Gengxing Song

In Brassicaceae cross-breeding, asynchronous flowering and geographic separation often cause pollen shortages that severely constrain hybridization. Although pollen cryopreservation offers an effective solution, Brassicaceae pollen is typically short-lived due to its tricellular structure, thin exine, and high desiccation sensitivity, necessitating optimized cryopreservation protocols. In this study, we optimized a pollen cryopreservation protocol for three representatives Brassicaceae species: Brassica rapa L. (Chinese cabbage), Brassica oleracea L. (cabbage), and Barbarea vulgaris R. Br. (European rockcress). An in vitro pollen germination system was optimized to reliably assess pollen viability before and after cryopreservation. Key parameters including pollen collection time, drying duration, freezing procedure, and thawing conditions were systematically evaluated. The optimal protocol comprised: pollen collection at 8:00–10:00, drying at 28 °C and 2% relative humidity for 1 h, precooling at −20 °C for 30 min, storage at −80 °C, and thawing under running tap water (ca. 25 °C). Following 30-day cryopreservation, pollen maintained high germination rates (75.19% for Brassica rapa L., 71.18% for Brassica oleracea L., 80.33% for Barbarea vulgaris) and produced comparable silique development, seed quality, and seed germination rates to those of fresh pollen following pollination. This study established a reliable and efficient cryopreservation system for Brassicaceae pollen that effectively overcomes asynchronous flowering and geographic barriers in hybridization, thereby improving breeding efficiency and facilitating germplasm innovation for Brassicaceae crops.

Background: Formin proteins are crucial regulators of actin filament assembly and elongation in eukaryotic cells, playing important roles in plant development and abiotic stress responses. However, the functional characterization of formins in Brassica rapa L. remains undiscovered. Methods: A total of 27 formin family members (BrFHs) were identified through genome-wide alignment with Arabidopsis thaliana (L.) Heynh. Results: Phylogenetic analysis classified BrFH gene family into two distinct clades, designated Group I and Group II, which exhibit divergent protein architectures. Promoter analysis revealed that BrFHs contain multiple cis-regulatory elements related to growth and development, stress responses, and phytohormone signaling. These findings suggest that BrFHs may have diversified functions. Tissue-specific expression analysis revealed that BrFHs exhibit distinct expression patterns across various tissues. Notably, BrFH15 and BrFH18 are highly expressed in flowers, displaying expression profiles similar to those of floral development genes such as AP3, AGL10 and so on. Additionally, many BrFHs show dynamic expression patterns in response to cold stresses. In particular, BrFH2, BrFH19 and BrFH27 were up-regulated, and their co-expression within the gene network suggests potential roles in regulating cold stress. Conclusions: These results clarify the functional roles of BrFHs and shed light on the molecular mechanisms underlying their regulation of tissue development and responses to cold stress in Brassica rapa.

Prezygotic interspecific incompatibility prevents hybridization between species, which limits interbreeding strategies for crop improvement using wild relatives. The Brassica rapa female self-incompatibility determinant, S -locus receptor kinase (SRK), recognizes interspecific pollen. Here, we report the discovery of a pan-Brassicaceae SRK-interacting interspecific pollen signal (SIPS). On B. rapa stigmas, SIPSs from Arabidopsis and other Brassicaceae species target Br SRK and recruit the female fertility regulator FERONIA receptor kinase to increase stigmatic reactive oxygen species and reduce interspecific pollen viability. Arabidopsis thaliana sips mutant pollen failed to trigger interspecific incompatibility responses. Unlike self-incompatibility, which is controlled by the polymorphic S locus, different genetic variants of SRK interacted comparably with SIPS. This study establishes SIPS-SRK as a Brassicaceae-specific ligand-receptor pair that broadly maintains the stigmatic interspecific barrier in self-incompatible species.

Background: Formin proteins are crucial regulators of actin filament assembly and elongation in eukaryotic cells, playing important roles in plant development and abiotic stress responses. However, the functional characterization of formins in Brassica rapa remains uncover. Methods: A total of 27 formin family members (BrFHs) were identified through genome-wide alignment with Arabidopsis thaliana. Results: Phylogenetic analysis classified BrFH gene family into two distinct clades, designated Group I and Group II, which exhibit divergent protein architectures. Promoter analysis revealed that BrFHs contain multiple cis-regulatory elements related to growth and development, stress responses, and phytohormone signaling. These findings suggest that BrFHs may have diversified functions. Tissue-specific expression analysis revealed that BrFHs exhibit distinct expression patterns across various tissues. Notably, BrFH15 and BrFH18 are highly expressed in flowers, displaying expression profiles similar to those of floral development genes such as AP3, AGL10 and so on. Additionally, many BrFHs show dynamic expression patterns in response to cold stresses. In particular, BrFH2, BrFH19 and BrFH27 were up-regulated, and their co-expression within the gene network suggests potential roles in regulating cold stress. Conclusions: These results clarify the functional roles of BrFHs and shed light on the molecular mechanisms underlying their regulation of tissue development and responses to abiotic stress in Brassica rapa.

Mutations in BrMYB31 were responsible for glossy phenotype, which was verified in two allelic mutants and gene silencing analysis. BrMYB31 regulated wax biosynthesis by modulating BrCER4 expression in Chinese cabbage. Plant cuticular wax plays a crucial role in resisting both biotic and abiotic stresses, but its deficiency is beneficial for improving the commercial properties of certain leafy vegetables. The glossy appearance resulting from the absence of epidermal wax crystals is a striking product feature of Chinese cabbage (Brassica rapa L. ssp. pekinensis). In this study, we identified two allelic mutants with wax crystal deficiency (wdm2 and wdm5) derived from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage. Genetic analysis indicated that a recessive nuclear gene is responsible for the glossy phenotype. Based on MutMap sequencing combined with the kompetitive allele-specific PCR (KASP) genotyping approach, BraA02g022420.3C, a gene belonging to MYB transcription factor family, was predicted as the candidate gene and designated it as BrMYB31. Allelic mutant sanger sequencing analysis revealed a single-nucleotide polymorphism (SNP) (G/A) in the 2nd exon of BrMYB31 in wdm2, resulting in the substitution of lysine (K) with arginine (R), and also a SNP (G/A) in the 3rd exon of BrMYB31 in wdm5, causing tryptophan (W) to be converted into a terminator (X). Further virus-induced gene silencing analysis demonstrated that suppression of BrMYB31 exhibited a glossy phenotype. Transcriptome analysis revealed that BrMYB31 likely regulates glossy leaf characteristics probably by impacting wax biosynthesis-related genes expression. Transcriptional activity assays confirmed that BrMYB31 functions as a transcriptional activator. Yeast one-hybrid assays, luciferase reporter assays, and GUS activity analyses indicated that BrMYB31 could directly bind to BrCER4 promoter. This study illustrates the effectiveness of integrating MutMap and KASP in mining candidate genes associated with glossy leaf traits in EMS mutants and provides new insights into the regulatory network of cuticle wax biosynthesis in Chinese cabbage.