Increase in lysophosphatidate acyltransferase activity in oilseed rape (Brassica napus) increases seed triacylglycerol content despite its low intrinsic flux control coefficient.

Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT. We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential. Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14 C-glycerol between phosphoglycerides. Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products.

Trichostatin A increases embryo and green plant regeneration in wheat.

KEY MESSAGE: Chemical agents such as trichostatin A (TSA) can assist in optimization of doubled haploidy for rapid improvements in wheat germplasm and addressing recalcitrance issues in cell culture responses. In wheat, plant regeneration through microspore culture is an integral part of doubled haploid (DH) production. However, low response to tissue culture and genotype specificity are two major constraints in the broad deployment of this breeding tool. Recently, the structure of chromatin was shown to be linked with cell transitions during tissue culture. Specifically, repression of genes that are required for cell morphogenesis, through acetylation of histones, may play an important role in this process. Reduction of histone acetylation by chemical inhibition may increase tissue culture efficiency. Here, the role of trichostatin A (TSA) in inducing microspore-derived embryos was investigated in wheat. The optimal dose of TSA was determined for wheat cultivars and subsequently validated in F1 hybrids. A significant increase in the efficiency of DH production was observed in both cultivated varieties and F1 hybrids. Thus, the inclusion of TSA in DH protocols for wheat breeding programs is advocated.

Increased mtPDH Activity Through Antisense Inhibition of Mitochondrial Pyruvate Dehydrogenase Kinase Enhances Inflorescence Initiation, and Inflorescence Growth and Harvest Index at Elevated CO2 in Arabidopsis thaliana.

Mitochondrial pyruvate dehydrogenase (mtPDH) is a key respiratory enzyme that links glycolysis and the tricarboxylic acid cycle, and it is negatively regulated by mtPDH kinase (mtPDHK). Arabidopsis lines carrying either a constitutive or seed-specific antisense construct for mtPDHK were used to test the hypothesis that alteration of mtPDH activity in a tissue- and dosage-dependent manner will enhance reproductive growth particularly at elevated CO2 (EC) through a combined enhancement of source and sink activities. Constitutive transgenic lines showed increased mtPDH activity in rosette leaves at ambient CO2 (AC) and EC, and in immature seeds at EC. Seed-specific transgenic lines showed enhanced mtPDH activity in immature seeds. A strong relationship existed between seed mtPDH activity and inflorescence initiation at AC, and at EC inflorescence stem growth, silique number and seed harvest index were strongly related to seed mtPDH activity. Leaf photosynthetic rates showed an increase in rosette leaves of transgenic lines at AC and EC that correlated with enhanced inflorescence initiation. Collectively, the data show that mtPDHK plays a key role in regulating sink and source activities in Arabidopsis particularly during the reproductive phase.

A desaturase-like protein from white spruce is a Delta(9) desaturase.

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.

Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli.

A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.

Increased levels of erucic acid in Brassica carinata by co-suppression and antisense repression of the endogenous FAD2 gene.

Erucic acid and its derivatives represent important industrial feedstock compounds, and there is an increasing demand for the production of high erucate oils in this regard. Our goal therefore, is to develop high erucic acid (HEA) Brassicaceae lines with increased proportions of erucic acid and very long-chain fatty acids (VLCFAs). We proposed that oleate availability may be a rate-limiting factor in the biosynthesis of erucic acid. We have tried to address this question by manipulating the expression of the endogenous FAD2 gene in B. carinata using co-supression and antisense approaches. Both methods resulted in transgenic lines exhibiting decreased proportions of polyunsaturated C18 fatty acids (18:2+18:3) and concomitant and significantly increased proportions of 18:1, 22:1 and total VLCFAs. Co-suppressed FAD2 B. carinata lines exhibited 3-18% decreases in 18:2, 22-49% decreases in 18:3 and significantly increased proportions of 18:1 (36-99%), 22:1 (12-27%) and VLCFAs (6-15%). Transgenic B. carinata lines developed using an antisense FAD2 approach exhibited decreased proportions of 18:2 and 18:3 (9-39% and 33-48%, respectively) and significantly increased proportions of 18:1 (54-130%), 22:1 (5-19%) and VLCFAs (6-21%). The possibility of using these approaches to produce prototype transgenic germplasm of the Brassicaceae accumulating seed oils with improved proportions of erucic and other VLCFAs is discussed.

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.