Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.     

Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.     

The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways

The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.     

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.Communicated by J.W. Snape     

Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.     

Qualitative simulation of the initiation of sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Under conditions of nutrient deprivation, the Gram positive soil bacterium Bacillus subtilis can abandon vegetative growth and form a dormant, environmentally-resistant spore instead. The decision to either divide or sporulate is controlled by a large and complex genetic regulatory network integrating various environmental, cell-cycle, and metabolic signals. Although sporulation in B. subtilis is one of the best-understood model systems for prokaryotic development, very little quantitative data on kinetic parameters and molecular concentrations are available. A qualitative simulation method is used to model the sporulation network and simulate the response of the cell to nutrient deprivation. Using this method, we have been able to reproduce essential features of the choice between vegetative growth and sporulation, in particular the role played by competing positive and negative feedback loops.     

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303

phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.