Self-incompatibility (SI) is a mechanism in plants that prevents self-fertilization and promotes outcrossing. SI is also widely utilized in the breeding of Brassicaceae crops. Understanding the regulatory mechanisms of SI is essential but has been greatly restrained in most Brassicaceae crops due to inefficient transformation. In this study, we developed methods for examining signaling pathways and genes of pollen–stigma interactions in Brassicaceae crops lacking an efficient genetic transformation system. We pretreated excised stigmas of Brassica rapa (B. rapa L. ssp. Pekinensis) in vitro with chemicals to modify signaling pathways or with phosphorothioate antisense oligodeoxyribonucleotides (AS-ODNs) to modify the expression of the corresponding genes involved in pollen–stigma interactions. Using this method, we first determined the involvement of reactive oxygen species (ROS) in SI with the understanding that the NADPH oxidase inhibitor diphenyleneiodonium chloride, which inhibits ROS production, eliminated the SI of B. rapa. We further identified the key gene for ROS production in SI and used AS-ODNs targeting BrRBOHF (B. rapa RESPIRATORY-BURST OXIDASE HOMOLOGF), which encodes one of the NADPH oxidases, to effectively suppress its expression, reduce stigmatic ROS, and promote the growth of self-pollen in B. rapa stigmas. Moreover, pistils treated in planta with the ROS scavenger sodium salicylate disrupted SI and resulted in enlarged ovules with inbred embryos 12 d after pollination. This method will enable the functional study of signaling pathways and genes regulating SI and other pollen–stigma interactions in different Brassicaceae plants.

The ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) gene family plays a pivotal role in plant growth, induction of phytohormones, and the abiotic stress response. However, the AS2 gene family in Brassica rapa has yet to be investigated. In this study, we identified 62 AS2 genes in the B. rapa genome, which were classified into six subfamilies and distributed across 10 chromosomes. Sequence analysis of BrAS2 promotors showed that there are several typical cis-elements involved in abiotic stress tolerance and stress-related hormone response. Tissue-specific expression analysis showed that BrAS2-47 exhibited ubiquitous expression in all tissues, indicating it may be involved in many biological processes. Gene expression analysis showed that the expressions of BrAS2-47 and BrAS2-10 were significantly downregulated under cold stress, heat stress, drought stress, and salt stress, while BrAS2-58 expression was significantly upregulated under heat stress. RT-qPCR also confirmed that the expression of BrAS2-47 and BrAS2-10 was significantly downregulated under cold stress, drought stress, and salt stress, and in addition BrAS2-56 and BrAS2-4 also changed significantly under the three stresses. In addition, protein–protein interaction (PPI) network analysis revealed that the Arabidopsis thaliana genes AT5G67420 (homologous gene of BrAS2-47 and BrAS2-10) and AT3G49940 (homologous gene of BrAS2-58) can interact with NIN-like protein 7 (NLP7), which has been previously reported to play a role in resistance to adverse environments. In summary, our findings suggest that among the BrAS2 gene family, BrAS2-47 and BrAS2-10 have the most potential for the regulation of abiotic stress tolerance. These results will facilitate future functional investigations of BrAS2 genes in B. rapa.

Nitrate transporter 2 (NRT2) proteins play vital roles in both nitrate (NO₃⁻) uptake and translocation as well as abiotic stress responses in plants. However, little is known about the NRT2 gene family in Brassica rapa. In this study, 14 NRT2s were identified in the B. rapa genome. The BrNRT2 family members contain the PLN00028 and MATE_like superfamily domains. Cis-element analysis indicated that regulatory elements related to stress responses are abundant in the promoter sequences of BrNRT2 genes. BrNRT2.3 expression was increased after drought stress, and BrNRT2.1 and BrNRT2.8 expression were significantly upregulated after salt stress. Furthermore, protein interaction predictions suggested that homologs of BrNRT2.3, BrNRT2.1, and BrNRT2.8 in Arabidopsis thaliana may interact with the known stress-regulating proteins AtNRT1.1, AtNRT1.5, and AtNRT1.8. In conclusion, we suggest that BrNRT2.1, BrNRT2.3, and BrNRT2.8 have the greatest potential for inducing abiotic stress tolerance. Our findings will aid future studies of the biological functions of BrNRT2 family genes.

CCT MOTIF FAMILY (CMF) genes belong to the CCT gene family and have been shown to play a role in diverse processes, such as flowering time and yield regulation, as well as responses to abiotic stresses. CMF genes have not yet been identified in Brassica rapa. A total of 25 BrCMF genes were identified in this study, and these genes were distributed across eight chromosomes. Collinearity analysis revealed that B. rapa and Arabidopsis thaliana share many homologous genes, suggesting that these genes have similar functions. According to sequencing analysis of promoters, several elements are involved in regulating the expression of genes that mediate responses to abiotic stresses. Analysis of the tissue-specific expression of BrCMF14 revealed that it is highly expressed in several organs. The expression of BrCMF22 was significantly downregulated under salt stress, while the expression of BrCMF5, BrCMF7, and BrCMF21 was also significantly reduced under cold stress. The expression of BrCMF14 and BrCMF5 was significantly increased under drought stress, and the expression of BrCMF7 was upregulated. Furthermore, protein–protein interaction network analysis revealed that A. thaliana homologs of BrCMF interacted with genes involved in the abiotic stress response. In conclusion, BrCMF5, BrCMF7, BrCMF14, BrCMF21, and BrCMF22 appear to play a role in responses to abiotic stresses. The results of this study will aid future investigations of CCT genes in B. rapa.

High temperature negatively affects reproductive process significantly, leading to tremendous losses in crop quality and yield. Zhinengcong (ZNC), a crude extract from the endophytic fungus Paecilomyces variotii, has been shown to improve plant growth and resistance to biotic and abiotic stresses. We show here that ZNC can also alleviate heat stress-induced reproductive defects in Solanum lycopersicum, such as short-term heat-induced inhibition on pollen viability, germination and tube growth, and long-term heat stress-induced pollen developmental defects. We further demonstrated that ZNC alleviates heat stress by downregulating the expressions of ROS production-related genes, RBOHs, and upregulating antioxidant related genes and the activities of the corresponding enzymes, thus preventing the over accumulation of heat-induced reactive oxygen species (ROS) in anther, pollen grain and pollen tube. Furthermore, spraying application of ZNC onto tomato plants under long-term heat stress promotes fruit and seed bearing in the field. In summary, plant endophytic fungus extract ZNC promotes the reproductive process and yield of tomato plants under heat stress and presents excellent potential in agricultural applications.

The bile acid sodium symporter (BASS) family plays an important role in transporting substances and coordinating plants’ salt tolerance. However, the function of BASS in Brassica rapa has not yet been elucidated. In this study, eight BrBASS genes distributed on five chromosomes were identified that belonged to four subfamilies. Expression profile analysis showed that BrBASS7 was highly expressed in roots, whereas BrBASS4 was highly expressed in flowers. The promoter element analysis also identified several typical homeopathic elements involved in abiotic stress tolerance and stress-related hormonal responses. Notably, under salt stress, the expression of BrBASS2 was significantly upregulated; under osmotic stress, that of BrBASS4 increased and then decreased; and under cold stress, that of BrBASS7 generally declined. The protein–protein interaction analysis revealed that the BrBASS2 homologous gene AtBASS2 interacted with Nhd1 (N-mediated heading date-1) to alleviate salt stress in plants, while the BrBASS4 homologous gene AtBASS3 interacted with BLOS1 (biogenesis of lysosome-related organelles complex 1 subunit 1) via co-regulation with SNX1 (sorting nexin 1) to mitigate an unfavorable growing environment for roots. Further, Bra-miR396 (Bra-microRNA396) targeting BrBASS4 and BrBASS7 played a role in the plant response to osmotic and cold stress conditions, respectively. This research demonstrates that BrBASS2, BrBASS4, and BrBASS7 harbor great potential for regulating abiotic stresses. The findings will help advance the study of the functions of the BrBASS gene family.