Identification and characterization of the plasma membrane H+-ATPase genes in Brassica napus and functional analysis of BnHA9 in salt tolerance.

As a primary proton pump, plasma membrane (PM) H+-ATPase plays critical roles in regulating plant growth, development, and stress responses. PM H+-ATPases have been well characterized in many plant species. However, no comprehensive study of PM H+-ATPase genes has been performed in Brassica napus (rapeseed). In this study, we identified 32 PM H+-ATPase genes (BnHAs) in the rapeseed genome, and they were distributed on 16 chromosomes. Phylogenetical and gene duplication analyses showed that the BnHA genes were classified into five subfamilies, and the segmental duplication mainly contributed to the expansion of the rapeseed PM H+-ATPase gene family. The conserved domain and subcellular analyses indicated that BnHAs encoded canonical PM H+-ATPase proteins with 14 highly conserved domains and localized on PM. Cis-acting regulatory element and expression pattern analyses indicated that the expression of BnHAs possessed tissue developmental stage specificity. The 25 upstream open reading frames with the canonical initiation codon ATG were predicted in the 5' untranslated regions of 11 BnHA genes and could be used as potential target sites for improving rapeseed traits. Protein interaction analysis showed that BnBRI1.c associated with BnHA2 and BnHA17, indicating that the conserved activity regulation mechanism of BnHAs may be present in rapeseed. BnHA9 overexpression in Arabidopsis enhanced the salt tolerance of the transgenic plants. Thus, our results lay a foundation for further research exploring the biological functions of PM H+-ATPases in rapeseed.

Lipidomics studies reveal dynamic changes in polar lipids of developing endosperm of oat and wheat varieties with differing oil contents.

Polar lipids have biosynthetic pathways which intersect and overlap with triacylglycerol biosynthesis; however, polar lipids have not been well characterized in the developing endosperms of oat with high oil accumulation. The polar lipids in endosperms of oat and wheat varieties having different oil contents were analyzed and compared at different developmental stages. Our study shows that the relative contents of polar lipid by mass were decreased more slowly in wheat than in oat. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, which showed similar abundance and gradual decreases during endosperm development in oat and wheat, while lysophospholipids were noticeably higher in oat. Monogalactosyldiacylglycerol showed a gradual increase in wheat and a decrease in oat during endosperm development. The relative contents of some polar lipid species and their unsaturation index were significantly different in their endosperms. These characteristics of polar lipids might indicate an adaption of oat to accommodate oil accumulation.

FUSCA3-induced AINTEGUMENTA-like 6 manages seed dormancy and lipid metabolism.

FUSCA 3 (FUS3), a seed master regulator, plays critical role in seed dormancy and oil accumulation. However, its downstream regulation mechanisms remain poorly understood. Here, we explored the roles of AINTEGUMENTA-like 6 (AIL6), a seed transcription factor, in these processes. The activation of AIL6 by FUS3 was demonstrated by dual-LUC assay. Seeds of ail6 mutants showed alterations in fatty acid compositions, and both AtAIL6 (AIL6 from Arabidopsis thaliana) and BnaAIL6 (AIL6 from Brassica napus) rescued the phenotype. Over-expression (OE) of AIL6s reversed changes in seed fatty acid composition. Notably, OE lines showed low seed germination rates down to 12% compared to 100% of wild-type Col-0. Transcriptome analysis of the mutant and an OE line indicated widespread expression changes of genes involved in lipid metabolism and phytohormone pathways. In OE mature seeds, GA4 content decreased more than 15-fold, while abscisic acid and indole-3-acetic acid (IAA) contents clearly increased. Exogenous GA3 treatments did not effectively rescue the low germination rate. Nicking seed coats increased germination rates from 25% to nearly 80% while the wild-type rdr6-11 is 100% and 98% respectively, and elongation of storage time also improved seed germination. Furthermore, dormancy imposed by AIL6 was fully released in the della quintuple mutant. Together, our results indicate AIL6 acts as a manager downstream of FUS3 in seed dormancy and lipid metabolism.

A Set of Molecular Markers to Accelerate Breeding and Determine Seed Purity of CMS Three-Line Hybrids in Brassica napus.

Cytoplasmic male sterility (CMS) is the main mechanism employed to utilize the heterosis of Brassica napus. CMS three-line rapeseed hybrids have dramatically enhanced yield and brought about the global revolution of hybrid varieties, replacing conventional crop varieties. Over the last half century, China has led the development of hybrid Brassica napus varieties. Two sterile lines, polima (pol) and shaan 2A, were of particular importance for the establishment of three-line hybrid systems in rapeseed, which has opened up a new era of heterosis utilization. However, in current breeding practices, it takes up to three years to identify the restorer or maintainer relationship and the cytoplasmic type of any inbred material. This greatly affects the breeding speed of new varieties and inhibits the rapid development of the rapeseed industry. To address this problem, we developed a set of molecular markers for the identification of fertile cytoplasmic gene N and sterile cytoplasmic gene S, as well as for the fertile nucleus gene R and sterile nucleus gene r, based on differences in the gene sequences between the CMS line, maintainer line and restorer line of Brassica napus. Combining these markers can accurately identify the CMS line, maintainer and restorer of both the pol and shaan systems, as well as their hybrids. These markers can not only be used to identify of the maintainer and restorer relationship of inbred materials; they can also be used as general molecular markers to identify the CMS-type hybrid purity of pol and shaan systems.

Multiple caleosins have overlapping functions in oil accumulation and embryo development.

Caleosins are lipid droplet- and endoplasmic reticulum-associated proteins. To investigate their functions in oil accumulation, expression levels of caleosins in developing seeds of Arabidopsis thaliana were examined and four seed-expressed caleosins (CLO1, CLO2, CLO4, and CLO6) were identified. The four single mutants showed similar minor changes of fatty acid composition in seeds. Two double mutants (clo1 clo2 and clo1×clo2) demonstrated distinct changes of fatty acid composition, a 16-23% decrease of oil content, and a 10-13% decrease of seed weight. Moreover, a 40% decrease of oil content, further fatty acid changes, and misshapen membranes of smaller lipid droplets were found in seeds of quadruple CLO RNAi lines. Notably, ~40% of quadruple CLO RNAi T1 seeds failed to germinate, and deformed embryos and seedlings were also observed. Complementation experiments showed that CLO1 rescued the phenotype of clo1 clo2. Overexpression of CLO1 in seedlings and BY2 cells increased triacylglycerol content up to 73.6%. Transcriptome analysis of clo1 clo2 developing seeds showed that expression levels of some genes related to lipid, embryo development, calcium signaling, and stress responses were affected. Together, these results suggest that the major seed-expressed caleosins have overlapping functions in oil accumulation and show pleiotropic effects on embryo development.

Systematic analysis of MADS-box gene family in the U's triangle species and targeted mutagenesis of BnaAG homologs to explore its role in floral organ identity in Brassica napus.

MADS-box transcription factors play an important role in regulating floral organ development and participate in environmental responses. To date, the MADS-box gene family has been widely identified in Brassica rapa (B. rapa), Brassica oleracea (B. oleracea), and Brassica napus (B. napus); however, there are no analogous reports in Brassica nigra (B. nigra), Brassica juncea (B. juncea), and Brassica carinata (B. carinata). In this study, a whole-genome survey of the MADS-box gene family was performed for the first time in the triangle of U species, and a total of 1430 MADS-box genes were identified. Based on the phylogenetic relationship and classification of MADS-box genes in Arabidopsis thaliana (A. thaliana), 1430 MADS-box genes were categorized as M-type subfamily (627 genes), further divided into Mα, Mß, Mγ, and Mδ subclades, and MIKC-type subfamily (803 genes), further classified into 35 subclades. Gene structure and conserved protein motifs of MIKC-type MADS-box exhibit diversity and specificity among different subclades. Comparative analysis of gene duplication events and syngenic gene pairs among different species indicated that polyploidy is beneficial for MIKC-type gene expansion. Analysis of transcriptome data within diverse tissues and stresses in B. napus showed tissue-specific expression of MIKC-type genes and a broad response to various abiotic stresses, particularly dehydration stress. In addition, four representative floral organ mutants (wtl, feml, aglf-2, and aglf-1) in the T0 generation were generated by editing four AGAMOUS (BnaAG) homoeologs in B. napus that enriched the floral organ variant phenotype. In brief, this study provides useful information for investigating the function of MADS-box genes and contributes to revealing the regulatory mechanisms of floral organ development in the genetic improvement of new varieties.

ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation.

ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.

Transcriptome Analysis Reveals Candidate Genes for Petroselinic Acid Biosynthesis in Fruits of Coriandrum sativum L.

Petroselinic acid (18:1Δ6), a monounsaturated cis Δ-6 fatty acid, has many prospective applications in functional foods and for the nutraceutical and pharmaceutical industries. Up to 80% of petroselinic acid has been found in the oil from fruits of coriander (Coriandrum sativum L.), which make it an ideal source for investigating the biosynthesis of petroselinic acid. A coriander acyl-acyl carrier protein desaturase was identified to be involved in its biosynthesis more than two decades ago, but since then little further progress in this area has been reported. In this study, the fatty acid profiles of coriander fruits at six developmental stages were analyzed. Fruit samples from three developmental stages with rapid accumulation of petroselinic acid were used for RNA sequencing using the Illumina Hiseq4000 platform. The transcriptome analysis presented 93 323 nonredundant unigenes and 8545 differentially expressed genes. Functional annotation and combined gene expression data revealed candidate genes potentially involved in petroselinic acid biosynthesis and its incorporation into triacylglycerols. Tissue-specific examination of q-PCR validation further suggested that ACPD1/3, KAS I-1, FATB-1/3, and DGAT2 may be highly involved. Bioinformatic analysis of CsFATB and CsDGAT2 identified their putative key amino acids or functional motifs. These results provide a molecular foundation for petroselinic acid biosynthesis in coriander fruit and facilitate its genetic engineering in other hosts.

Evaluating Targeted Intervention on Coal Miners' Unsafe Behavior.

Miners' unsafe behavior is the main cause of roof accidents in coal mines, and behavior intervention plays a significant role in reducing the occurrence of miners' unsafe behavior. However, traditional behavior intervention methods lack pertinence. In order to improve the intervention effect and reduce the occurrence of coal mine roof accidents more effectively, this study proposed a targeted intervention method for unsafe behavior. The process of targeted intervention node locating was constructed, and based on the analysis of 331 coal mine roof accidents in China, three kinds of targeted intervention nodes were located. The effectiveness of targeted intervention nodes was evaluated by using structural equation model (SEM) through randomly distributing questionnaires to miners of Pingdingshan coal. The results show that, in preventing roof accidents of coal mines, the targeted intervention nodes have a significant positive impact on the intervention effect. The method can also be applied to the safety management of other industries by adjusting the node location and evaluation process.

Strong co-suppression impedes an increase in polyunsaturated fatty acids in seeds overexpressing FAD2.

Fatty acid desaturase2 (FAD2) catalyses the conversion of oleic acid to linoleic acid and is the main determinant of the levels of essential poly-unsaturated fatty acids (PUFAs) in seed oils. The very limited number of successful examples of overexpression of FAD2 over the last two decades and a shortage of reports on co-suppression make it uncertain whether FAD2 can increase PUFAs effectively across a broad range of oil crops. In this study, strong co-suppression was observed in about 80% of over 100 transgenic lines when FAD2 was overexpressed in three oilseed crops, namely flax (Linum usitatissimum), carinata (Brassica carinata), and camelina (Camelina sativa), as well as in the model plant Arabidopsis. Further analyses of Arabidopsis transgenic lines revealed both endogenous and transgenic FAD2 gene-silencing. Thus, the commonality and potency of FAD2 co-suppression seemingly imposes an obstacle to engineering oilseed PUFA enhancement by direct FAD2 overexpression. AtFAD2, driven by the 35S promoter, also caused co-suppression in Arabidopsis roots. The FAD2 co-suppression was unstable and PUFA phenotypes of T4 lines were similar to the wild-type, further indicating that high PUFA content cannot be achieved by screening advanced generations. However, we demonstrate that the obstacle of FAD2 co-suppression can be overcome in the Arabidopsis rdr6 mutant, which is impaired in post-transcriptional gene-silencing, and that lines with high PUFA content are stable through four generations.

An Interactive Model among Potential Human Risk Factors: 331 Cases of Coal Mine Roof Accidents in China.

In order to explore optimal strategies for managing potential human risk factors, this paper developed an interactive model among potential human risk factors based on the development processes of accidents. This model was divided into four stages, i.e., risk latency stage, risk accumulation stage, risk explosion stage and risk residue stage. Based on this model, this paper analyzed risk management procedures and relevant personal's responsibility in each stage, and then probed into the interactive mechanism among human risk factors in three aspects, i.e., knowledge, information and communication. The validity and feasibility of the model was validated by analyzing a coal mine roof accident in China. In addition, the contribution of different functional levels' personnel in risk evolution was discussed. It showed that this model can effectively reveal the interactive mechanism of potential human risk factors, and can thus give significant insights into the development of risk management theories and practices. It also proves that the contribution of different functional levels' personnel in the model is different. This can further help practitioners design enhanced Behavioral-Based Safety (BBS) intervention approaches which can have a more sustainable and persistent impact on corporate personnel's safety behavior. Specific recommendations and suggestions are provided fundamentally for future BBS practices in the coal mine industry.

Investigation of Plant Species with Identified Seed Oil Fatty Acids in Chinese Literature and Analysis of Five Unsurveyed Chinese Endemic Species.

Diverse fatty acid structures from different plant species are important renewable resources for industrial raw materials and as liquid fuels with high energy density. Because of its immense geographical and topographical variations, China is a country with enormous diversity of plant species, including large numbers of plants endemic to China. The richness of this resource of species provides a wide range of fatty acids in seeds or other tissues, many of which have been identified by Chinese scientists. However, in the past, most publications describing analysis of these plants were written in Chinese, making access for researchers from other countries difficult. In this study, we investigated reports on seed and fruit oil fatty acids as described in Chinese literature. Six books and more than one thousand papers were collected and the identified fatty acids and relevant plant species were summarized. In total, about 240 fatty acids from almost 1,500 plant species were identified from available Chinese literature. Only about one third of these species were retrieved in the PhyloFAdb and SOFA online databases of plant fatty acids. By referring to a summary of plant species endemic to China, 277 Chinese endemic species from 68 families have been surveyed for seed fatty acids. These account for <2% of total Angiosperm species endemic to China indicating the scope of species yet to be surveyed. To discover additional new fatty acid structures that might benefit society, it is important in the future to study oilseed fatty acids of the many other Chinese endemic plants. As an example, seeds of five unsurveyed species were collected and their fatty acids were analyzed. Ricinoleic acid was detected for the first time in the Salicaceae family.

FUSCA3 activates triacylglycerol accumulation in Arabidopsis seedlings and tobacco BY2 cells.

Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3 g26790, FUS3), are associated with embryo maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50-fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased three- to four-fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased the expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation.

Characteristic of the pepper CaRGA2 gene in defense responses against Phytophthora capsici Leonian.

The most significant threat to pepper production worldwide is the Phytophthora blight, which is caused by the oomycete pathogen, Phytophthora capsici Leonian. In an effort to help control this disease, we isolated and characterized a P. capsici resistance gene, CaRGA2, from a high resistant pepper (C. annuum CM334) and analyzed its function by the method of real-time PCR and virus-induced gene silencing (VIGS). The CaRGA2 has a full-length cDNA of 3,018 bp with 2,874 bp open reading frame (ORF) and encodes a 957-aa protein. The protein has a predicted molecular weight of 108.6 kDa, and the isoelectric point is 8.106. Quantitative real-time PCR indicated that CaRGA2 expression was rapidly induced by P. capsici. The gene expression pattern was different between the resistant and susceptible cultivars. CaRGA2 was quickly expressed in the resistant cultivar, CM334, and reached to a peak at 24 h after inoculation with P. capsici, five-fold higher than that of susceptible cultivar. Our results suggest that CaRGA2 has a distinct pattern of expression and plays a critical role in P. capsici stress tolerance. When the CaRGA2 gene was silenced via VIGS, the resistance level was clearly suppressed, an observation that was supported by semi-quantitative RT-PCR and detached leave inoculation. VIGS analysis revealed their importance in the surveillance to P. capsici in pepper. Our results support the idea that the CaRGA2 gene may show their response in resistance against P. capsici. These analyses will aid in an effort towards breeding for broad and durable resistance in economically important pepper cultivars.