Saleh Shah

Increasing the flow of carbon into seed oil

The demand for vegetable oils for food, fuel (bio-diesel) and bio-product applications is increasing rapidly. In Canada alone, it is estimated that a 50 to 75% increase in canola oil production will be required to meet the demand for seed oil in the next 7-10years. Plant breeding and genetics have demonstrated that seed oil content is a quantitative trait based on a number of contributing factors including embryo genetic effects, cytoplasmic effects, maternal genetic effects, and genotype-environment interactions. Despite the involvement of numerous quantitative trait loci in determining seed oil content, genetic engineering to over-express/repress specific genes encoding enzymes and other proteins involved in the flow of carbon into seed oil has led to the development of transgenic lines with significant increases in seed oil content. Proteins encoded by these genes include enzymes catalyzing the production of building blocks for oil assembly, enzymes involved in oil assembly, enzymes regulating metabolic carbon partitioning between oil, carbohydrate and secondary metabolite fractions, and transcription factors which orchestrate metabolism at a more general level.

Phytoremediation of arsenate contaminated soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2

It was previously observed that transgenic tomato plants that express the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC 4.1.99.4) gene, and thereby produce lower levels of ethylene, were partially protected from the deleterious effects of six different metals. However, since tomato plants are unlikely to be utilized in the phytoremediation of contaminated terrestrial sites, transgenic canola (Brassica napus) plants that constitutively express the same gene were generated and tested for their ability to proliferate in the presence of high levels of arsenate in the soil and to accumulate it in plant tissues. The ability of the plant growth-promoting bacterium E. cloacae CAL2 to facilitate the growth of both non-transformed and ACC deaminase-expressing canola plants was also tested. In the presence of arsenate, in both the presence and absence of the added plant growth-promoting bacterium, transgenic canola plants grew to a significantly greater extent than non-transformed canola plants.

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.

Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditions

The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabido...

A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus

Eight reference genes of Brassica napus were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) data, focusing on vegetative tissues and developing embryos. Analyses of expression stability indicated that UP1, UBC9, UBC21, and TIP41 were the top four choices as stably expressed reference genes for vegetative tissues, whereas ACT7, UBC21, TIP41, and PP2A were the top four choices for maturing embryos. In addition, radiolabeling of overall messenger RNA (mRNA) of maturing embryos indicated that the expression patterns of the top four ranked reference genes reflected the overall mRNA content changes in maturing embryos.

The use of transgenic canola (Brassica napus) and plant growth-promoting bacteria to enhance plant biomass at a nickel-contaminated field site

The applicability of transgenic plants and plant growth-promoting bacteria to improve plant biomass accumulation as a phytoremediation strategy at a nickel (Ni)-contaminated field site was examined. Two crops of 4-day old non-transformed and transgenic canola (Brassica napus) seedlings in the presence and absence of Pseudomonas putida strain UW4 (crop #1) or P. putida strain HS-2 (crop #1 and 2) were transplanted at a Ni-contaminated field site in 2005. Overall, transgenic canola had increased growth but decreased shoot Ni concentrations compared to non-transformed canola, resulting in similar total Ni per plant. Under optimal growth conditions (crop #2), the addition of P. putida HS-2 significantly enhanced growth for non-transformed canola. Canola with P. putida HS-2 had trends of higher total Ni per plant than canola without P. putida HS-2, indicating the potential usefulness of this bacterium in phytoremediation strategies. Modifications to the planting methods may be required to increase plant Ni uptake.

Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus.

Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.

Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Δ mutant strain of Saccharomyces cerevisiae

The identification of the yeast phosphatidate phosphohydrolase (PAH1) gene encoding an enzyme with phosphatidate phosphatase (PAP; 3‐sn‐phosphatidate phosphohydrolase, EC 3.1.3.4) activity led to the discovery of mammalian Lipins and subsequently to homologous genes from plants. In the present study, we describe the functional characterization of Arabidopsis and Brassica napus homologs of PAH1. Recombinant expression studies confirmed that homologous PAHs from plants can rescue different phenotypes exhibited by the yeast pah1Δ strain, such as temperature growth sensitivity and atypical neutral lipid composition. Using this expression system, we examined the role of the putative catalytic motif DXDXT and other conserved residues by mutational analysis. Mutants within the carboxy‐terminal lipin domain displayed significantly decreased PAP activity, which was reflected by their limited ability to complement different phenotypes of pah1Δ. Subcellular localization studies using a green fluorescent protein fusion protein showed that Arabidopsis PAH1 is mostly present in the cytoplasm of yeast cells. However, upon oleic acid stimulation, green fluorescent protein fluorescence was predominantly found in the nucleus, suggesting that plant PAH1 might be involved in the transcriptional regulation of gene expression. In addition, we demonstrate that mutation of conserved residues that are essential for the PAP activity of the Arabidopsis PAH1 enzyme did not impair its nuclear localization in response to oleic acid. In conclusion, the present study provides evidence that Arabidopsis and B. napus PAHs restore lipid synthesis in yeast and that DXDXT is a functional enzymic motif within plant PAHs.

Characterization of Defense Signaling Pathways of Brassica napus and Brassica carinata in Response to Sclerotinia sclerotiorum Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus)

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it’s in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.