Functional disruption of the pentatricopeptide protein SLG1 affects mitochondrial RNA editing, plant development, and responses to abiotic stresses in Arabidopsis

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.     

Design of New Genome- and Gene-Sourced Primers and Identification of QTL for Seed Oil Content in a Specially High-Oil Brassica napus Cultivar

Rapeseed (Brassica napus L.) is one of most important oilseed crops in the world. There are now various rapeseed cultivars in nature that differ in their seed oil content because they vary in oil-content alleles and there are high-oil alleles among the high-oil rapeseed cultivars. For these experiments, we generated doubled haploid (DH) lines derived from the cross between the specially high-oil cultivar zy036 whose seed oil content is approximately 50% and the specially low-oil cultivar 51070 whose seed oil content is approximately 36%. First, to address the deficiency in polymorphic markers, we designed 5944 pairs of newly developed genome-sourced primers and 443 pairs of newly developed primers related to oil-content genes to complement the 2244 pairs of publicly available primers. Second, we constructed a new DH genetic linkage map using 527 molecular markers, consisting of 181 publicly available markers, 298 newly developed genome-sourced markers and 48 newly developed markers related to oil-content genes. The map contained 19 linkage groups, covering a total length of 2,265.54 cM with an average distance between markers of 4.30 cM. Third, we identified quantitative trait loci (QTL) for seed oil content using field data collected at three sites over 3 years, and found a total of 12 QTL. Of the 12 QTL associated with seed oil content identified, 9 were high-oil QTL which derived from the specially high-oil cultivar zy036. Two high-oil QTL on chromosomes A2 and C9 co-localized in two out of three trials. By QTL mapping for seed oil content, we found four candidate genes for seed oil content related to four gene markers: GSNP39, GSSR161, GIFLP106 and GIFLP046. This information will be useful for cloning functional genes correlated with seed oil content in the future.     

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F₂ plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F₂ plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F₂ plants were confirmed in the next generation by analysing F₄ seeds harvested from five F₃ plants per selected F₂ plant. F₃ lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.     

sn-1-Acylglycerol-3-phosphate acyltransferase of Escherichia coli causes insertion of cis-11 eicosenoic acid into the sn-2 position of transgenic rapeseed oil

The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3 end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.     

油菜油脂研究进展

油菜(Brassica napus)是世界范围内重要的油料作物, 是植物油脂的第三大来源, 其种植面积和总产量在油料作物中占有相当大的比例。我国油菜品种油脂含量普遍较国外低2-5个百分点, 而油脂含量每增加1个百分点对产油量提高的贡献, 相当于菜籽产量提高2.5个百分点。因此提高油菜油脂含量是解决油菜生产效益低的重要途径之一。本文综述了油菜油脂研究的状况, 包括油脂积累的遗传学基础、油脂合成途径和调控、油脂含量的QTL定位及油脂含量与品质性状的遗传相关性, 同时展望了油菜油脂研究前景, 以期为油菜油脂含量的品种改良提供科学指导。     

Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil.

Lysophosphatidic acid acyltransferase acylates the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid, a precursor to triacylglycerol. A cDNA encoding lysophosphatidic acid acyltransferase was isolated from developing seeds of meadowfoam (Limnanthes alba alba). The cDNA encodes a 281-amino acid protein with a molecular mass of 32 kD. The cDNA was expressed in developing seeds of transgenic high-erucic-acid rapeseed (Brassica napus) using a napin expression cassette. Erucic acid was present at the sn-2 position of triacylglycerols from transgenic plants but was absent from that position of seed oil extracted from control plants. Trierucin was present in the transgenic oil. Alteration of the sn-2 erucic acid composition did not affect the total erucic acid content. These experiments demonstrate the feasibility of using acyltransferases to alter the stereochemical composition of transgenic seed oils and also represent a necessary step toward increasing the erucic acid content of rapeseed oil.     

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

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.     

Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed -ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the -ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.     

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F₁ plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.     

油菜油脂研究进展

油菜（Brassica napus）是世界范围内重要的油料作物,是植物油脂的第三大来源,其种植面积和总产量在油料作物中占有相当大的比例。我国油菜品种油脂含量普遍较国外低2—5个百分点,而油脂含量每增加1个百分点对产油量提高的贡献,相当于菜籽产量提高2．5个百分点。因此提高油菜油脂含量是解决油菜生产效益低的重要途径之一。本文综述了油菜油脂研究的状况,包括油脂积累的遗传学基础、油脂合成途径和调控、油脂含量的QTL定位及油脂含量与品质性状的遗传相关性,同时展望了油菜油脂研究前景,以期为油菜油脂含量的品种改良提供科学指导。     

Effects of Heat Stress during Seed Filling Stage on <i>Brassica napus</i> Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise, the threat of heat stress to rapeseed production is becoming more obvious. Exploring the response characteristics of two important biological pathways, oil accumulation and photosynthesis, to heat stress during B. napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed. The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B. napus germplasms with different oil content and environmental sensitivity, including 6 rapeseed varieties which exhibited environment-sensitive/insensitive and with high, medium or low oil content, were tested by whole plant heat stress or the in vitro silique culture system. Both assay exhibited similar trend on oil content of the rapeseed germplasms. The heat effect on the chlorophyll fluorescence kinetic parameters F_v/F_m, ETR and Y(II) were also consistent. Heat stress significantly decreased oil content, although there was abundant genetic variation on heat tolerance among the genotypes. Correlation analysis showed that the decrease rate of F_v/F_m of silique heat-stressed B. napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed (R = 0.9214, P-value < 0.01). Overall, the results indicated that heat stress inhibited oil accumulation and photosynthesis in B. napus developing seed. The decrease rate of chlorophyll fluorescence parameter F_v/F_m of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification. Further, two heat insensitive rapeseed varieties with high oil content were identified.     

Important photosynthetic contribution of silique wall to seed yield-related traits in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In plants, green non-foliar organs are able to perform photosynthesis just as leaves do, and the seed-enclosing pod acts as an essential photosynthetic organ in legume and Brassica species. To date, the contribution of pod photosynthesis to seed yield and related components still remains largely unexplored, and in Arabidopsis thaliana, the photosynthetic activity of the silique (pod) is unknown. In this study, an Arabidopsis glk1/glk2 mutant defective in both leaf and silique photosynthesis was used to create tissue-specific functional complementation lines. These lines were used to analyze the contribution of silique wall photosynthesis to seed yield and related traits, and to permit the comparison of this contribution with that of leaf photosynthesis. Our results showed that, together with leaves, the photosynthetic assimilation of the silique wall greatly contributed to total seed yield per plant. As for individual components of yield traits, leaf photosynthesis alone contributed to the seed number per silique and silique length, while silique wall photosynthesis alone contributed to thousand-seed weight. In addition, enhancing the photosynthetic capacity of the silique wall by overexpressing the photosynthesis-related RCA gene in this tissue resulted in significantly increased seed weight and oil content in the wild-type (WT) background. These results reveal that silique wall photosynthesis plays an important role in seed-related traits, and that enhancing silique photosynthesis in WT plants can further improve seed yield-related traits and oil production. This finding may have significant implications for improving the seed yield and oil production of oilseed crops and other species with pod-like organs.     

An ultra scale-down process study for the production of polyhydroxybutyrate from transgenic rapeseed

A study of the processing of 10 g quantities of wild-type and transgenic rapeseed is described to remove oil prior to PHB separation from meal. Processing of transgenic seed with 0.54% polyhydroxybutyrate (PHB) is compared with natural rapeseed containing zero, 0.5%, 5% and 20% PHB derived from a commercial microbial process. The 20% material was in the form of 1-2 wm particles and in the form of 80 wm aggregates of the smaller particles. An ultra scaled-down process was operated to match the conditions in commercial oil extraction involving mechanical and hexane extraction. The rapeseed extraction differed for the 20% PHB in the wild-type. This was evidently caused by entrappment of oil in the 80 wm aggregates and by loss to hexane of the 1-2 wm PHB particles. The transgenic seeds required a higher mechanical force to yield the same level of oil. Whereas natural seed with added microbial PHB released this to the oil during pressing and to the hexane during solvent extraction, the transgenic seed evidently did not which would be of importance during large-scale processing. Though with a scale-down of 10⁸ from the likely industrial level the results cannot be quantitatively predictive of production performance, they define process issues and do so several seasons before the quantities needed for pilot trials can be available or the final concentrations of PHB attained.     

油菜高含油量基因工程研究

油菜是世界上重要油料作物之一,是世界食用植物油的重要来源。近十年来,随着其种植面积的不断扩大,目前已成为世界第二大植物油来源,因此提高油菜种子含油量具有重大的经济利用价值。近年来,基因工程技术的飞速发展带来了优化油菜品种资源的新方法。三酰甘油对种子油脂的形成十分重要,它是油菜种子最主要的储藏脂类。将三酰甘油合成代谢途径中的关键酶基因及一些转录因子转入到油菜组基因中,一方面增加种子中关键酶基因的表达;另一方面增加转录因子表达以增强糖酵解和三酰甘油形成的相关基因表达,增加底物浓度和三酰甘油合成的速度,期待获得高含油量的转基因油菜。本文综述了国内外关于油菜油酯代谢关键酶基因及调控基因的研究进展,并展望了未来提高油菜含油量的发展思路。     

Suppression of the SUGAR‐DEPENDENT1 triacylglycerol lipase family during seed development enhances oil yield in oilseed rape (Brassica napus L.)

Increasing the productivity of oilseed crops is an important challenge for plant breeders and biotechnologists. To date, attempts to increase oil production in seeds via metabolic pathway engineering have focused on boosting synthetic capacity. However, in the tissues of many organisms, it is well established that oil levels are determined by both anabolism and catabolism. Indeed, the oil content of rapeseed (Brassica napus L.) has been reported to decline by approximately 10% in the final stage of development, as the seeds desiccate. Here, we show that RNAi suppression of the SUGAR‐DEPENDENT1 triacylglycerol lipase gene family during seed development results in up to an 8% gain in oil yield on either a seed, plant or unit area basis in the greenhouse, with very little adverse impact on seed vigour. Suppression of lipolysis could therefore constitute a new method for enhancing oil yield in oilseed crops.     

<Emphasis Type="Italic">MAM</Emphasis> gene silencing leads to the induction of C3 and reduction of C4 and C5 side-chain aliphatic glucosinolates in <Emphasis Type="Italic">Brassica napus</Emphasis>

Methylthioalkylmalate (MAM) synthases and their associated genes that have been extensively investigated in Arabidopsis control the side-chain elongation of methionine during the synthesis of aliphatic glucosinolates. A Brassica homolog of the Arabidopsis MAM genes was used in this study to analyze the role of MAM genes in B. napus through RNA interference (RNAi). The silencing of the MAM gene family in B. napus canola and B. napus rapeseed resulted in the reduction of aliphatic glucosinolates and total glucosinolate content. The results indicated that RNAi has potential for reducing glucosinolate content and improving meal quality in B. napus canola and rapeseed cultivars. Interestingly, MAM gene silencing in B. napus significantly induced the production of 2-propenyl glucosinolate, a 3-carbon side-chain glucosinolate commonly found in B. juncea mustard. Most transgenic plants displayed induction of 2-propenyl glucosinolate; however, the absolute content of this glucosinolate in transgenic B. napus canola was relatively low (less than 1.00 μmol g⁻¹ seed). In the high glucosinolate content progenies derived from the crosses of B. napus rapeseed and transgenic B. napus canola, MAM gene silencing strongly induced the production of 2-propenyl glucosinolate to high levels (up to 4.45 μmol g⁻¹ seed).