Seed-specific expression of the class 2 Phytoglobin (Pgb2) increases seed oil in Brassica napus.

To examine the function of phytoglobin 2 (Pgb2) on seed oil level in the oil-producing crop Brassica napus L., we generated transgenic plants in which BnPgb2 was over-expressed in the seeds using the cruciferin1 promoter. Over-expression of BnPgb2 elevated the amount of oil, which showed a positive relationship with the level of BnPgb2, without altering the oil nutritional value, as evidenced by the lack of major changes in composition of fatty acids (FA), and key agronomic traits. Two key transcription factors, LEAFY COTYLEDON1 (LEC1) and WRINKLED1 (WRI1), known to promote the synthesis of fatty acids (FA) and potentiate oil accumulation, were induced in BnPgb2 over-expressing seeds. The concomitant induction of several enzymes of sucrose metabolism, SUCROSE SYNTHASE1 (SUS) 1 and 3, FRUCTOSE BISPHOSPHATE ALDOLASE (FPA), and PHOSPHOGLYCERATE KINASE (PGK), and starch synthesis, ADP-GLUCOSE PHOSPHORYLASE (AGPase) suggests that BnPgb2 favors sugar mobilization for FA production. The two plastid FA biosynthetic enzymes SUBUNIT A OF ACETYL-CoA CARBOXYLASE (ACCA2), and MALONYL-CoA:ACP TRANSACYLASE (MCAT) were also up-regulated by the over-expression of BnPgb2. The requirement of BnPgb2 for oil deposition was further evidenced in natural germplasm by the higher levels of BnPgb2 in seeds of high-oil genotypes relative to their low-oil counterparts.

Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1.

Over the last few decades, research focusing on canola (Brassica napus L.) seed oil content and composition has expanded. Oil production and accumulation are influenced by genes participating in embryo and seed development. The Arabidopsis LEAFY COTYLEDON1 (LEC1) is a well characterized regulator of embryo development that also enhances the expression of genes involved in fatty acid (FA) synthesis. B. napus lines over-expressing or down-regulating BnLEC1 were successfully generated by Agrobacterium-mediated transformation. The constitutive expression of BnLEC1 in B. napus var. Polo, increased seed oil content by 7-16%, while the down-regulation of BnLEC1 in B. napus var. Topas reduced oil content by 9-12%. Experimental manipulation of BnLEC1 caused transcriptional changes in enzymes participating in sucrose metabolism, glycolysis, and FA biosynthesis, suggesting an enhanced carbon flux towards FA biosynthesis in tissues over-expressing BnLEC1. The increase in oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value or glucosinolate (GLS) levels. Suppression of BnLEC1 reduced seed oil accumulation and elevated the level of GLS possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. Collectively, these findings demonstrate that experimental alterations of BnLEC1 expression can be used to influence oil production and quality in B. napus.

Decreased seed oil production in FUSCA3 Brassica napus mutant plants.

Canola (Brassica napus L.) oil is extensively utilized for human consumption and industrial applications. Among the genes regulating seed development and participating in oil accumulation is FUSCA3 (FUS3), a member of the plant-specific B3-domain family of transcription factors. To evaluate the role of this gene during seed storage deposition, three BnFUSCA3 (BnFUS3) TILLING mutants were generated. Mutations occurring downstream of the B3 domain reduced silique number and repressed seed oil level resulting in increased protein content in developing seeds. BnFUS3 mutant seeds also had increased levels of linoleic acid, possibly due to the reduced expression of ω-3 FA DESATURASE (FAD3). These observed phenotypic alterations were accompanied by the decreased expression of genes encoding transcription factors stimulating fatty acid (FA) synthesis: LEAFY COTYLEDON1 and 2 (LEC1 and 2) ABSCISIC ACID-INSENSITIVE 3 (BnABI3) and WRINKLED1 (WRI1). Additionally, expression of genes encoding enzymes involved in sucrose metabolism, glycolysis, and FA modifications were down-regulated in developing seeds of the mutant plants. Collectively, these transcriptional changes support altered sucrose metabolism and reduced glycolytic activity, diminishing the carbon pool available for the synthesis of FA and ultimately seed oil production. Based on these observations, it is suggested that targeted manipulations of BnFUS3 can be used as a tool to influence oil accumulation in the economically important species B. napus.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Distinct fluctuations in nucleotide metabolism accompany the enhanced in vitro embryogenic capacity of Brassica cells over-expressing SHOOTMERISTEMLESS.

Besides regulating meristem formation and maintenance in vivo, SHOOTMERISTEMLESS (STM) has been shown to affect embryogenesis. While the over-expression of Brassica napus (Bn)STM enhances the number of microspore-derived embryos produced in culture and their ability to regenerate viable plants, a down-regulation of this gene represses the embryogenic process (Elhiti et al., J Exp Bot, 61:4069-4085, 2010). Synthesis and degradation of pyrimidine and purine nucleotides were measured in developing microspore-derived embryos (MDEs) generated from B. napus lines ectopically expressing or down-regulating BnSTM. Pyrimidine metabolism was investigated by following the metabolic fate of exogenously supplied (14)C-uridine, uracil and orotic acid, whereas purine metabolism was estimated by using (14)C-adenine, adenosine and inosine. The improvement in embryo number and quality affected by the ectopic expression of BnSTM was linked to the increased pyrimidine and purine salvage activity during the early phases of embryogenesis and the enlargement of the adenylate pool (ATP + ADP) required for the active growth of the embryos. This was due to an increase in transcriptional and enzymatic activity of several salvage enzymes, including adenine phosphoribosyltransferase (APRT) and adenosine kinase (ADK). The highly operative salvage pathway induced by the ectopic expression of BnSTM was associated with a slow catabolism of nucleotides, suggesting the presence of an antagonist mechanism controlling the rate of salvage and degradation pathways. During the second half of embryogenesis utilization of uridine for UTP + UDPglucose (UDPG) synthesis increased in the embryos over-expressing BnSTM, and this coincided with a better post-germination performance. All these events were precluded by the down-regulation of BnSTM which repressed the formation of the embryos and their post-embryonic performance. Overall, this work provides evidence that precise metabolic changes are associated with proper embryo development in culture.

Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways.

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of (14)C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apical meristems in Brassica napus microspore-derived embryos.

Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4x10(-6) M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.