Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Nontransformation methods for studying signaling pathways and genes involved in Brassica rapa pollen-stigma interactions.

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 RopGEF Gene Family and Their Potential Roles in Responses to Abiotic Stress in Brassica rapa.

Guanine nucleotide-exchange factors (GEFs) genes play key roles in plant root and pollen tube growth, phytohormone responses, and abiotic stress responses. RopGEF genes in Brassica rapa have not yet been explored. Here, GEF genes were found to be distributed across eight chromosomes in B. rapa and were classified into three subfamilies. Promoter sequence analysis of BrRopGEFs revealed the presence of cis-elements characteristic of BrRopGEF promoters, and these cis-elements play a role in regulating abiotic stress tolerance and stress-related hormone responses. Organ-specific expression profiling demonstrated that BrRopGEFs were ubiquitously expressed in all organs, especially the roots, suggesting that they play a role in diverse biological processes. Gene expression analysis revealed that the expression of BrRopGEF13 was significantly up-regulated under osmotic stress and salt stress. RT-qPCR analysis revealed that the expression of BrRopGEF13 was significantly down-regulated under various types of abiotic stress. Protein-protein interaction (PPI) network analysis revealed interactions between RopGEF11, the homolog of BrRopGEF9, and the VPS34 protein in Arabidopsis thaliana, as well as interactions between AtRopGEF1, the homolog of BrRopGEF13 in Arabidopsis, and the ABI1, HAB1, PP2CA, and CPK4 proteins. VPS34, ABI1, HAB1, PP2CA, and CPK4 have previously been shown to confer resistance to unfavorable environments. Overall, our findings suggest that BrRopGEF9 and BrRopGEF13 play significant roles in regulating abiotic stress tolerance. These findings will aid future studies aimed at clarifying the functional characteristics of BrRopGEFs.

Analysis and cloning of the synthetic pathway of the phytohormone indole-3-acetic acid in the plant-beneficial Bacillus amyloliquefaciens SQR9.

BACKGROUND: The plant growth-promoting rhizobacteria (PGPR) strain Bacillus amyloliquefaciens SQR9, isolated from the cucumber rhizosphere, protects the host plant from pathogen invasion and promotes plant growth through efficient root colonization. The phytohormone indole-3-acetic acid (IAA) has been suggested to contribute to the plant-growth-promoting effect of Bacillus strains. The possible IAA synthetic pathways in B. amyloliquefaciens SQR9 were investigated in this study, using a combination of chemical and genetic analysis. RESULTS: Gene candidates involved in tryptophan-dependent IAA synthesis were identified through tryptophan response transcriptional analysis, and inactivation of genes ysnE, dhaS, yclC, and yhcX in SQR9 led to 86, 77, 55, and 24 % reductions of the IAA production, respectively. The genes patB (encoding a conserved hypothetical protein predicted to be an aminotransferase), yclC (encoding a UbiD family decarboxylase), and dhaS (encoding indole 3-acetaldehyde dehydrogenase), which were proposed to constitute the indole-3-pyruvic acid (IPyA) pathway for IAA biosynthesis, were separately expressed in SQR9 or co-expressed as an entire IAA synthesis pathway cluster in SQR9 and B. subtilis 168, all these recombinants showed increased IAA production. These results suggested that gene products of dhaS, patB, yclB, yclC, yhcX and ysnE were involved in IAA biosynthesis. Genes patB, yclC and dhaS constitute a potential complete IPyA pathway of IAA biosynthesis in SQR9. CONCLUSIONS: In conclusion, biosynthesis of IAA in B. amyloliquefaciens SQR9 occurs through multiple pathways.

Spatially differential regulation of ATF2 phosphorylation contributes to warning coloration of gregarious locusts.

Warning coloration are common defense strategies used by animals to deter predators. Pestilential gregarious locusts exhibit a notable black-brown pattern as a form of warning coloration. However, the mechanisms regulating this distinctive pattern remain largely unknown. Here, we revealed that the black and brown integuments of locusts are governed by varying amounts of ß-carotene and ß-carotene-binding protein (ßCBP) complexes. ßCBP expression is regulated by the bZIP transcription factor activation transcription factor 2 (ATF2), which is activated by protein kinase C alpha in response to crowding. Specifically, ATF2 is phosphorylated at Ser327 and translocates to the nucleus, where it binds to the ßCBP promoter and stimulates overexpression. Differential phosphorylation of ATF2 leads to the divergent black and brown coloration in gregarious locusts. The accumulation of red pigments vital for creating the brown sternum depends on ßCBP overexpression. The spatial variation in ATF2 phosphorylation enables locusts to rapidly adapt to changing environment for aposematism.

Effects of sporulation conditions on the growth, germination, and resistance of Clostridium perfringens spores.

Clostridium perfringens can form metabolically dormant spores that can survive in meat preservation processes and cause food spoilage and human disease upon germination and outgrowth. The characteristics of spores in food products are closely related to the sporulation environment. To control or inactivate C. perfringens spores in food industry, the effects of sporulation conditions on the spores characteristics should be examined. This study aimed to investigate the effects of temperature (T), pH, and water activity (aw) on the growth, germination, and wet-heat resistance of C. perfringens C1 spores isolated from food product. The results showed that C. perfringens C1 spores produced at T = 37 °C, pH = 8, and aw = 0.997 had the highest sporulation rate and germination efficiency and lowest wet-heat resistance. A further increase in pH and sporulation temperature reduced the spore counts and germination efficiency, but enhanced spores' wet-heat resistance. By using air-drying method and Raman spectroscopy analysis, the water content, composition, and levels of calcium dipicolinic acid, proteins, and nucleic acids in spores produced under different sporulation conditions were determined. The results obtained revealed that sporulation conditions should be carefully considered during food production and processing, thus providing a novel insight into prevention and control of spores in food industry.

Endophytic extract Zhinengcong alleviates heat stress-induced reproductive defect in Solanum lycopersicum.

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.

Role of Probiotics in Mycoplasma pneumoniae Pneumonia in Children: A Short-Term Pilot Project.

Mycoplasma pneumoniae is one of the most common pathogens causing community-acquired pneumonia in children. Mycoplasma pneumoniae pneumonia (MPP) can be successfully treated with azithromycin; however, antibiotic-associated diarrhea (AAD) is a common adverse effect. Increasing evidence suggests that some probiotics may prevent the development of AAD. The present study determined the effects of probiotics (live Clostridium butyricum plus Bifidobacterium infantis) on the prevention and treatment of AAD in children with MPP when co-administered with intravenous azithromycin. Fifty-five children with MPP were enrolled and received azithromycin (10 mg/kg/day; once daily for 7 days) combined with probiotics (starting on the third day of azithromycin treatment; 1,500 mg three times daily); 50 healthy children served as controls. At the end of the trial, the incidence of AAD, fecal microbiota, intestinal mucosal barriers, and systemic inflammation were analyzed using recommended systems biology techniques. No cases of AAD or other adverse events occurred in children with MPP after co-administration of probiotics with azithromycin. A live C. butyricum plus B. infantis preparation partly reconstructed the gut microbiota, especially restoration of bacterial diversity. The indicators of intestinal mucosal barrier function, such as D-lactate, endotoxin, and diamine oxidase, were significantly improved and the systemic inflammation (interleukin 10) was attenuated after probiotic therapy. The present study indicated that co-administration of probiotics with azithromycin is a promising therapy for MPP treatment which could prevent and treat AAD effectively.