Precise Determination of Selenium Forms and Contents in Selenium-Enriched Rapeseed Seedlings and Flowering Stalks by HPLC-ICP-MS.

Rapeseed (Brassica napus L.) has the ability of selenium (Se) enrichment. Identification of selenides in Se-rich rapeseed products will promote the development and utilization of high value. By optimizing the Se species extraction process (protease type, extraction reagent, enzyme sample ratio, extraction time, etc.) and chromatographic column, an efficient, stable, and accurate method was established for the identification of Se species and content in rapeseed seedlings and flowering stalks, which were cultured by inorganic Se hydroponics. Five Se compounds, including selenocystine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet), selenite (SeIV), and selenate (SeVI) were qualitatively and quantitatively identified. Organoselenium was absolutely dominant in both seedlings and flowering stalks among the detected rapeseed varieties, with 64.18-90.20% and 94.38-98.47%, respectively. Further, MeSeCys, a highly active selenide, predominated in rapeseed flowering stalks with a proportion of 56.36-72.93% and a content of 1707.3-5030.3 µg/kg. This study provides a new source of MeSeCys supplementation for human Se fortification.

Nitric oxide affects seed oil accumulation and fatty acid composition through protein S-nitrosation.

Nitric oxide (NO) is a key signaling molecule regulating several plant developmental and stress responses. Here, we report that NO plays an important role in seed oil content and fatty acid composition. RNAi silencing of Arabidopsis S-nitrosoglutathione reductase 1 (GSNOR1) led to reduced seed oil content. In contrast, nitrate reductase double mutant nia1nia2 had increased seed oil content, compared with wild-type plants. Moreover, the concentrations of palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) were higher, whereas those of stearic acid (C18:0), oleic acid (C18:1), and arachidonic acid (C20:1) were lower, in seeds of GSNOR1 RNAi lines. Similar results were obtained with rapeseed embryos cultured in vitro with the NO donor sodium nitroprusside (SNP), and the NO inhibitor NG-Nitro-L-arginine Methyl Ester (L-NAME). Compared with non-treated embryos, the oil content decreased in SNP-treated embryos, and increased in L-NAME-treated embryos. Relative concentrations of C16:0, C18:2 and C18:3 were higher, whereas C18:1 concentration decreased in rapeseed embryos treated with SNP. Proteomics and transcriptome analysis revealed that three S-nitrosated proteins and some key genes involved in oil synthesis, were differentially regulated in SNP-treated embryos. Therefore, regulating NO content could be a novel approach to increasing seed oil content in cultivated oil crops.

Seed structure characteristics to form ultrahigh oil content in rapeseed.

BACKGROUND: Rapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis.

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Maternal control of seed oil content in Brassica napus: the role of silique wall photosynthesis.

Seed oil content is an important agronomic trait in rapeseed. However, our understanding of the regulatory processes controlling oil accumulation is still limited. Using two rapeseed lines (zy036 and 51070) with contrasting oil content, we found that maternal genotype greatly affects seed oil content. Genetic and physiological evidence indicated that difference in the local and tissue-specific photosynthetic activity in the silique wall (a maternal tissue) was responsible for the different seed oil contents. This effect was mimicked by in planta manipulation of silique wall photosynthesis. Furthermore, the starch content and expression of the important lipid synthesis regulatory gene WRINKLED1 in developing seeds were linked with silique wall photosynthetic activity. 454 pyrosequencing was performed to explore the possible molecular mechanism for the difference in silique wall photosynthesis between zy036 and 51070. Interestingly, the results suggested that photosynthesis-related genes were over-represented in both total silique wall expressed genes and genes that were differentially expressed between genotypes. A potential regulatory mechanism for elevated photosynthesis in the zy036 silique wall is proposed on the basis of knowledge from Arabidopsis. Differentially expressed ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-related genes were used for further investigations. Oil content correlated closely with BnRBCS1A expression levels and Rubisco activities in the silique wall, but not in the leaf. Taken together, our results highlight an important role of silique wall photosynthesis in the regulation of seed oil content in terms of maternal effects.

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content.

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.

[Studies on immunoregulation of polysaccharides-la from Radix Cynanchi Bungei].

OBJECTIVE: To study the effects of PRCB1a (one component of polysaccharides from Radix Cynanchi Bungei) on transformation of T lymphocytes of rabbit in vitro and immune function in mice. METHOD: Three doses of PRCB1a (2,4,6 g x L(-1)) were respectively put in bottle with PHA and blood of rabbit. The effect of PRCB1a on immunity in vitro was studied by observing transformation of T lymphocytes; The dosage of PRCB1a (50,100,150 mg x kg(-1) x d(-1)) was given orally for seven days. The effects on immune function were investigated in mice. RESULT: Three doses of PRCB1a could significantly promote (P < 0.01) the ability of T lymphocytes proliferation; PRCB1a could improve the mouse thymus and spleen index, the celiac macrophage ability of engulfing CRBC, the delayed type hypersensitivity ability and the macrophage engulfing carbon granula ability. CONCLUSION: The results indicate PRCB1a can enhance nonspectific and specific cellular immune function.