Chao Zhang

HD-Zip III transcription factors are known to play roles in regulating shoot apical meristem (SAM) and axillary meristem (AM) development, exerting direct effects on plant architecture and branching. However, the HD-Zip III (BjuHDZ) gene family has not been systematically characterized in Brassica juncea L. (B. juncea). Therefore, in this study, we performed a comprehensive bioinformatics analysis of the BjuHDZ gene family and identified a total of 20 members. These genes were randomly and unevenly distributed across the 16 chromosomes of B. juncea, and their encoded proteins exhibited divergent physicochemical properties. Through phylogenetic analysis, the 20 BjuHDZ proteins were divided into five subgroups (REV, HB8, HB9, HB14, and HB15). Conserved exon–intron organization and motif profiles were observed within each BjuHDZ gene subgroup. Furthermore, their promoter sequences harbored a wide array of cis-elements implicated in light responsiveness, hormonal regulation, stress tolerance, and growth/development. The qRT-PCR analysis showed that the expression levels of BjuHDZ7, BjuHDZ13, and BjuHDZ15 were significantly upregulated in the middle and upper axillary buds of two pairs of multi-branching (Bj02 and, Bj08) accessions. Meanwhile, BjuHDZ17 was highly expressed in the upper, middle, and lower axillary buds as well as in the SAM of both multi-branching materials. Taken together, these findings lay a foundation for further investigation into the biological functions of BjuHDZ genes in B. juncea.

Rapeseed (Brassica napus L.) has been cultivated as an ornamental plant in recent years. However, the metabolic and regulatory processes involved in pigment accumulation in. B. napus flowers are poorly understood. To address this knowledge gap, we conducted a multi-omics analysis of the orange-red-flowered ‘OrP’ and the yellow-flowered ‘ZS11’ rapeseed cultivars. The total anthocyanin content of ‘OrP’ petals was 5.420-fold and 3.345-fold higher than ‘ZS11’ petals at the S2 and S4 developmental stages, respectively. The red coloration of ‘OrP’ flowers resulted primarily from the presence of anthocyanin pigment derivatives. The up-regulated differentially expressed genes (DEGs) of four stages in ‘OrP’ were found to be significantly enriched in phenylpropanoid, flavonoid, and anthocyanin metabolism-associated GO and KEGG terms. Weighted Gene Co-expression Network Analysis (WGCNA) revealed that 51 DEGs were linked to anthocyanin metabolism, including several structural genes such as BnaCHS, BnaF3H, BnaF3′H, BnaCHS, BnaDFR, BnaANS, BnaUGTs, and the transcription factor (TF) genes BnaHY5, BnaBBX22, BnaPIL1, BnaPAP2, BnaTT8, BnaTTG2, and BnaMYBL2. Furthermore, we found that three main factors affecting the relative content of anthocyanins in petals were likely responsible for the fading of ‘OrP’ petals, namely the significantly down-regulated expression of genes (BnaDFR, BnaANS, BnaPAP2, BnaTT8, and BnaTTG2) related to anthocyanin biosynthesis, the significantly up-regulated expression of genes (Bna.BGLUs, Bna.PRXs, and BnaMYBL2) related to anthocyanin degradation or the negative regulation of anthocyanin biosynthesis, and the rapidly increasing petals area.

Growth-regulating factors (GRFs) are plant-specific transcription factors involved in the regulation of plant growth, development, and abiotic stress processes. However, the function of Brassica juncea (L.) Czern & Coss GRFs remain largely unknown. In this study, 34 BjGRF genes were identified in B. juncea. BjGRF members of the same subfamily were found to share a similar motif composition and gene structure. In total, 663 cis-acting elements were found in the promoter regions of BjGRF genes, which were related to light response, hormone response, environmental stresses, and plant growth/development. Additionally, 48 pairs of segmental duplication genes were identified during gene duplication events, and no tandemly duplicated genes were identified. qRT-PCR analysis showed that the 34 BjGRF genes were primarily expressed in the roots, followed by the leaves. Furthermore, the 10 BjGRF genes were screened in response to drought stress, and the expression patterns of the genes were relatively consistent, with a maximum expression level at 3 or 24 h, and the selected BjGRF03 and BjGRF32 might be involved in drought stress. This preliminary study clarifies the response of the BjGRF gene family to drought stress and provides ideas for further analyses of the biological functions of BjGRF genes.

Rapeseed (Brassica napus L.) is an important oilseed crop widely cultivated worldwide, and drought is the main environmental factor limiting its yield enhancement and the expansion of planted areas. SIMILAR TO RCD ONE (SRO) is a plant-specific small gene family that plays a crucial role in plant growth, development, and responses to abiotic stresses such as drought. However, the functional role of SROs in rapeseed remains poorly understood. In this study, 19 BnaSROs were identified from the rapeseed genome, with 9, 10, 10, 18, and 20 members identified from the genomes of Brassica rapa, Brassica nigra, Brassica oleracea, Brassica juncea, and Brassica carinata, respectively. We then analyzed their sequence characteristics, phylogenetic relationships, gene structures, and conserved domains, and explored the collinearity relationships of the SRO members in Brassica napus and Brassica juncea. Next, we focused on the analysis of tissue expression and stress-responsive expression patterns of rapeseed SRO members and examined their expression profiles under ABA, MeJA and water-deficit drought treatments using qPCR. Transcriptome data analysis and qPCR detection indicated that BnaSROs exhibit multiple stress-responsive expression patterns. BnaSRO1 and BnaSRO11, which are likely to function through interactions with NAC transcription factors, were screened as major drought-regulated members. Our results provide a solid foundation for functional analysis of the role of the SRO gene family in abiotic stress responses, especially drought stress responses, 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.

Harvest index (HI) is an important agronomic trait, and the HI of rapeseed (Brassica napus L.) remains much lowerer than that of other major crops, indicating there is large scope for improvement of this trait. It is important to explore the genetic mechanism of rapeseed harvest index for its genetic improvement. In this study, QTLs for HI using the 186 recombinant inbred lines (RIL) in the three environments (2016, 2017, and 2018) and best linear unbiased prediction (BLUP) were detected based on the high-density genetic linkage map. A total of 12 HI QTLs located on chromosomes A03, A05, A06, A07, A09, C04, and C05_random were detected, with the explained phenotypic variation of individual QTL range from 1.27% to 14.20%. GWAS for HI was performed using 588 resequencing natural populations established by our previous study. A total of 6 SNPs associated with HI were detected in three years (2016, 2017, and 2019) and BLUP, which located on chromosomes A09, C01, and C03. The S9_25882060 on chromosome A09 detected by the environment in 2019 overlapped with S9_25961704, and was close to S9_24834640 on chromosome A09 detected by the environment in 2016. In addition, 2016HI (S9_24834640) detected in 2016 was located in the interval region of q2017HI-9. Among these 18 loci, one locus was overlapped with reported HI-related QTLs, and six loci were contiguous to reported yield-related QTLs. Combining the results of transcriptome sequencing data of our laboratory, 36 primary candidate genes were selected and these genes involved in the photosynthesis, transmembrane transport, storage components synthesis, transcriptional regulation and so on. These results provide important theoretical basis for genetic improvement of HI in rapeseed.

The allotetraploid species Brassica juncea (mustard) is grown worldwide as oilseed and vegetable crops; the yellow seed-color trait is particularly important for oilseed crops. Here, to examine the factors affecting seed coat color, we performed a metabolic and transcriptomic analysis of yellow- and dark-seeded B. juncea seeds. In this study, we identified 236 compounds, including 31 phenolic acids, 47 flavonoids, 17 glucosinolates, 38 lipids, 69 other hydroxycinnamic acid compounds, and 34 novel unknown compounds. Of these, 36 compounds (especially epicatechin and its derivatives) accumulated significantly different levels during the development of yellow- and dark-seeded B. juncea. In addition, the transcript levels of BjuDFR, BjuANS,BjuBAN, BjuTT8, and BjuTT19 were closely associated with changes to epicatechin and its derivatives during seed development, implicating this pathway in the seed coat color determinant in B. juncea. Furthermore, we found numerous variations of sequences in the TT8A genes that may be associated with the stability of seed coat color in B. rapa, B. napus, and B. juncea, which might have undergone functional differentiation during polyploidization in the Brassica species. The results provide valuable information for understanding the accumulation of metabolites in the seed coat color of B. juncea and lay a foundation for exploring the underlying mechanism.

Sugars are important throughout a plant’s lifecycle. Monosaccharide transporters (MST) are essential sugar transporters that have been identified in many plants, but little is known about the evolution or functions of MST genes in rapeseed (Brassica napus). In this study, we identified 175 MST genes in B. napus, 87 in Brassica oleracea, and 83 in Brassica rapa. These genes were separated into the sugar transport protein (STP), polyol transporter (PLT), vacuolar glucose transporter (VGT), tonoplast monosaccharide transporter (TMT), inositol transporter (INT), plastidic glucose transporter (pGlcT), and ERD6-like subfamilies, respectively. Phylogenetic and syntenic analysis indicated that gene redundancy and gene elimination have commonly occurred in Brassica species during polyploidization. Changes in exon-intron structures during evolution likely resulted in the differences in coding regions, expression patterns, and functions seen among BnMST genes. In total, 31 differentially expressed genes (DEGs) were identified through RNA-seq among materials with high and low harvest index (HI) values, which were divided into two categories based on the qRT-PCR results, expressed more highly in source or sink organs. We finally identified four genes, including BnSTP5, BnSTP13, BnPLT5, and BnERD6-like14, which might be involved in monosaccharide uptake or unloading and further affect the HI of rapeseed. These findings provide fundamental information about MST genes in Brassica and reveal the importance of BnMST genes to high HI in B. napus.

Alternative splicing (AS) is a post-transcriptional regulatory process that enhances transcriptome diversity, thereby affecting plant growth, development, and stress responses. To identify the new transcripts and changes in the isoform-level AS landscape of rapeseed (Brassica napus) infected with the fungal pathogen Leptosphaeria maculans, we compared eight RNA-seq libraries prepared from mock-inoculated and inoculated B. napus cotyledons and stems. The AS events that occurred in stems were almost the same as those in cotyledons, with intron retention representing the most common AS pattern. We identified 1892 differentially spliced genes between inoculated and uninoculated plants. We performed a weighted gene co-expression network analysis (WGCNA) to identify eight co-expression modules and their Hub genes, which are the genes most connected with other genes within each module. There are nine Hub genes, encoding nine transcription factors, which represent key regulators of each module, including members of the NAC, WRKY, TRAF, AP2/ERF-ERF, C2H2, C2C2-GATA, HMG, bHLH, and C2C2-CO-like families. Finally, 52 and 117 alternatively spliced genes in cotyledons and stems were also differentially expressed between mock-infected and infected materials, such as HMG and C2C2-Dof; which have dual regulatory mechanisms in response to L. maculans. The splicing of the candidate genes identified in this study could be exploited to improve resistance to L. maculans.

Cytokinin oxidase/dehydrogenases (CKXs) play a critical role in the irreversible degradation of cytokinins, thereby regulating plant growth and development. Brassica napus is one of the most widely cultivated oilseed crops worldwide. With the completion of whole-genome sequencing of B. napus, genome-wide identification and expression analysis of the BnCKX gene family has become technically feasible. In this study, we identified 23 BnCKX genes and analyzed their phylogenetic relationships, gene structures, conserved motifs, protein subcellular localizations, and other properties. We also analyzed the expression of the 23 BnCKX genes in the B. napus cultivar Zhong Shuang 11 (‘ZS11’) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), revealing their diverse expression patterns. We selected four BnCKX genes based on the results of RNA-sequencing and qRT-PCR and compared their expression in cultivated varieties with extremely long versus short siliques. The expression levels of BnCKX5-1, 5-2, 6-1, and 7-1 significantly differed between the two lines and changed during pod development, suggesting they might play roles in determining silique length and in pod development. Finally, we investigated the effects of treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA) and the auxin indole-3-acetic acid (IAA) on the expression of the four selected BnCKX genes. Our results suggest that regulating BnCKX expression is a promising way to enhance the harvest index and stress resistance in plants.