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Background: Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. Results: In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. Conclusion: The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii. [ABSTRACT FROM AUTHOR]

Background: Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar 'Iwa' using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent. Results: Following 5 days' exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type 'Iwa' potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants. Conclusions: Somatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae is able to biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine and pseudoephedrine, by the action of pyruvate decarobxylase (PDC) enzyme. This biotransformation can alternatively be performed by acetohydroxyacid synthase (AHAS) which is a mitochondrial enzyme. In the yeast petite mutants, AHAS accumulates in the cytosol. In the current study, wild-type yeast cells and yeast petite mutants were examined for L-PAC biosynthesis. The results showed higher L-PAC titers in the yeast petite mutants. In addition, the effect of cell immobilization and carbon source (glucose or molasses) on L-PAC production was investigated. It was found that cell immobilization enhances L-PAC formation. The highest L-PAC concentration (2.4 g/l) was obtained at 2 g/l of benzaldehyde using the immobilized petite mutants grown on molasses. [ABSTRACT FROM AUTHOR]

Background: Herbicide resistance in agrestal weeds is a global problem threatening food security. Non-target-site resistance (NTSR) endowed by mechanisms neutralising the herbicide or compensating for its action is considered the most agronomically noxious type of resistance. Contrary to target-site resistance, NTSR mechanisms are far from being fully elucidated. A part of weed response to herbicide stress, NTSR is considered to be largely driven by gene regulation. Our purpose was to establish a transcriptome resource allowing investigation of the transcriptomic bases of NTSR in the major grass weed Alopecurus myosuroides L. (Poaceae) for which almost no genomic or transcriptomic data was available. Results: RNA-Seq was performed from plants in one F2 population that were sensitive or expressing NTSR to herbicides inhibiting acetolactate-synthase. Cloned plants were sampled over seven time-points ranging from before until 73 h after herbicide application. Assembly of over 159M high-quality Illumina reads generated a transcriptomic resource (ALOMYbase) containing 65,558 potentially active contigs (N50 = 1240 nucleotides) predicted to encode 32,138 peptides with 74 % GO annotation, of which 2017 were assigned to protein families presumably involved in NTSR. Comparison with the fully sequenced grass genomes indicated good coverage and correct representation of A. myosuroides transcriptome in ALOMYbase. The part of the herbicide transcriptomic response common to the resistant and the sensitive plants was consistent with the expected effects of acetolactate-synthase inhibition, with striking similarities observed with published Arabidopsis thaliana data. A. myosuroides plants with NTSR were first affected by herbicide action like sensitive plants, but ultimately overcame it. Analysis of differences in transcriptomic herbicide response between resistant and sensitive plants did not allow identification of processes directly explaining NTSR. Five contigs associated to NTSR in the F2 population studied were tentatively identified. They were predicted to encode three cytochromes P450 (CYP71A, CYP71B and CYP81D), one peroxidase and one disease resistance protein. Conclusions: Our data confirmed that gene regulation is at the root of herbicide response and of NTSR. ALOMYbase proved to be a relevant resource to support NTSR transcriptomic studies, and constitutes a valuable tool for future research aiming at elucidating gene regulations involved in NTSR in A. myosuroides. [ABSTRACT FROM AUTHOR]

Background: Herbicide resistance in agrestal weeds is a global problem threatening food security. Non-target-site resistance (NTSR) endowed by mechanisms neutralising the herbicide or compensating for its action is considered the most agronomically noxious type of resistance. Contrary to target-site resistance, NTSR mechanisms are far from being fully elucidated. A part of weed response to herbicide stress, NTSR is considered to be largely driven by gene regulation. Our purpose was to establish a transcriptome resource allowing investigation of the transcriptomic bases of NTSR in the major grass weed Alopecurus myosuroides L. (Poaceae) for which almost no genomic or transcriptomic data was available. Results: RNA-Seq was performed from plants in one F2 population that were sensitive or expressing NTSR to herbicides inhibiting acetolactate-synthase. Cloned plants were sampled over seven time-points ranging from before until 73 h after herbicide application. Assembly of over 159M high-quality Illumina reads generated a transcriptomic resource (ALOMYbase) containing 65,558 potentially active contigs (N50 = 1240 nucleotides) predicted to encode 32,138 peptides with 74 % GO annotation, of which 2017 were assigned to protein families presumably involved in NTSR. Comparison with the fully sequenced grass genomes indicated good coverage and correct representation of A. myosuroides transcriptome in ALOMYbase. The part of the herbicide transcriptomic response common to the resistant and the sensitive plants was consistent with the expected effects of acetolactate-synthase inhibition, with striking similarities observed with published Arabidopsis thaliana data. A. myosuroides plants with NTSR were first affected by herbicide action like sensitive plants, but ultimately overcame it. Analysis of differences in transcriptomic herbicide response between resistant and sensitive plants did not allow identification of processes directly explaining NTSR. Five contigs associated to NTSR in the F2 population studied were tentatively identified. They were predicted to encode three cytochromes P450 (CYP71A, CYP71B and CYP81D), one peroxidase and one disease resistance protein. Conclusions: Our data confirmed that gene regulation is at the root of herbicide response and of NTSR. ALOMYbase proved to be a relevant resource to support NTSR transcriptomic studies, and constitutes a valuable tool for future research aiming at elucidating gene regulations involved in NTSR in A. myosuroides. [ABSTRACT FROM AUTHOR]

Background: Weed management is critical to global crop production and is complicated by rapidly evolving herbicide resistance in weeds. New sources of herbicide resistance are needed for crop plants so that applied herbicides can be rotated or combined to thwart the evolution of resistant weeds. The diverse family of cytochrome P450 proteins has been suggested to be a source of detoxifying herbicide metabolism in both weed and crop plants, and greater understanding of these genes will offer avenues for crop improvement and novel weed management practices. Results: Here, we report the identification of CYP749A16 (Gh_D10G1401) which is responsible for the natural tolerance exhibited by most cotton, Gossypium hirsutum L., cultivars to the herbicide trifloxysulfuron sodium (TFS, CGA 362622, commercial formulation Envoke). A 1-bp frameshift insertion in the third exon of CYP749A16 results in the loss of tolerance to TFS. The DNA marker designed from this insertion perfectly co-segregated with the phenotype in 2145 F2 progeny of a cross between the sensitive cultivar Paymaster HS26 and tolerant cultivar Stoneville 474, and in 550 recombinant inbred lines of a multi-parent advanced generation inter-cross population. Marker analysis of 382 additional cotton cultivars identified twelve cultivars containing the 1-bp frameshift insertion. The marker genotypes matched perfectly with phenotypes in 188 plants from the selected twelve cultivars. Virus-induced gene silencing of CYP749A16 generated sensitivity in the tolerant cotton cultivar Stoneville 474. Conclusions: CYP749A16 located on chromosome D10 is required for TFS herbicide tolerance in cotton. This finding should add to the repertoire of tools available to farmers and breeders for the advancement of agricultural productivity. [ABSTRACT FROM AUTHOR]