Bourlaye Fofana

Gene expression of stearoyl-ACP desaturase and Δ12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum)

Flaxs recent popularity in human and animal foods is mostly due to its desirable FA composition. Flax is an excellent source of omega-3 FA, which have been shown to have many health benefits. To date, little is known about the genetic and environmental factors that control the FA composition of flax seeds. To elucidate some of the important genetic components, reverse transcriptase (RT)-PCR and real-time PCR were used to determine the expression profiles of two key FA biosynthetic genes during seed development. Plants of flax cultivar AC McDuff were grown under field conditions, and RNA was extracted from ovaries and developing bolls collected from 2 d after anthesis (DAA) to maturity. Desaturation enzymes stearoyl-ACP desaturase (SAD) and Δ12 FA desaturase 2 (FAD2) were both expressed in ovaries, and their expression was differentially modulated throughout seed development. SAD was most highly expressed in ovaries. Its expression quickly decreased until 4 DAA; this was followed by a slight peak at 8 DAA, only to return to relatively low levels of expression in maturing bolls, ranging from 2.1% to 4.5% relative to the level observed in ovaries. FAD2 expression displayed a different temporal pattern. While expression of FAD2 did decrease in the early stages of seed development, expression increased starting at 8 DAA, peaking at 16 DAA, when it was 158% relative to the level observed in ovaries. FAD2, which desaturates oleic acid (18∶1 cisΔ9) into linoleic acid (18∶2 cisΔ9, 12), is therefore controlled at the transcription level. To relate enzyme expression with FA profile, GC was performed on the same subsamples used for RT-PCR and real-time PCR, and proportions of palmitic, stearic, oleic, linoleic, and linolenic acids were determined for the same developmental stages. Although FAD2 expression increased from 8 to 16 DAA, relative changes in linoleic acid (18∶2 cis Δ9, 12) were not observed. However, linolenic acid (ALA; α-18∶3; 18∶3 cisΔ9, 12, 15) levels increased steadily, meaning that linoleic acid (18∶2 cisΔ9, 12) is a transient substrate converted by FAD3 as quickly as it is produced by FAD2. Phenotypes are the result of genotypes, environment, and the interaction of the two. To evaluate the environmental impact on the production of FA in flax, FA profiles were assessed in a total of four environments (two locations, two years). Warm and dry environmental conditions resulted in lower levels of PUFA 18∶2 cisΔ9, 12 and 18∶3 cisΔ9, 12, 15, and higher levels of 18∶1 cisΔ9. FAD2 expression and/or activity may therefore be affected by the environment.

Temporal Gene Expression Profiling of the Wheat Leaf Rust Pathosystem Using cDNA Microarray Reveals Differences in Compatible and Incompatible Defence Pathways

In this study, we detail the construction of a custom cDNA spotted microarray containing 7728 wheat ESTs and the use of the array to identify host genes that are differentially expressed upon challenges with leaf rust fungal pathogens. Wheat cultivar RL6003 (Thatcher Lr1) was inoculated with Puccinia triticina virulence phenotypes BBB (incompatible) or TJB (7-2) (compatible) and sampled at four different time points (3, 6, 12, and 24 hours) after inoculation. Transcript expression levels relative to a mock treatment were measured. One hundred ninety two genes were found to have significantly altered expression between the compatible and incompatible reactions. Among those were genes involved in photosynthesis, the production of reactive oxygen species, ubiquitination, signal transduction, as well as in the shikimate/phenylpropanoid pathway. These data indicate that various metabolic pathways are affected, some of which might be used by RL6003 to mount a coordinated defense against an incompatible fungal pathogen.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG)

BackgroundLignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.ResultsFive UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.ConclusionWe have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Assessment of molecular diversity at QTLs for preharvest sprouting resistance in wheat using microsatellite markers

Breeding for preharvest sprouting (PHS) resistance is of great interest in wheat-growing areas where high rainfall occurs during grain ripening and harvest. We have characterized 32 wheat accessions using 33 microsatellite markers flanking PHS quantitative trait loci (QTLs) previously identified on group 3, 4, 5, and 6 chromosomes of hexaploid wheat. A total of 229 alleles, with an average of 6.94 alleles per marker, were observed among the 32 wheat lines. The polymorphic information content (PIC) was estimated and ranged between 0.25 and 0.90, with an average of 0.67. A cluster analysis revealed 3 main clusters and 3 singlet wheat lines, which is in agreement with pedigree-based relationships, seed coat colour, and origin. Canadian wheat accessions were subdivided into 4 sub-clusters based on pedigree and wheat classes. Grouping of preharvest sprouting germplasm into clusters was consistent with cluster-specific allele diversity observed in the PHS-resistant lines AUS1408, Red-RL4137, White-RL4137, and Kenya321. The implications of these findings in white wheat breeding for PHS tolerance are discussed.

Histidine 352 (His352) and tryptophan 355 (Trp355) are essential for flax UGT74S1 glucosylation activity toward secoisolariciresinol.

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357 , and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352 , and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks

In 1893, Charles Barnes (1858-1910) proposed to designate the biological process for “synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light” as “photosyntax” or “photosynthesis”. His preference went for the word “photosyntax”, but “photosynthesis” came into common usage as the term of choice (Gest, 2002). Although this definition is still widely used today, the Oxford English Dictionary (OED, 1989) has defined the biological photosynthesis as “the process by which carbon dioxide is converted into organic matter in the presence of the chlorophyll of plants under the influence of light, which in all plants except some bacteria involves the production of oxygen from water”. By considering the photosynthetic bacteria that do not produce oxygen and do not necessarily require carbonic acid as carbon source, Gest (Gest, 1993) has refined these definitions as followed: “photosynthesis is a series of processes in which electromagnetic energy is converted to chemical energy used for biosynthesis of organic cell materials; a photosynthetic organism is one in which a major fraction of the energy required for cellular syntheses is supplied by light”. Photosynthesis is therefore a unique source for carbon sequestration and allows the aerobic organisms to survive based upon not only on its released oxygen but also its synthesized organic compounds. As such, plants are an ideal host for pathogenic microorganisms such as fungi, bacteria and viruses and also the only food source for herbivores. Theoretically, the photosynthetic capacity of plants is unlimited when the optimal growing conditions are met. Unfortunately, terrestrial plants are constantly challenged by abiotic (UV, water, salinity, temperature) (Baker et al., 1988, Baldry et al., 1966, Barhoumi et al., 2007, Barrow and Cockburn, 1982, Bassham, 1977, Batista-Santos et al., 2011, Bauerle et al., 2007, Berry, 1975, Bischof et al., 2000, Ripley et al., 2008, Ripley et al., 2007, Roberntz and Stockfors, 1998) and biotic (pathogens, pests, animal and human) stresses that reduce their productivity and even threaten their survival (Bilgin et al., 2010, Bonfig et al., 2006, Erickson and Hawkins, 1980, Garavaglia et al., 2010, Kocal et al., 2008, Tang et al., 2009). While the regulation of plant defence responses has been extensively investigated, the effects of pathogen infection on primary metabolism, including photosynthesis, are however less known. Currently, interest in this research area is growing and some aspects of photosynthesis, assimilate

UGT74S1 is the key player in controlling secoisolariciresinol diglucoside (SDG) formation in flax

BackgroundFlax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known.ResultsFlax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4–153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1.ConclusionsWe showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying the SMG precursor to UGT74S1.

Genetic, Agronomy, and Metabolomics of Prince Edwards Island Wild Rose Collection and Promise for Cultivar Development

Agriculture – the control of plants for human consumption – is believed to have appeared and developed during the paleolitic/neolitic period, ~ 10,000 years ago [1]. The first agriculture had no single or simple origin since a wide variety of plants and animals have been independently domesticated at different times and different places [1-4]. The origin of agriculture and crops domestication is intertwined. Plant domestication involves changes in the plant’s genetic makeup and morphological appearance following successive selections within wild plants and based upon on the variations that are best suitable for humans needs [5]. Domestication is therefore an artificial selection process conducted by humans for the production of plants showing fewer undesirable traits compared to its wild related plants, and making them more dependent on the new artificial environments for their continued survival and development. The concept of selection assumes the existence of a population or group of individuals from which choices can be made. Thus, the diversity of morphotypes or genetic diversity is considered as the backbone for plant domestication and crop improvement. Nonetheless, the way this genetic diversity was probed across time has constantly evolved while being a continuum from the first day. Moreover, while the selection criteria for the desired traits and purposes in the ancient domestication process were certainly exclusively based on morphology (size, color, shape of leaves and fruits, easiness for identification) and to satisfy man’s energy supply needs (taste and flavour, satiety potential), today, the required traits and purposes for plant domestication (seen as continuum) have been refined and expanded. Indeed, new technologies have been developed for probing the genetic diversity whereas human needs

Genetic analyses using GGE model and a mixed linear model approach, and stability analyses using AMMI bi-plot for late-maturity alpha-amylase activity in bread wheat genotypes

Low falling number and discounting grain when it is downgraded in class are the consequences of excessive late-maturity α-amylase activity (LMAA) in bread wheat (Triticum aestivum L.). Grain expressing high LMAA produces poorer quality bread products. To effectively breed for low LMAA, it is necessary to understand what genes control it and how they are expressed, particularly when genotypes are grown in different environments. In this study, an International Collection (IC) of 18 spring wheat genotypes and another set of 15 spring wheat cultivars adapted to South Dakota (SD), USA were assessed to characterize the genetic component of LMAA over 5 and 13 environments, respectively. The data were analysed using a GGE model with a mixed linear model approach and stability analysis was presented using an AMMI bi-plot on R software. All estimated variance components and their proportions to the total phenotypic variance were highly significant for both sets of genotypes, which were validated by the AMMI model analysis. Broad-sense heritability for LMAA was higher in SD adapted cultivars (53%) compared to that in IC (49%). Significant genetic effects and stability analyses showed some genotypes, e.g. ‘Lancer’, ‘Chester’ and ‘LoSprout’ from IC, and ‘Alsen’, ‘Traverse’ and ‘Forefront’ from SD cultivars could be used as parents to develop new cultivars expressing low levels of LMAA. Stability analysis using an AMMI bi-plot revealed that ‘Chester’, ‘Lancer’ and ‘Advance’ were the most stable across environments, while in contrast, ‘Kinsman’, ‘Lerma52’ and ‘Traverse’ exhibited the lowest stability for LMAA across environments.

Induced Mutagenesis in UGT74S1 Gene Leads to Stable New Flax Lines with Altered Secoisolariciresinol Diglucoside (SDG) Profiles

Flax secoisolariciresinol (SECO) diglucoside (SDG) lignan is an emerging natural product purported to prevent chronic diseases in humans. SECO, the aglycone form of SDG, has shown higher intestinal cell absorption but it is not accumulated naturally in planta. Recently, we have identified and characterized a UDP-glucosyltransferase gene, UGT74S1, that glucosylates SECO into its monoglucoside (SMG) and SDG forms when expressed in yeast. However, whether this gene is unique in controlling SECO glucosylation into SDG in planta is unclear. Here, we report on the use of UGT74S1 in reverse and forward genetics to characterize an ethyl methane sulfonate (EMS) mutagenized flax population from cultivar CDC Bethune and consisting of 1996 M2 families. EMS mutagenesis generated 73 SNP variants causing 79 mutational events in the UGT74S1 exonic regions of 93 M2 families. The mutation frequency in the exonic regions was determined to be one per 28 Kb. Of these mutations, 13 homozygous missense mutations and two homozygous nonsense mutations were observed and all were transmitted into the M3 and M4 generations. Forward genetics screening of the population showed homozygous nonsense mutants completely lacking SDG biosynthesis while the production of SMG was observed only in a subset of the M4 lines. Heterozygous or homozygous M4 missense mutants displayed a wide range of SDG levels, some being greater than those of CDC Bethune. No additional deleterious mutations were detected in these mutant lines using a panel of 10 other genes potentially involved in the lignan biosynthesis. This study provides further evidence that UGT74S1 is unique in controlling SDG formation from SECO and this is the first report of non-transgenic flax germplasm with simultaneous knockout of SDG and presence of SMG in planta.