Gaomiao Zhan

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus.

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10-40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.

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.

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.

Discovery of Pod Shatter-Resistant Associated SNPs by Deep Sequencing of a Representative Library Followed by Bulk Segregant Analysis in Rapeseed

Background Single nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. As of yet, there is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species. Methodology/Principal Findings We demonstrate a novel method for identifying SNP markers in rapeseed by deep sequencing a representative library and performing bulk segregant analysis. With this method, SNPs associated with rapeseed pod shatter-resistance were discovered. Firstly, a reduced representation of the rapeseed genome was used. Genomic fragments ranging from 450–550 bp were prepared from the susceptible bulk (ten F2 plants with the silique shattering resistance index, SSRI <0.10) and the resistance bulk (ten F2 plants with SSRI >0.90), and also Solexa sequencing-produced 90 bp reads. Approximately 50 million of these sequence reads were assembled into contigs to a depth of 20-fold coverage. Secondly, 60,396 ‘simple SNPs’ were identified, and the statistical significance was evaluated using Fishers exact test. There were 70 associated SNPs whose –log10 p value over 16 were selected to be further analyzed. The distribution of these SNPs appeared a tight cluster, which consisted of 14 associated SNPs within a 396 kb region on chromosome A09. Our evidence indicates that this region contains a major quantitative trait locus (QTL). Finally, two associated SNPs from this region were mapped on a major QTL region. Conclusions/Significance 70 associated SNPs were discovered and a major QTL for rapeseed pod shatter-resistance was found on chromosome A09 using our novel method. The associated SNP markers were used for mapping of the QTL, and may be useful for improving pod shatter-resistance in rapeseed through marker-assisted selection and map-based cloning. This approach will accelerate the discovery of major QTLs and the cloning of functional genes for important agronomic traits in rapeseed and other crop species.

Cosuppression of RBCS3B in Arabidopsis leads to severe photoinhibition caused by ROS accumulation

Key messageCosuppression of anArabidopsisRubisco small subunit geneRBCS3BatArabidopsisresulted in albino or pale green phenotypes which were caused by ROS accumulationAbstractAs the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.

Cloning and functions analysis of a pyruvate dehydrogenase kinase in Brassica napus

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), which plays a key role in intermediary metabolism. In this study, a 1,490-bp PDK in Brassica napus (BnPDK1) was isolated and cloned from Brassica cDNA library. BnPDK1 has an 1,104 open reading frame encoding 367 amino acids. Genomic DNA gel blot analysis result indicated that BnPDK1 is a multi-copy gene. RNA gel blot analysis and RNA in situ hybridization were used to determine the expression of BnPDK1 in different organs. BnPDK1 gene was ubiquitously expressed in almost all the tissues tested, having the highest expression in the stamen and the young silique. Over-expression of BnPDK1 in transgenic Arabidopsis lines would repress the PDC activity, and resulted in the decrease of seed oil content and leaf photosynthesis. These results implied that BnPDK1 was involved in the regulation of fatty acid biosynthesis in developing seeds.

Molecular Analysis and Expression Patterns of Four 14-3-3 Genes from Brassica napus L.

In eukaryotes, 14-3-3 proteins constitute a family of ubiquitous regulatory molecules. They play very important roles in many cell processes. However, their enconding genes and roles in plants remain to be elucidated. In this paper, four 14-3-3 genes from Brassica napus L. were obtained by randomly sequencing a full-length cDNA library and named as Bn1433-1-4 respectively. The phylogenetic comparison with Arabidopsis 14-3-3 family showed that Bn1433-1 and Bn1433-2 belonged to Episilon group while Bn1433-3 and Bn1433-4 belonged to non-Episilon group. The transcript levels of four Bn1433 genes were analyzed in different organs, various stress conditions and some hormone treatments by real-time PCR. The result showed that all the Bn1433s were expressed constitutively in roots, stems, leaves and immature seeds except that Bn1433-4 exhibited strong expression in immature seeds of 14 and 28 DAF (day after flowering). By the analysis of real-time PCR, different expression patterns of Bn1433s were studied in various stress conditions and hormone treatments. It was suggested that the functions of Bn1433s are diverse and they are involved in the regulation of various stresses and hormones treatments.

A Simple Method for Isolating Chloroplast DNA and Mitochondria DNA from the Same Rapeseed Green Leaf Tissue

Abstract In the study, we present a fast, simple and inexpensive protocol for isolating chloroplast and mitochondrial DNA from one rapeseed leaf tissue sample. The chloroplast and mitochondria were separated from the same green leaf tissue by differential centrifugations. The protocol is the first report that isolates plant chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from the same sample homogenate. The organelle DNA yield is 2–10 micrograms per gram of tissue; the DNA was fully restrictable and was successfully used for sequencing.

Important photosynthetic contribution of silique wall to seed yield-related traits in Arabidopsis thaliana

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.