Natural variation in BnaA9.NF-YA7 contributes to drought tolerance in Brassica napus L.

Rapeseed (Brassica napus) is one of the important oil crops worldwide. Its production is often threatened by drought stress. Here, we identify a transcription factor (BnaA9.NF-YA7) that negatively regulates drought tolerance through genome-wide association study in B. napus. The presence of two SNPs within a CCAAT cis element leads to downregulation of BnaA9.NF-YA7 expression. In addition, the M63I (G-to-C) substitution in the transactivation domain can activate low level expression of BnaA4.DOR, which is an inhibitory factor of ABA-induced stomatal closure. Furthermore, we determine that Bna.ABF3/4s directly regulate the expression of BnaA9.NF-YA7, and BnaA9.NF-YA7 indirectly suppresses the expression of Bna.ABF3/4s by regulation of Bna.ASHH4s. Our findings uncover that BnaA9.NF-YA7 serves as a supplementary role for ABA signal balance under drought stress conditions, and provide a potential molecular target to breed drought-tolerant B. napus cultivars.

Identification of the High-Affinity Potassium Transporter Gene Family (HKT) in Brassica U-Triangle Species and Its Potential Roles in Abiotic Stress in Brassica napus L.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

LESION MIMIC MUTANT 1 confers basal resistance to Sclerotinia sclerotiorum in rapeseed via a salicylic acid-dependent pathway.

Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia-resistant and light-dependent lesion mimic mutant from an ethyl methanesulfonate-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, named LESION MIMIC MUTANT 1 (LMM1), which mapped onto chromosome C04 by bulked segregant analysis within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance, which is consistent with the higher salicylic acid levels in lmm1. Combining bulked segregant analysis and transcriptome analysis, we identified a significantly up-regulated gene, BnaC4.PR2, which encodes ß-1,3-glucanase, as the candidate gene for LMM1. Overexpression of BnaC4.PR2 may induce a reactive oxygen species burst to trigger partial cell death and systemic acquired resistance. Our study provides a new genetic resource for S. sclerotiorum resistance as well as new insights into disease resistance breeding in B. napus.

Identification of Trehalose-6-Phosphate Synthase (TPS) Genes Associated with Both Source-/Sink-Related Yield Traits and Drought Response in Rapeseed (Brassica napus L.).

Trehalose-6-phosphate synthase (TPS) is an important enzyme for the synthesis of Trehalose-6-phosphate (T6P). In addition to being a signaling regulator of carbon allocation that improves crop yields, T6P also plays essential roles in desiccation tolerance. However, comprehensive studies, such as evolutionary analysis, expression analysis, and functional classification of the TPS family in rapeseed (Brassica napus L.) are lacking. Here, we identified 35 BnTPSs, 14 BoTPSs, and 17 BrTPSs in cruciferous plants, which were classified into three subfamilies. Phylogenetic and syntenic analysis of TPS genes in four cruciferous species indicated that only gene elimination occurred during their evolution. Combined phylogenetic, protein property, and expression analysis of the 35 BnTPSs suggested that changes in gene structures might have led to changes in their expression profiles and further functional differentiation during their evolution. In addition, we analyzed one set of transcriptome data from Zhongshuang11 (ZS11) and two sets of data from extreme materials associated with source-/sink-related yield traits and the drought response. The expression levels of four BnTPSs (BnTPS6, BnTPS8, BnTPS9, and BnTPS11) increased sharply after drought stress, and three differentially expressed genes (BnTPS1, BnTPS5, and BnTPS9) exhibited variable expression patterns among source and sink tissues between yield-related materials. Our findings provide a reference for fundamental studies of TPSs in rapeseed and a framework for future functional research of the roles of BnTPSs in both yield and drought resistance.

Comparative transcriptome analysis identifies candidate genes related to seed coat color in rapeseed.

Yellow seed coat in rapeseed (Brassica napus) is a desirable trait that can be targeted to improve the quality of this oilseed crop. To better understand the inheritance mechanism of the yellow-seeded trait, we performed transcriptome profiling of developing seeds in yellow- and black-seeded rapeseed with different backgrounds. The differentially expressed genes (DEGs) during seed development showed significant characteristics, these genes were mainly enriched for the Gene Ontology (GO) terms carbohydrate metabolic process, lipid metabolic process, photosynthesis, and embryo development. Moreover, 1206 and 276 DEGs, which represent candidates to be involved in seed coat color, were identified between yellow- and black-seeded rapeseed during the middle and late stages of seed development, respectively. Based on gene annotation, GO enrichment analysis, and protein-protein interaction network analysis, the downregulated DEGs were primarily enriched for the phenylpropanoid and flavonoid biosynthesis pathways. Notably, 25 transcription factors (TFs) involved in regulating flavonoid biosynthesis pathway, including known (e.g., KNAT7, NAC2, TTG2 and STK) and predicted TFs (e.g., C2H2-like, bZIP44, SHP1, and GBF6), were identified using integrated gene regulatory network (iGRN) and weight gene co-expression networks analysis (WGCNA). These candidate TF genes had differential expression profiles between yellow- and black-seeded rapeseed, suggesting they might function in seed color formation by regulating genes in the flavonoid biosynthesis pathway. Thus, our results provide in-depth insights that facilitate the exploration of candidate gene function in seed development. In addition, our data lay the foundation for revealing the roles of genes involved in the yellow-seeded trait in rapeseed.

Comparative transcriptome and co-expression network analysis revealed the genes associated with senescence and polygalacturonase activity involved in pod shattering of rapeseed.

BACKGROUND: The pod shattering (PS) trait negatively affects the crop yield in rapeseed especially under dry conditions. To better understand the trait and cultivate higher resistance varieties, it's necessary to identify key genes and unravel the PS mechanism thoroughly. RESULTS: In this study, we conducted a comparative transcriptome analysis between two materials significantly different in silique shatter resistance lignin deposition and polygalacturonase (PG) activity. Here, we identified 10,973 differentially expressed genes at six pod developmental stages. We found that the late pod development stages might be crucial in preparing the pods for upcoming shattering events. GO enrichment results from K-means clustering and weighed gene correlation network analysis (WGCNA) both revealed senescence-associated genes play an important role in PS. Two hub genes Bna.A05ABI5 and Bna.C03ERF/AP2-3 were selected from the MEyellow module, which possibly regulate the PS through senescence-related mechanisms. Further investigation found that senescence-associated transcription factor Bna.A05ABI5 upregulated the expression of SAG2 and ERF/AP2 to control the shattering process. In addition, the upregulation of Bna.C03ERF/AP2-3 is possibly involved in the transcription of downstream SHP1/2 and LEA proteins to trigger the shattering mechanism. We also analyzed the PS marker genes and found Bna.C07SHP1/2 and Bna.PG1/2 were significantly upregulated in susceptible accession. Furthermore, the role of auxin transport by Bna.WAG2 was also observed, which could reduce the PG activity to enhance the PS resistance through the cell wall loosening process. CONCLUSION: Based on comparative transcriptome evaluation, this study delivers insights into the regulatory mechanism primarily underlying the variation of PS in rapeseed. Taken together, these results provide a better understanding to increase the yield of rapeseed by reducing the PS through better engineered crops.

Genome-Wide Association Study of Glucosinolate Metabolites (mGWAS) in Brassica napus L.

Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates (m145 gluconapin, m150 glucobrassicanapin and m151 progoitrin), one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis (e.g., BnaMAM1, BnaGGP1, BnaSUR1 and BnaMYB51) and novel genes (e.g., BnaMYB44, BnaERF025, BnaE2FC, BnaNAC102 and BnaDREB1D) were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species.

Genome-Wide Identification and Posttranscriptional Regulation Analyses Elucidate Roles of Key Argonautes and Their miRNA Triggers in Regulating Complex Yield Traits in Rapeseed.

Argonautes (AGOs) interact with microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), which can posttranscriptionally regulate the expression of targeted genes. To date, however, the AGOs and their miRNA triggers remain elusive in rapeseed (Brassica napus). Here, we systematically performed a phylogenetic analysis and examined the collinear relationships of the AGOs among four Brassicaceae species. Their physicochemical properties, gene structures, and expression patterns among 81 tissues from multiple materials and developmental stages were further analyzed. Additionally, their posttranscriptional regulation was analyzed using psRNATarget prediction, miRNA-/mRNA-Seq analyses, and a qRT-PCR verification. We finally identified 10 AtAGOs, 13 BolAGOs, 11 BraAGOs, and 24 BnaAGOs. An expression analysis of the BnaAGOs in the B. napus cultivar ZS11, as well as genotypes with extreme phenotypes in various yield-related traits, revealed the conservation and diversity of these genes. Furthermore, we speculated the posttranscriptional regulation of the B. napus miR168a-AGO1s and miR403-AGO2s modules. Combining miRNA-Seq and mRNA-Seq analyses, we found that the B. napus miR168a-AGO1s module may play an essential role in negatively regulating yield traits, whereas the miR403-AGO2s module positively impacts yield. This is the first attempt to comprehensively analyze the AGOs and their miRNA triggers in B. napus and provides a theoretical basis for breeding high-yielding varieties through the manipulation of the miRNA-AGOs modules.

Multi-omics revolution to promote plant breeding efficiency.

Crop production is the primary goal of agricultural activities, which is always taken into consideration. However, global agricultural systems are coming under increasing pressure from the rising food demand of the rapidly growing world population and changing climate. To address these issues, improving high-yield and climate-resilient related-traits in crop breeding is an effective strategy. In recent years, advances in omics techniques, including genomics, transcriptomics, proteomics, and metabolomics, paved the way for accelerating plant/crop breeding to cope with the changing climate and enhance food production. Optimized omics and phenotypic plasticity platform integration, exploited by evolving machine learning algorithms will aid in the development of biological interpretations for complex crop traits. The precise and progressive assembly of desire alleles using precise genome editing approaches and enhanced breeding strategies would enable future crops to excel in combating the changing climates. Furthermore, plant breeding and genetic engineering ensures an exclusive approach to developing nutrient sufficient and climate-resilient crops, the productivity of which can sustainably and adequately meet the world's food, nutrition, and energy needs. This review provides an overview of how the integration of omics approaches could be exploited to select crop varieties with desired traits.

Identification of candidate genes regulating seed oil content by QTL mapping and transcriptome sequencing in Brassica napus.

Increasing oil production is a major goal in rapeseed (Brassica napus) molecular breeding programs. Identifying seed oil content (SOC)-related candidate genes is an important step towards achieving this goal. We performed quantitative trait locus (QTL) mapping of SOC in B. napus using a high-density SNP genetic map constructed from recombinant inbred lines and the Illumina InfiniumTM 60K SNP array. A total of 26 QTLs were detected in three years on A01, A03, A05, A06, A09, C01, C03 and C05, which accounted for 3.69%~18.47% of the phenotypic variation in SOC. Of these, 13 QTLs are reported here for the first time. 1713 candidate genes in the 26 QTLs confidence interval were obtained. We then identified differentially expressed genes (DEGs) between the high- and low-SOC accessions, to narrow down our focus to 21 candidate genes (Y1-Y21) related to SOC, and we will focus on 11 (Y1-Y11) candidate genes that contribute to the formation of high-SOC. In addition to providing insight into the genetic basis of SOC in B. napus, the loci identified and candidate genes in this study can be used in molecular breeding strategies to increase SOC in this important seed crop.

Biomolecular Strategies for Vascular Bundle Development to Improve Crop Yield.

The need to produce crops with higher yields is critical due to a growing global population, depletion of agricultural land, and severe climate change. Compared with the "source" and "sink" transport systems that have been studied a lot, the development and utilization of vascular bundles (conducting vessels in plants) are increasingly important. Due to the complexity of the vascular system, its structure, and its delicate and deep position in the plant body, the current research on model plants remains basic knowledge and has not been repeated for crops and applied to field production. In this review, we aim to summarize the current knowledge regarding biomolecular strategies of vascular bundles in transport systems (source-flow-sink), allocation, helping crop architecture establishment, and influence of the external environment. It is expected to help understand how to use sophisticated and advancing genetic engineering technology to improve the vascular system of crops to increase yield.

BnKAT2 Positively Regulates the Main Inflorescence Length and Silique Number in Brassica napus by Regulating the Auxin and Cytokinin Signaling Pathways.

Brassica napus is the dominant oil crop cultivated in China for its high quality and high yield. The length of the main inflorescence and the number of siliques produced are important traits contributing to rapeseed yield. Therefore, studying genes related to main inflorescence and silique number is beneficial to increase rapeseed yield. Herein, we focused on the effects of BnKAT2 on the main inflorescence length and silique number in B. napus. We explored the mechanism of BnKAT2 increasing the effective length of main inflorescence and the number of siliques through bioinformatics analysis, transgenic technology, and transcriptome sequencing analysis. The full BnKAT2(BnaA01g09060D) sequence is 3674 bp, while its open reading frame is 2055 bp, and the encoded protein comprises 684 amino acids. BnKAT2 is predicted to possess two structural domains, namely KHA and CNMP-binding domains. The overexpression of BnKAT2 effectively increased the length of the main inflorescence and the number of siliques in B. napus, as well as in transgenic Arabidopsis thaliana. The type-A Arabidopsis response regulator (A-ARR), negative regulators of the cytokinin, are downregulated in the BnKAT2-overexpressing lines. The Aux/IAA, key genes in auxin signaling pathways, are downregulated in the BnKAT2-overexpressing lines. These results indicate that BnKAT2 might regulate the effective length of the main inflorescence and the number of siliques through the auxin and cytokinin signaling pathways. Our study provides a new potential function gene responsible for improvement of main inflorescence length and silique number, as well as a candidate gene for developing markers used in MAS (marker-assisted selection) breeding to improve rapeseed yield.

Spatio-temporal transcriptome profiling and subgenome analysis in Brassica napus.

Brassica napus is an important oil crop and an allotetraploid species. However, the detailed analysis of gene function and homoeologous gene expression in all tissues at different developmental stages was not explored. In this study, we performed a global transcriptome analysis of 24 vegetative and reproductive tissues at six developmental stages (totally 111 tissues). These samples were clustered into eight groups. The gene functions of silique pericarp were similar to roots, stems and leaves. In particular, glucosinolate metabolic process was associated with root and silique pericarp. Genes involved in protein phosphorylation were often associated with stamen, anther and the early developmental stage of seeds. Transcription factor (TF) genes were more specific than structural genes. A total of 17 100 genes that were preferentially expressed in one tissue (tissue-preferred genes, TPGs), including 889 TFs (5.2%), were identified in the 24 tissues. Some TPGs were identified as hub genes in the co-expression network analysis, and some TPGs in different tissues were involved in different hormone pathways. About 67.0% of the homoeologs showed balanced expression, whereas biased expression of homoeologs was associated with structural divergence. In addition, the spatiotemporal expression of homoeologs was related to the presence of transposable elements (TEs) and regulatory elements (REs); more TEs and fewer REs in the promoters resulted in divergent expression in different tissues. This study provides a valuable transcriptional map for understanding the growth and development of B. napus, for identifying important genes for future crop improvement, and for exploring gene expression patterns in the B. napus.

Rapeseed (Brassica napus) Mitogen-Activated Protein Kinase 1 Enhances Shading Tolerance by Regulating the Photosynthesis Capability of Photosystem II.

Rapeseed (Brassica napus) is the third-largest source of vegetable oil in the world with an edible, medicinal, and ornamental value. However, insufficient light or high planting density directly affects its growth, development, yield, and quality. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that play key roles in regulating the responses to biotic and abiotic stresses in plants. In this study, we found that the promoter of BnaMAPK1 contained several light-responsive elements (including the AT1-motif, G-Box, and TCT-motif), consistent with its shading stress-induced upregulation. Compared with the wild type under shading stress, BnaMAPK1-overexpressing plants showed higher light capture efficiency and carbon assimilation capacity, enhancing their shading tolerance. Using RNA sequencing, we systematically investigated the function of BnaMAPK1 in shading stress on photosynthetic structure, Calvin cycle, and light-driven electron transport. Notably, numerous genes encoding light-harvesting chlorophyll a/b-binding proteins (BnaLHCBs) in photosystem II-light-harvesting complex (LHC) II supercomplex were significantly downregulated in the BnaMAPK1-overexpressing lines relative to the wild type under shading stress. Combining RNA sequencing and yeast library screening, a candidate interaction partner of BnaMAPK1 regulating in shading stress, BnaLHCB3, was obtained. Moreover, yeast two-hybrid and split-luciferase complementation assays confirmed the physical interaction relationship between BnaLHCB3 and BnaMAPK1, suggesting that BnaMAPK1 may involve in stabilizing the photosystem II-LHC II supercomplex. Taken together, our results demonstrate that BnaMAPK1 positively regulates photosynthesis capability to respond to shading stress in rapeseed, possibly by controlling antenna proteins complex in photosystem II, and could provide valuable information for further breeding for rapeseed stress tolerance.

Genome-Wide Association Study of Phenylalanine Derived Glucosinolates in Brassica rapa.

Glucosinolates (GSLs) are sulfur-containing bioactive compounds usually present in Brassicaceae plants and are usually responsible for a pungent flavor and reduction of the nutritional values of seeds. Therefore, breeding rapeseed varieties with low GSL levels is an important breeding objective. Most GSLs in Brassica rapa are derived from methionine or tryptophan, but two are derived from phenylalanine, one directly (benzylGSL) and one after a round of chain elongation (phenethylGSL). In the present study, two phenylalanine (Phe)-derived GSLs (benzylGSL and phenethylGSL) were identified and quantified in seeds by liquid chromatography and mass spectrometry (LC-MS) analysis. Levels of benzylGSL were low but differed among investigated low and high GSL genotypes. Levels of phenethylGSL (also known as 2-phenylethylGSL) were high but did not differ among GSL genotypes. Subsequently, a genome-wide association study (GWAS) was conducted using 159 B. rapa accessions to demarcate candidate regions underlying 43 and 59 QTNs associated with benzylGSL and phenethylGSL that were distributed on 10 chromosomes and 9 scaffolds, explaining 0.56% to 70.86% of phenotypic variations, respectively. Furthermore, we find that 15 and 18 known or novel candidate genes were identified for the biosynthesis of benzylGSL and phenethylGSL, including known regulators of GSL biosynthesis, such as BrMYB34, BrMYB51, BrMYB28, BrMYB29 and BrMYB122, and novel regulators or structural genes, such as BrMYB44/BrMYB77 and BrMYB60 for benzylGSL and BrCYP79B2 for phenethylGSL. Finally, we investigate the expression profiles of the biosynthetic genes for two Phe-derived GSLs by transcriptomic analysis. Our findings provide new insight into the complex machinery of Phe-derived GSLs in seeds of B. rapa and help to improve the quality of Brassicaceae plant breeding.

Multi-omics analysis reveals the mechanism of seed coat color formation in Brassica rapa L.

KEYMESSAGE: Multi-omics analysis of the transcriptome, metabolome and genome identified major and minor loci and candidate genes for seed coat color and explored the mechanism of flavonoid metabolites biosynthesis in Brassica rapa. Yellow seed trait is considered an agronomically desirable trait with great potential for improving seed quality of Brassica crops. Mechanisms of the yellow seed trait are complex and not well understood. In this study, we performed an integrated metabolome, transcriptome and genome-wide association study (GWAS) on different B. rapa varieties to explore the mechanisms underlying the seed coat color formation. A total of 2,499 differentially expressed genes and 116 differential metabolites between yellow and black seeds with strong association with the flavonoid biosynthesis pathway was identified. In addition, 330 hub genes involved in the seed coat color formation, and the most significantly differential flavonoids biosynthesis were detected based on weighted gene co-expression network analysis. Metabolite GWAS analysis using the contents of 42 flavonoids in developing seeds of 159 B. rapa lines resulted in the identification of 1,626 quantitative trait nucleotides (QTNs) and 37 chromosomal intervals, including one major locus on chromosome A09. A combination of QTNs detection, transcriptome and functional analyses led to the identification of 241 candidate genes that were associated with different flavonoid metabolites. The flavonoid biosynthesis pathway in B. rapa was assembled based on the identified flavonoid metabolites and candidate genes. Furthermore, BrMYB111 members (BraA09g004490.3C and BraA06g034790.3C) involved in the biosynthesis of taxifolin were functionally analyzed in vitro. Our findings lay a foundation and provide a reference for systematically investigating the mechanism of seed coat color in B. rapa and in the other plants.

Metabolite Characteristics Analysis of Siliques and Effects of Lights on the Accumulation of Glucosinolates in Siliques of Rapeseed.

Glucosinolates (GSLs) are naturally occurring secondary metabolites found in the Brassicaceae family, which mainly synthesize in the siliques with a wide range of functions. In this study, we investigated the effects of lights on metabolites in siliques of rapeseed through ultra high-performance liquid chromatography (UPLC)-heated electrospray ionization (HESI)-tandem mass spectrometry (MS/MS). A total of 249 metabolites, including 29 phenolic acids, 38 flavonoids, 22 GSLs, 93 uncalculated and 67 unknown compounds, were identified in siliques of rapeseed. Meanwhile, 62 metabolites showed significant differences after shading treatment, which were mainly GSLs and unknown compounds. Interestingly, the amounts of 10 GSLs had high accumulation levels in siliques, while the expression levels of their corresponding biosynthetic genes (AOP, GSL-OH, IGMT, and ST5a) were obviously reduced after shading treatment. Further evidence showed that the amounts of GSLs were significantly reduced in seeds, in accordance with the expression profiles of transporter genes (BnaGTRs). Our findings indicated that lights could affect the accumulation and transportation of GSLs from siliques to seeds in rapeseed. Therefore, this study facilitates a better understanding of metabolic characteristics of siliques and provides insight into the importance of light for GSLs accumulation and transportation in siliques and seeds of rapeseed.

Genome-Wide Identification of the TIFY Gene Family in Brassiceae and Its Potential Association with Heavy Metal Stress in Rapeseed.

The TIFY gene family plays important roles in various plant biological processes and responses to stress and hormones. The chromosome-level genome of the Brassiceae species has been released, but knowledge concerning the TIFY family is lacking in the Brassiceae species. The current study performed a bioinformatics analysis on the TIFY family comparing three diploid (B. rapa, B. nigra, and B. oleracea) and two derived allotetraploid species (B. juncea, and B. napus). A total of 237 putative TIFY proteins were identified from five Brassiceae species, and classified into ten subfamilies (six JAZ types, one PPD type, two TIFY types, and one ZML type) based on their phylogenetic relationships with TIFY proteins in A. thaliana and Brassiceae species. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the TIFY family genes during the process of polyploidization, and most of these TIFY family genes (TIFYs) were subjected to purifying selection after duplication based on Ka/Ks values. The spatial and temporal expression patterns indicated that different groups of BnaTIFYs have distinct spatiotemporal expression patterns under normal conditions and heavy metal stresses. Most of the JAZIII subfamily members were highest in all tissues, but JAZ subfamily members were strongly induced by heavy metal stresses. BnaTIFY34, BnaTIFY59, BnaTIFY21 and BnaTIFY68 were significantly upregulated mostly under As3+ and Cd2+ treatment, indicating that they could be actively induced by heavy metal stress. Our results may contribute to further exploration of TIFYs, and provided valuable information for further studies of TIFYs in plant tolerance to heavy metal stress.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.