the affinity of stathmin for tubulin 50-fold (and vice versa). These results are the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and reveal a new mechanism of action for VLB.     

Randomized controlled trial of dietary intervention: association between level of urinary phenolics and anti-mutagenicity

We have undertaken a randomized trial to confirm the ability of a class of phenolics, flavonoids, to increase urinary anti-mutagenicity in smokers. Ninety heavy smokers were recruited and randomly assigned to three groups, who were given three different diets. One diet was rich in flavonoids, but not based on supplementation ('flavonoid'), one was a normal iso-caloric diet with an adequate administration of fruit and vegetables ('normal'), and one was based on supplementation of flavonoids in the form of green tea and soy products ('supplement'). The urinary anti-mutagenicity-as inhibiting effect of the urinary extracts on the mutations induced by MeIQx-was measured in Salmonella typhimurium YG1024 in the presence of liver S9 from male Sprague-Dawley rats treated with Aroclor 1254. The amount of total phenolics in the urinary extracts was measured by use of spectrometric analysis. We found that important dietary modifications can be achieved through special recipes and instructions given by a cook during an intensive course. The intervention was focused on increasing the flavonoid intake, and it was successful in that respect. In fact, differences in flavonoid intake were appreciated mainly between the first group (normal diet) and the other two (flavonoid-rich and supplemented diet), suggesting that dietary modification can be as effective as supplementation. However, both urinary anti-mutagenicity and the amounts of urinary phenolics did not change as a consequence of the trial. These results suggest that only a small fraction of urinary phenolics is influenced by dietary changes in the intake of flavonoids, and that most urinary anti-mutagens and phenolics are metabolites of dietary flavonoids, whose formation is more affected by the activity and diversity of bacterial flora in the colon than by the quantity and type of intake. A strong correlation was found between urinary phenolics and anti-mutagenicity in all the groups involved in the trial. Such correlation was not explained by dietary variables.     

Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi₃, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi₃ gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi₃ mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.     

3Increasing the source/sink ratio in Vitis vinifera (cv Sangiovese) induces extensive transcriptome reprogramming and modifies berry ripening

Background

The genomic architecture of bud phenology and height growth remains poorly known in most forest trees. In non model species, QTL studies have shown limited application because most often QTL data could not be validated from one experiment to another. The aim of our study was to overcome this limitation by basing QTL detection on the construction of genetic maps highly-enriched in gene markers, and by assessing QTLs across pedigrees, years, and environments.

Results

Four saturated individual linkage maps representing two unrelated mapping populations of 260 and 500 clonally replicated progeny were assembled from 471 to 570 markers, including from 283 to 451 gene SNPs obtained using a multiplexed genotyping assay. Thence, a composite linkage map was assembled with 836 gene markers. For individual linkage maps, a total of 33 distinct quantitative trait loci (QTLs) were observed for bud flush, 52 for bud set, and 52 for height growth. For the composite map, the corresponding numbers of QTL clusters were 11, 13, and 10. About 20% of QTLs were replicated between the two mapping populations and nearly 50% revealed spatial and/or temporal stability. Three to four occurrences of overlapping QTLs between characters were noted, indicating regions with potential pleiotropic effects. Moreover, some of the genes involved in the QTLs were also underlined by recent genome scans or expression profile studies. Overall, the proportion of phenotypic variance explained by each QTL ranged from 3.0 to 16.4% for bud flush, from 2.7 to 22.2% for bud set, and from 2.5 to 10.5% for height growth. Up to 70% of the total character variance could be accounted for by QTLs for bud flush or bud set, and up to 59% for height growth.

Conclusions

This study provides a basic understanding of the genomic architecture related to bud flush, bud set, and height growth in a conifer species, and a useful indicator to compare with Angiosperms. It will serve as a basic reference to functional and association genetic studies of adaptation and growth in Picea taxa. The putative QTNs identified will be tested for associations in natural populations, with potential applications in molecular breeding and gene conservation programs. QTLs mapping consistently across years and environments could also be the most important targets for breeding, because they represent genomic regions that may be least affected by G × E interactions.