Integrating GWAS, RNA-Seq and functional analysis revealed that BnaA02.SE mediates silique elongation by affecting cell proliferation and expansion in Brassica napus.

Rapeseed (Brassica napus) silique is the major carbohydrate source for seed development, and the final silique length has attracted great attention from breeders. However, no studies had focused on the dynamic character of silique elongation length (SEL). Here, the dynamic SEL investigation in a natural population including 588 lines over two years indicate that dynamic SEL during 0-20 days after flowering was the most essential stage associated with seed number per silique (SPS) and thousand seed weight (TSW). Then, nine loci were identified to be associated with SEL based on GWAS analysis, among which five SNPs (over 50%) distributed on the A02 chromosome within 6.08 to 6.48 Mb. Subsequently, we screened 5078 differentially expressed genes between two extreme materials. An unknown protein, BnaA02.SE, was identified combining with GWAS and RNA-Seq analysis. Subcellular localization and expression profiles analysis demonstrated that BnaA02.SE is a chloroplast- and nucleus-localized protein mainly expressed in pericarps and leaves. Furthermore, transgenic verification and dynamic cytological observation reveal that overexpressed BnaA02.SE can promote silique elongation by regulating JA and IAA contents, affecting cell proliferation and expansion, respectively, and finally enhance seed yield by influencing SPS and TSW. Haplotype analysis reveal that the homologs of BnaA02.SE may also be involved in silique elongation regulation. Our findings provided comprehensive insights into a newly SEL trait, and cloned the first gene (BnaA02.SE) controlling silique elongation in B. napus. The identified BnaA02.SE and its homologs can offer a valuable target for improving B. napus yield.

Molecular mapping and candidate gene identification of two major quantitative trait loci associated with silique length in oilseed rape (Brassica napus L.).

Rapeseed is a significant global source of plant oil. Silique size, particularly silique length (SL), impacts rapeseed yield. SL is a typical quantitative trait controlled by multiple genes. In our previous study, we constructed a DH population of 178 families known as the 158A-SGDH population. In this study, through SL QTL mapping, we identified twenty-six QTL for SL across five replicates in two environments. A QTL meta-analysis revealed eight consensus QTL, including two major QTL: cqSL.A02-1 (11.32-16.44% of PVE for SL), and cqSL.C06-1 (10.90-11.95% of PVE for SL). Based on biparental resequencing data and microcollinearity analysis of target regions in Brassica napus and Arabidopsis, we identified 11 candidate genes at cqSL.A02-1 and 6 candidate genes at cqSL.C06-1, which are potentially associated with silique development. Furthermore, transcriptome analysis of silique valves from both parents on the 14th, 21st, and 28th days after pollination (DAP) combined with gene function annotation revealed three significantly differentially expressed genes at cqSL.A02-1, BnaA02G0058500ZS, BnaA02G0060100ZS, and BnaA02G0060900ZS. Only the gene BnaC06G0283800ZS showed significant differences in parental transcription at cqSL.C06-1. Two tightly linked insertion-deletion markers for the cqSL.A02-1 and cqSL.C06-1 loci were developed. Using these two QTL, we generated four combinations: A02SGDH284C06158A, A02SGDH284C06SGDH284, A02158AC06158A, and A02158AC06SGDH284. Subsequent analysis identified an ideal QTL combination, A02158AC06SGDH284, which exhibited the longest SL of this type, reaching 6.06 ± 0.10 cm, significantly surpassing the other three combinations. The results will provide the basis for the cloning of SL-related genes of rapeseed, along with the development of functional markers of target genes and the breeding of rapeseed varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01464-x.

Exploring silique number in Brassica napus L.: Genetic and molecular advances for improving yield.

Silique number is a crucial yield-related trait for the genetic enhancement of rapeseed (Brassica napus L.). The intricate molecular process governing the regulation of silique number involves various factors. Despite advancements in understanding the mechanisms regulating silique number in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), the molecular processes involved in controlling silique number in rapeseed remain largely unexplored. In this review, we identify candidate genes and review the roles of genes and environmental factors in regulating rapeseed silique number. We use genetic regulatory networks for silique number in Arabidopsis and grain number in rice to uncover possible regulatory pathways and molecular mechanisms involved in regulating genes associated with rapeseed silique number. A better understanding of the genetic network regulating silique number in rapeseed will provide a theoretical basis for the genetic improvement of this trait and genetic resources for the molecular breeding of high-yielding rapeseed.

AUX1, PIN3, and TAA1 collectively maintain fertility in Arabidopsis.

MAIN CONCLUSION: We found that auxin synthesis gene TAA1 and auxin polar transport genes AUX1 and PIN3 collectively maintain fertility and seed size in Arabidopsis. Auxin plays a vital role in plant gametophyte development and embryogenesis. The auxin synthesis gene TAA1 and the auxin polar transport genes AUX1 and PIN3 are expressed during Arabidopsis gametophyte and seed development. However, aux1, pin3, and taa1 single mutants only exhibit mild reproductive defects. We, therefore, generated aux1-T pin3 taa1-k2 and aux1-T pin3-2 taa1-k1 triple mutants by crossing or CRISPR/Cas9 technique. These triple mutants displayed severe reproductive defects with approximately 70% and 77%, respectively, of the siliques failing to elongate after anthesis. Reciprocal crosses and microscopy analyses showed that the development of pollen and ovules in the aux1 pin3 taa1 mutants was normal, whereas the filaments were remarkably short, which might be the cause of the silique sterility. Further analyses indicated that the development and morphology of aux1 pin3 taa1 seeds were normal, but their size was smaller compared with that of the wild type. These results indicate that AUX1, PIN3, and TAA1 act in concert to maintain fertility and seed size in Arabidopsis.

Analysis of Altered Flowering Related Genes in a Multi-Silique Rapeseed (Brassica napus L.) Line zws-ms Based on Combination of Genome, Transcriptome and Proteome Data.

Based on previous researches, we further investigated the multi-silique trait in rapeseed (Brassica napus L.) line zws-ms. In this study, we used a relatively comprehensive list of flowering related genes in rapeseed and compared them between zws-ms and its near-isogenic line (NIL) zws-217. Genes were studied on genome, transcriptome and proteome levels and then we focused on genes with non-synonymous single nucleotide polymorphism (SNP) or frame-shift insertion-deletion (InDel), finding some genes on the list which changes their sequences. Then, combined with their annotation and the information of their orthologs, certain genes such as BnaA09g05900D, ortholog of AGAMOUS-LIKE 42 (AGL42), which encodes an MADS-box protein, were assumed as probably responsible for the multi-silique trait. Also, we analyzed the Differentially Accumulated Proteins (DAPs) between zws-ms and zws-217, revealing some genes involved in homologous recombination and mismatch repair pathways. Since the development of flowers/siliques is crucial to crops and it influences the yield of rapeseed, this study paved a way to deeply understand the mechanism of the multi-pistil flower formation, which may facilitate researches on rapeseed production in future.

Point clouds segmentation of rapeseed siliques based on sparse-dense point clouds mapping.

In this study, we propose a high-throughput and low-cost automatic detection method based on deep learning to replace the inefficient manual counting of rapeseed siliques. First, a video is captured with a smartphone around the rapeseed plants in the silique stage. Feature point detection and matching based on SIFT operators are applied to the extracted video frames, and sparse point clouds are recovered using epipolar geometry and triangulation principles. The depth map is obtained by calculating the disparity of the matched images, and the dense point cloud is fused. The plant model of the whole rapeseed plant in the silique stage is reconstructed based on the structure-from-motion (SfM) algorithm, and the background is removed by using the passthrough filter. The downsampled 3D point cloud data is processed by the DGCNN network, and the point cloud is divided into two categories: sparse rapeseed canopy siliques and rapeseed stems. The sparse canopy siliques are then segmented from the original whole rapeseed siliques point cloud using the sparse-dense point cloud mapping method, which can effectively save running time and improve efficiency. Finally, Euclidean clustering segmentation is performed on the rapeseed canopy siliques, and the RANSAC algorithm is used to perform line segmentation on the connected siliques after clustering, obtaining the three-dimensional spatial position of each silique and counting the number of siliques. The proposed method was applied to identify 1457 siliques from 12 rapeseed plants, and the experimental results showed a recognition accuracy greater than 97.80%. The proposed method achieved good results in rapeseed silique recognition and provided a useful example for the application of deep learning networks in dense 3D point cloud segmentation.

Expression Characterization of ABCDE Class MADS-Box Genes in Brassica rapa with Different Pistil Types.

MADS-box is a vital transcription factor family that functions in plant growth and development. Apart from APETALA2, all genes in the ABCDE model that explain the molecular mechanism of floral organ development belong to the MADS-box family. Carpel and ovule numbers in plants are essential agronomic traits that determine seed yield, and multilocular siliques have great potential for the development of high-yield varieties of Brassica. In this study, ABCDE genes in the MADS-box family from Brassica rapa were identified and characterized. Their tissue-specific expression patterns in floral organs and their differential expression in different pistil types of B. rapa were revealed by qRT-PCR. A total of 26 ABCDE genes were found to belong to the MADS-box family. Our proposed ABCDE model of B. rapa is consistent with that of Arabidopsis thaliana, indicating that ABCDE genes are functionally conserved. These results of qRT-PCR showed that the expression levels of class C and D genes were significantly different between the wild-type (wt) and tetracarpel (tetrac) mutant of B. rapa. Interestingly, the expression of the homologs of class E genes was imbalanced. Therefore, it is speculated that class C, D, and E genes are involved in developing the carpel and ovule of B. rapa. Our findings reveal the potential for the selection of candidate genes to improve yield traits in Brassica crops.

The Tartary buckwheat bHLH gene ALCATRAZ contributes to silique dehiscence in Arabidopsis thaliana.

Tartary buckwheat is popular because of its rich nutrients. However, the difficulty in shelling restricts food production. The gene ALCATRAZ (AtALC) plays a key role in silique dehiscence in Arabidopsis thaliana. In this study, an atalc mutant was obtained by CRISPR/Cas9, and a FtALC gene homologous to AtALC was complemented into the atalc mutant to verify its function. Phenotypic observations showed that three atalc mutant lines did not dehiscence, while ComFtALC lines recovered the dehiscence phenotype. The contents of lignin, cellulose, hemicellulose, and pectin in the siliques of all the atalc mutant lines were significantly higher than those in the wild-type and ComFtALC lines. Moreover, FtALC was found to regulate the expression of cell wall pathway genes. Finally, the interaction of FtALC with FtSHP and FtIND was verified by yeast two-hybrid, bimolecular fluorescent complimentary (BIFC) and firefly luciferase completion imaging assays (LCIs). Our findings enrich the silique regulatory network and lay the foundation for the cultivation of easily shelled tartary buckwheat varieties.

Fine Mapping of a Pleiotropic Locus (BnUD1) Responsible for the Up-Curling Leaves and Downward-Pointing Siliques in Brassica napus.

Leaves and siliques are important organs associated with dry matter biosynthesis and vegetable oil accumulation in plants. We identified and characterized a novel locus controlling leaf and silique development using the Brassica napus mutant Bnud1, which has downward-pointing siliques and up-curling leaves. The inheritance analysis showed that the up-curling leaf and downward-pointing silique traits are controlled by one dominant locus (BnUD1) in populations derived from NJAU5773 and Zhongshuang 11. The BnUD1 locus was initially mapped to a 3.99 Mb interval on the A05 chromosome with a BC6F2 population by a bulked segregant analysis-sequencing approach. To more precisely map BnUD1, 103 InDel primer pairs uniformly covering the mapping interval and the BC5F3 and BC6F2 populations consisting of 1042 individuals were used to narrow the mapping interval to a 54.84 kb region. The mapping interval included 11 annotated genes. The bioinformatic analysis and gene sequencing data suggested that BnaA05G0157900ZS and BnaA05G0158100ZS may be responsible for the mutant traits. Protein sequence analyses showed that the mutations in the candidate gene BnaA05G0157900ZS altered the encoded PME in the trans-membrane region (G45A), the PMEI domain (G122S), and the pectinesterase domain (G394D). In addition, a 573 bp insertion was detected in the pectinesterase domain of the BnaA05G0157900ZS gene in the Bnud1 mutant. Other primary experiments indicated that the locus responsible for the downward-pointing siliques and up-curling leaves negatively affected the plant height and 1000-seed weight, but it significantly increased the seeds per silique and positively affected photosynthetic efficiency to some extent. Furthermore, plants carrying the BnUD1 locus were compact, implying they may be useful for increasing B. napus planting density. The findings of this study provide an important foundation for future research on the genetic mechanism regulating the dicotyledonous plant growth status, and the Bnud1 plants can be used directly in breeding.

Perilla frutescens PfFUL Regulates the Flowering and Silique Development in Transgenic Arabidopsis thaliana / 中国生物化学与分子生物学报

The FRUITFULL (FUL) gene belongs to the AP1/ FUL subfamily of the plant MADS-box family and has functions in regulating flowering time, floral meristem differentiation and fruit development. PfFUL gene sequence was derived from the perilla transcriptome data, and the basic physicochemical properties of PfFUL were analyzed by bioinformatics methods. Evolutionary relationships of PfFUL with other species of FUL were analyzed by phylogenetic tree. Plant expression vector 35S::PfFUL was constructed and used to transform wild type Col-0 and mutant ful-7 Arabidopsis to obtain overexpression 35S::PfFUL/ Col-0 and complemented mutation 35S::PfFUL / ful-7 plants respectively. Comparative phenotypic analysis was performed to preliminarily clarify the function of PfFUL gene in plant flowering and fruit development. The functions of the PfFUL gene during flowering and angular fruit development of the plants were initially clarified. The CDS of PfFUL gene is 738 bp and encodes 245 amino acids. Phylogenetic tree showed that the perilla PfFUL was closely related to Solanum lycopersicum, Salvia splendens and Salvia hispanica, but far related to Arabidopsis thaliana, Nicotiana tabacum and Vitis vinifera. Compared to Col-0 and ful-7, the transgenic plants showed early flowering (P0. 05), and the amount of wrinkled seed was significantly reduced (P<0. 01). In addition, phenotypic observations revealed that the transgenic plants also exhibited increased internode length and narrowed and curled cauline leaves. In conclusion, this study confirmed that the PfFUL gene regulates early flowering and fruit development in plants and participates in nutritional growth.

Combined Transcriptome and Metabolome Profiling Provide Insights into Cold Responses in Rapeseed (Brassica napus L.) Genotypes with Contrasting Cold-Stress Sensitivity.

Low temperature is a major environmental factor, which limits rapeseed (Brassica napus L.) growth, development, and productivity. So far, the physiological and molecular mechanisms of rapeseed responses to cold stress are not fully understood. Here, we explored the transcriptome and metabolome profiles of two rapeseed genotypes with contrasting cold responses, i.e., XY15 (cold-sensitive) and GX74 (cold-tolerant). The global metabolome profiling detected 545 metabolites in siliques of both genotypes before (CK) and after cold-stress treatment (LW). The contents of several sugar metabolites were affected by cold stress with the most accumulated saccharides being 3-dehydro-L-threonic acid, D-xylonic acid, inositol, D-mannose, D-fructose, D-glucose, and L-glucose. A total of 1943 and 5239 differentially expressed genes were identified from the transcriptome sequencing in XY15CK_vs_XY15LW and GX74CK_vs_GX74LW, respectively. We observed that genes enriched in sugar metabolism and biosynthesis-related pathways, photosynthesis, reactive oxygen species scavenging, phytohormone, and MAPK signaling were highly expressed in GX74LW. In addition, several genes associated with cold-tolerance-related pathways, e.g., the CBF-COR pathway and MAPK signaling, were specifically expressed in GX74LW. Contrarily, genes in the above-mentioned pathways were mostly downregulated in XY15LW. Thus, our results indicate the involvement of these pathways in the differential cold-stress responses in XY15 and GX74.

miR319-Regulated TCP3 Modulates Silique Development Associated with Seed Shattering in Brassicaceae.

Seed shattering is an undesirable trait that leads to crop yield loss. Improving silique resistance to shattering is critical for grain and oil crops. In this study, we found that miR319-targeted TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCPs) inhibited the process of post-fertilized fruits (silique) elongation and dehiscence via regulation of FRUITFULL (FUL) expression in Arabidopsis thaliana and Brassica napus. AtMIR319a activation resulted in a longer silique with thickened and lignified replum, whereas overexpression of an miR319a-resistant version of AtTCP3 (mTCP3) led to a short silique with narrow and less lignified replum. Further genetic and expressional analysis suggested that FUL acted downstream of TCP3 to negatively regulate silique development. Moreover, hyper-activation of BnTCP3.A8, a B. napus homolog of AtTCP3, in rapeseed resulted in an enhanced silique resistance to shattering due to attenuated replum development. Taken together, our findings advance our knowledge of TCP-regulated silique development and provide a potential target for genetic manipulation to reduce silique shattering in Brassica crops.

Genome-wide characterization of ovate family protein gene family associated with number of seeds per silique in Brassica napus.

Ovate family proteins (OFPs) were firstly identified in tomato as proteins controlling the pear shape of the fruit. Subsequent studies have successively proved that OFPs are a class of negative regulators of plant development, and are involved in the regulation of complex traits in different plants. However, there has been no report about the functions of OFPs in rapeseed growth to date. Here, we identified the OFPs in rapeseed at the genomic level. As a result, a total of 67 members were obtained. We then analyzed the evolution from Arabidopsis thaliana to Brassica napus, illustrated their phylogenetic and syntenic relationships, and compared the gene structure and conserved domains between different copies. We also analyzed their expression patterns in rapeseed, and found significant differences in the expression of different members and in different tissues. Additionally, we performed a GWAS for the number of seeds per silique (NSPS) in a rapeseed population consisting of 204 natural accessions, and identified a new gene BnOFP13_2 significantly associated with NSPS, which was identified as a novel function of OFPs. Haplotype analysis revealed that the accessions with haplotype 3 had a higher NSPS than other accessions, suggesting that BnOFP13_2 is associated with NSPS. Transcript profiling during the five stages of silique development demonstrated that BnOFP13_2 negatively regulates NSPS. These findings provide evidence for functional diversity of OFP gene family and important implications for oilseed rape breeding.

AtMYB31 is a wax regulator associated with reproductive development in Arabidopsis.

KEY MESSAGE: AtMYB31, a R2R3-MYB transcription factor that modulates wax biosynthesis in reproductive tissues, is involved in seed development in Arabidopsis. R2R3-MYB transcription factors play important roles in plant development; yet, the exact role of each of them remains to be resolved. Here we report that the Arabidopsis AtMYB31 is required for wax biosynthesis in epidermis of reproductive tissues, and is involved in seed development. AtMYB31 was ubiquitously expressed in both vegetative and reproductive tissues with higher expression levels in siliques and seeds, while AtMYB31 was localized to the nucleus and cytoplasm. Loss of function of AtMYB31 reduced wax accumulation in the epidermis of silique and flower tissues, disrupted seed coat epidermal wall development and mucilage production, altered seed proanthocyanidin and polyester content. AtMYB31 could direct activate expressions of several wax biosynthetic target genes. Altogether, AtMYB31, a R2R3-MYB transcription factor, regulates seed development in Arabidopsis.

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.

A proteomic analysis of Arabidopsis ribosomal phosphoprotein P1A mutant.

Ribosomal proteins are involved in the regulation of plant growth and development. However, the regulatory processes of most ribosomal proteins remain unclear. In this study, Arabidopsis plants with the mutation in ribosomal phosphoprotein P1A (RPP1A) produce larger and heavier seeds than wild-type plants. A comparative quantitative label-free proteomic analysis revealed that a total of 215 proteins were differentially accumulated between the young siliques of the wild type and rpp1a mutant. Knockout of RPP1A significantly reduced the abundance of proteins involved in carboxylic acid metabolism and lipid biosynthesis. Consistent with this, a metabolic analysis showed that the organic acids in the tricarboxylic acid cycle and the carbohydrates in the pentose phosphate pathway were severely reduced in the mature rpp1a mutant seeds. In contrast, the abundance of proteins related to seed maturation, especially seed storage proteins, was markedly increased during seed development. Indeed, seed storage proteins were accumulated in the mature rpp1a mutant seeds, and the seed nitrogen and sulfur contents were also increased. These results indicate that more carbon intermediates probably enter the nitrogen flow for the enhanced synthesis of seed storage proteins, which might subsequently contribute to the enlarged seed size in the rpp1a mutant. SIGNIFICANCE: Ribosomes are responsible for protein synthesis and are generally perceived as the housekeeping components in the cells. In this study, the knockout of RPP1A leads to an increased seed size through repressing carbon metabolism and lipid biosynthesis, and increasing the synthesis of seed storage proteins. Meanwhile, the abundance of seed storage proteins and the nitrogen and sulfur concentrations were increased in the mature rpp1a mutant seeds. The results provide a novel insight into the genetic regulatory networks for the control of seed size and seed storage protein accumulation, and this knowledge may facilitate the improvement of crop seed size.

Salt stress downregulates 2-hydroxybutyrylation in Arabidopsis siliques.

Lysine 2-hydroxyisobutyrylation (Khib) is one of the newly discovered post-translational modifications (PTMs) through protein acylation. It has been reported to be widely distributed in both eukaryotes and prokaryotes, and plays an important role in chromatin conformation change, gene transcription, protein subcellular localization, protein-protein interaction, signal transduction, and cellular proliferation. In this study, the Khib modification proteome of siliques from A. thaliana under salt stress (Ss) and those in the control (Cs) were compared. The results showed that Khib modification was abundant in siliques. Totally 3810 normalized Khib sites on 1254 proteins were identified, and the Khib modification showed a downregulation trend dramatically: it was down-regulated at 282 sites on 205 proteins while was up-regulated at 96 sites on 78 proteins in Ss siliques (Data are available via ProteomeXchange with identifier PXD028116 and PXD026643). Among them, 13 proteins, including F4IVN6, Q9M1P5, and Q9LF33, had sites with the most significant regulation of Khib modification. Bioinformatics analysis suggested that the differentially Khib-regulated proteins mainly participated in glycolysis/gluconeogenesis and endocytosis. In particular, there were differentially117 Khib-regulated proteins that were mapped to the protein-protein interaction database. In the KEGG pathway enrichment analysis, Khib-modified proteins were enriched in several pathways related to energy metabolism, including gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism. Overall, our work reveals the first systematic analysis of Khib proteome in Arabidopsis siliques under salt stress, and sheds a light on the future studies on the regulatory mechanisms of Khib during the salt stress response of plants. SIGNIFICANCE: In this study, we found the Khib-modified proteins in silique under salt stress and described the enrichment of Khib-modified proteins involved in the biological processes and cellular localization. Proteins undergoing 2-hydroxyisobutylation were mainly involved in the gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism, suggesting that 2-hydroxyisobutylation affects the energy metabolic pathway, and thus the development of the plant. In addition, specific candidate proteins that may affect plant development under salt stress were selected. This study will provide a theoretical basis for revealing the function and mechanism of these proteins and their 2-hydroxyisobutyryl modifications during the development of silique under salt stress.

BnaC7.ROT3, the causal gene of cqSL-C7, mediates silique length by affecting cell elongation in Brassica napus.

Siliques are a major carbohydrate source of energy for later seed development in rapeseed (Brassica napus). Thus, silique length has received great attention from breeders. We previously detected a novel quantitative trait locus cqSL-C7 that controls silique length in B. napus. Here, we further validated the cqSL-C7 locus and isolated its causal gene (BnaC7.ROT3) by map-based cloning. In 'Zhongshuang11' (parent line with long siliques), BnaC7.ROT3 encodes the potential cytochrome P450 monooxygenase CYP90C1, whereas in 'G120' (parent line with short siliques), a single nucleotide deletion in the fifth exon of BnaC7.ROT3 results in a loss-of-function truncated protein. Sub-cellular localization and expression pattern analysis revealed that BnaC7.ROT3 is a membrane-localized protein mainly expressed in leaves, flowers and siliques. Cytological observations showed that the cells in silique walls of BnaC7.ROT3-transformed positive plants were longer than those of transgene-negative plants in the background of 'G120', suggesting that BnaC7.ROT3 affects cell elongation. Haplotype analysis demonstrated that most alleles of BnaC7.ROT3 are favorable in B. napus germplasms, and its homologs may also be involved in silique length regulation. Our findings provide novel insights into the regulatory mechanisms of natural silique length variations and valuable genetic resources for the improvement of silique length in rapeseed.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.

Transcriptomic comparison of seeds and silique walls from two rapeseed genotypes with contrasting seed oil content.

Silique walls play pivotal roles in contributing photoassimilates and nutrients to fuel seed growth. However, the interaction between seeds and silique walls impacting oil biosynthesis is not clear during silique development. Changes in sugar, fatty acid and gene expression during Brassica napus silique development of L192 with high oil content and A260 with low oil content were investigated to identify key factors affecting difference of their seed oil content. During the silique development, silique walls contained more hexose and less sucrose than seeds, and glucose and fructose contents in seeds and silique walls of L192 were higher than that of A260 at 15 DAF, and sucrose content in the silique walls of L192 were lower than that of A260 at three time points. Genes related to fatty acid biosynthesis were activated over time, and differences on fatty acid content between the two genotypes occurred after 25 DAF. Genes related to photosynthesis expressed more highly in silique walls than in contemporaneous seeds, and were inhibited over time. Gene set enrichment analysis suggested photosynthesis were activated in L192 at 25 and 35 DAF in silique walls and at both 15 and 35 DAF in the seed. Expressions of sugar transporter genes in L192 was higher than that in A260, especially at 35 DAF. Expressions of genes related to fatty acid biosynthesis, such as BCCP2s, bZIP67 and LEC1s were higher in L192 than in A260, especially at 35 DAF. Meanwhile, genes related to oil body proteins were expressed at much lower levels in L192 than in A260. According to the WGCNA results, hub modules, such as ME.turquoise relative to photosynthesis, ME.green relative to embryo development and ME.yellow relative to lipid biosynthesis, were identified and synergistically regulated seed development and oil accumulation. Our results are helpful for understanding the mechanism of oil accumulation of seeds in oilseed rape for seed oil content improvement.