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.

FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Genome-Wide Identification and Expression Analysis under Abiotic Stress of BrAHL Genes in Brassica rapa.

The AT-hook motif nuclear localized (AHL) gene family is a highly conserved transcription factor critical for the growth, development, and stress tolerance of plants. However, the function of the AHL gene family in Brassica rapa (B. rapa) remains unclear. In this study, 42 AHL family members were identified from the B. rapa genome and mapped to nine B. rapa chromosomes. Two clades have formed in the evolution of the AHL gene family. The results showed that most products encoded by AHL family genes are located in the nucleus. Gene duplication was common and expanded the BrAHL gene family. According to the analysis of cis-regulatory elements, the genes interact with stress responses (osmotic, cold, and heavy metal stress), major hormones (abscisic acid), and light responses. In addition, the expression profiles revealed that BrAHL genes are widely expressed in different tissues. BrAHL16 was upregulated at 4 h under drought stress, highly expressed under cadmium conditions, and downregulated in response to cold conditions. BrAHL02 and BrAHL24 were upregulated at the initial time point and peaked at 12 h under cold and cadmium stress, respectively. Notably, the interactions between AHL genes and proteins under drought, cold, and heavy metal stresses were observed when predicting the protein-protein interaction network.

Genome-Wide Identification and Expression Analysis of AS2 Genes in Brassica rapa Reveal Their Potential Roles in Abiotic Stress.

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.

Genome-Wide Identification and Expression Analysis of BrGeBP Genes Reveal Their Potential Roles in Cold and Drought Stress Tolerance in Brassica rapa.

The GLABROUS1 Enhancer Binding Protein (GeBP) gene family is pivotal in regulating plant growth, development, and stress responses. However, the role of GeBP in Brassica rapa remains unclear. This study identifies 20 BrGeBP genes distributed across 6 chromosomes, categorized into 4 subfamilies. Analysis of their promoter sequences reveals multiple stress-related elements, including those responding to drought, low temperature, methyl jasmonate (MeJA), and gibberellin (GA). Gene expression profiling demonstrates wide expression of BrGeBPs in callus, stem, silique, and flower tissues. Notably, BrGeBP5 expression significantly decreases under low-temperature treatment, while BrGeBP3 and BrGeBP14 show increased expression during drought stress, followed by a decrease. Protein interaction predictions suggest that BrGeBP14 homolog, At5g28040, can interact with DES1, a known stress-regulating protein. Additionally, microRNA172 targeting BrGeBP5 is upregulated under cold tolerance. These findings underscore the vital role of BrGeBPs in abiotic stress tolerance. Specifically, BrGeBP3, BrGeBP5, and BrGeBP14 show great potential for regulating abiotic stress. This study contributes to understanding the function of BrGeBPs and provides valuable insights for studying abiotic stress in B. rapa.

Ethylene negatively mediates self-incompatibility response in Brassica rapa.

Self-incompatibility (SI) is a genetic mechanism most flowering plants adopted to reject self-pollen thus avoid inbreeding. In the Brassicaceae, self-pollen recognition triggers downstream signaling pathways to reject self-pollen. However, the downstream signaling pathways are not very clear. Here we show that ethylene negatively mediates self-incompatibility response of Chinese cabbage (Brassica rapa L. ssp. Pekinensis) via PCD in papilla cells. We found that ethylene signaling genes were upregulated after cross-pollination. Treating stigmas with ethylene, or suppressing the expression of a negative regulator of ethylene signaling, CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), caused PCD in papilla cells and broke down the self-incompatibility. On the other hand, treating stigmas with ethylene inhibitors, or suppressing the expression of ethylene-responsive factors (ERFs), inhibited PCD in papilla cells and the compatible pollination. Our study identified an additional signaling pathway mediating self-incompatibility responses in the Brassicaceae and also developed a new method in overcoming self-incompatibility to improve the efficiency of inbred line propagation in agriculture practice.

All-trans retinoic acid modulates the balance of ADAMTS13 and VWF in human microvascular endothelial cells.

PURPOSE: To better understand the antithrombotic property of All-trans retinoic acid (ATRA), we investigated whether ATRA may affect the balance between ADAMTS13 and von Willebrand factor (VWF) in human microvascular endothelial cell. METHODS: Compared to tumor necrosis factor-alpha (TNF-α), we observed the effects of ATRA on the expression of ADAMTS13 and VWF. ADAMTS13mRNA in human microvascular endothelial cell (HMEC-1 cell line) were detected by real-time polymerase chain reaction amplification (RT-PCR). The levels of ADAMTS13 and VWF antigen were detected by western blot or enzyme-linked immunosorbent assay (ELISA), and the proteolytic activity of ADAMTS13 was also determined by using GST-VWF73-His peptide as a specific substrate. RESULTS: ATRA significantly upregulated the expression of ADAMTS13mRNA in HMEC-1, while TNF-α inhibited ADAMTS13mRNA expression. ATRA could reverse the inhibition expression of ADAMTS13 by TNF-α. The results were confirmed from the levels of ADAMTS13 protein and its activity, while ATRA had no significant affection on triggering release of VWF. CONCLUSIONS: This study provides the evidence that ATRA modulates the balance of ADAMTS13 and VWF in human microvascular endothelial cell, which might be a very relevant compartment for the antithrombotic property of ATRA.

FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.

Plant growth and development are controlled by a delicate balance of hormonal cues. Growth-promoting hormones and growth-inhibiting counterparts often antagonize each other in their action, but the molecular mechanisms underlying these events remain largely unknown. Here, we report a cross-talk mechanism that enables a receptor-like kinase, FERONIA (FER), a positive regulator of auxin-promoted growth, to suppress the abscisic acid (ABA) response through activation of ABI2, a negative regulator of ABA signaling. The FER pathway consists of a FER kinase interacting with guanine exchange factors GEF1, GEF4, and GEF10 that, in turn, activate GTPase ROP11/ARAC10. Arabidopsis mutants disrupted in any step of the FER pathway, including fer, gef1gef4gef10, or rop11/arac10, all displayed an ABA-hypersensitive response, implicating the FER pathway in the suppression mechanism. In search of the target for the FER pathway, we found that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity, thereby linking the FER pathway with the inhibition of ABA signaling.

Genome-Wide Identification of BrCMF Genes in Brassica rapa and Their Expression Analysis under Abiotic Stresses.

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.

FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and play a crucial role in mediating RAC/ROP-regulated root hair development, a polarized cell growth process. We therefore screened T-DNA insertion mutants in these RLKs for root hair defects and found that mutations in one of them, At3g51550 encoding the FERONIA (FER) receptor-like kinase, induced severe root hair defects. We show that the fer phenotypes correlated with reduced levels of active RAC/ROPs and NADPH oxidase-dependent, auxin-regulated ROS accumulation in roots and root hairs and that up-regulating RAC/ROP signaling in fer countered the mutant phenotypes. Taken together, these observations strongly support FER as an upstream regulator for the RAC/ROP-signaled pathway that controls ROS-mediated root hair development. Moreover, FER was pulled down by ROP2 GTPase in a guanine nucleotide-regulated manner implying a dynamic signaling complex involving FER, a ROPGEF, and a RAC/ROP.

Correlation between the CD4+CD25high regulatory T cells and the outcome of chemotherapy in advanced esophageal carcinoma.

BACKGROUND/AIMS: In patients with esophageal carcinoma, local immune suppression and the expression of soluble immunosuppressive factors have been observed. We aimed to investigate the correlation between the level of CD4+CD25high regulatory T (Treg) cell and the outcome of chemotherapy in advanced esophageal carcinoma. METHODOLOGY: Forty-eight cases of advanced esophageal carcinoma patients were enrolled from June 2006 to December 2008. CD3+CD8+ T cell, CD3+CD4 T cell, CD4+CD25+ Treg cell and NK cell were determined before and after chemotherapy. After two cycles of chemotherapy, its effect was evaluated and the survival time was followed-up. RESULTS: Significant downregulation of CD4+CD25high Treg cell was noted in the advanced esophageal carcinoma patients after chemotherapy (p<0.05). However, there were no obvious differences in the CD3+CD8+ T cell, CD3+CD4+ T cell and NK cell before and after chemotherapy (p>0.05). Log-rank test showed age and the decrease of CD4+CD25high Treg cell after chemotherapy correlated with the median survival time (p<0.05). The COX multivariate analysis also suggested that the decrease of CD4+CD25high Treg cell after chemotherapy was an independent prognostic factor (p<0.05). CONCLUSIONS: Our results suggest that the downregulation of CD4+CD25high Treg cell after chemotherapy may be a predictor for the outcome of chemotherapy in advanced esophageal carcinoma patients.

Brassica rapa Nitrate Transporter 2 (BrNRT2) Family Genes, Identification, and Their Potential Functions in Abiotic Stress Tolerance.

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.

Comprehensive Analysis of BrHMPs Reveals Potential Roles in Abiotic Stress Tolerance and Pollen-Stigma Interaction in Brassica rapa.

Heavy metal-associated proteins (HMPs) participate in heavy metal detoxification. Although HMPs have been identified in several plants, no studies to date have identified the HMPs in Brassica rapa (B. rapa). Here, we identified 85 potential HMPs in B. rapa by bioinformatic methods. The promoters of the identified genes contain many elements associated with stress responses, including response to abscisic acid, low-temperature, and methyl jasmonate. The expression levels of BrHMP14, BrHMP16, BrHMP32, BrHMP41, and BrHMP42 were upregulated under Cu2+, Cd2+, Zn2+, and Pb2+ stresses. BrHMP06, BrHMP30, and BrHMP41 were also significantly upregulated after drought treatment. The transcripts of BrHMP06 and BrHMP11 increased mostly under cold stress. After applying salt stress, the expression of BrHMP02, BrHMP16, and BrHMP78 was induced. We observed increased BrHMP36 expression during the self-incompatibility (SI) response and decreased expression in the compatible pollination (CP) response during pollen-stigma interactions. These changes in expression suggest functions for these genes in HMPs include participating in heavy metal transport, detoxification, and response to abiotic stresses, with the potential for functions in sexual reproduction. We found potential co-functional partners of these key players by protein-protein interaction (PPI) analysis and found that some of the predicted protein partners are known to be involved in corresponding stress responses. Finally, phosphorylation investigation revealed many phosphorylation sites in BrHMPs, suggesting post-translational modification may occur during the BrHMP-mediated stress response. This comprehensive analysis provides important clues for the study of the molecular mechanisms of BrHMP genes in B. rapa, especially for abiotic stress and pollen-stigma interactions.

(Bortezomib plus lenalidomide/thalidomide)- vs. (bortezomib or lenalidomide/thalidomide)-containing regimens as induction therapy in newly diagnosed multiple myeloma: a meta-analysis of randomized controlled trials.

The aim of the study was to perform a meta-analysis of the efficacy and safety of (bortezomib plus lenalidomide/thalidomide)- vs. (bortezomib or lenalidomide/thalidomide)-containing regimens as induction therapy in newly diagnosed multiple myeloma. We searched electronic and printed sources for relevant articles published. Inclusion criteria was as follows: randomized controlled trials (RCT) of (bortezomib plus lenalidomide/thalidomide) vs. (bortezomib or lenalidomide/thalidomide)-containing regimens as induction therapy in newly diagnosed multiple myeloma. Two reviewers independently assessed potentially eligible studies and extracted relevant data. We retrieved five RCT studies including a total of 1,200 patients. Using the random-effects model to pool the five RCT with a statistically significant heterogeneity (P = 0.03; X² = 10.69; df = 4; I² = 63%), the weighted risk ratios of a complete response (CR) for (bortezomib plus lenalidomide/thalidomide)-containing regimens was 1.81 (P = 0.005; 95% CI: 1.20-2.73). When we excluded the study by Cavo et al. (Lancet 376:2075-2085, 2010), the pooled risk ratio for CR was 1.59 (P < 0.0001, 95% CI: 1.29-1.96) with no statistically significant heterogeneity (P = 0.54; X² = 2.14; df = 3; I² = 0%) among four RCT under the fixed effects mode. The pooled odds ratio for the main grade III/IV adverse events (the peripheral neuropathy, thrombotic events, and infections) were 1.76 (P = 0.32; 95% CI: 0.58-5.31), 0.92 (P = 0.76, 95% CI: 0.52-1.61), and 1.05 (P = 0.82, 95% CI: 0.70-1.57), respectively. Our analysis showed (bortezomib plus lenalidomide/thalidomide)-containing regimens as induction treatment in newly diagnosed multiple myeloma improved CR but did not increase the risk of major adverse events (the peripheral neuropathy, thrombotic events, and infections).

Genome-Wide Identification and Expression Analysis of BraGLRs Reveal Their Potential Roles in Abiotic Stress Tolerance and Sexual Reproduction.

Glutamate receptors (GLRs) are involved in multiple functions during the plant life cycle through affecting the Ca2+ concentration. However, GLRs in Brassica species have not yet been reported. In this study, 16 glutamate receptor-like channels (GLR) belonged to two groups were identified in the Brassica rapa (B. rapa) genome by bioinformatic analysis. Most members contain domains of ANF_receptor, Peripla_BP_6, Lig_chan, SBP_bac_3, and Lig_chan_Glu_bd that are closely related to glutamate receptor channels. This gene family contains many elements associated with drought stress, low temperature stress, methyl jasmonate (MeJA), salicylic acid (SA), and other stress resistance. Gene expression profiles showed that BraGLR genes were expressed in roots, stems, leaves, flowers, and siliques. BraGLR5 expression was elevated after drought stress in drought-sensitive plants. BraGLR1, BraGLR8, and BraGLR11 expression were significantly upregulated after salt stress. BraGLR3 expression is higher in the female sterile-line mutants than in the wild type. The expression levels of BraGLR6, BraGLR9, BraGLR12, and BraGLR13 were significantly higher in the male sterile-line mutants than in the wild type. The expression of most BraGLRs increased after self-pollination, with BraGLR9 exhibiting the greatest increase. These results suggest that BraGLRs play an important role in abiotic stress tolerance and sexual reproduction.

Pollen-pistil interactions: It takes two to tangle but a molecular cast of many to deliver.

Explosive advances have been made in the molecular understanding of pollen-pistil interactions that underlie reproductive success in flowering plants in the past three decades. Among the most notable is the discovery of pollen tube attractants [1∗,2∗]. The roles these molecules play in facilitating conspecific precedence thus promoting interspecific genetic isolation are also emerging [3-5]. Male-female interactions during the prezygotic phase and contributions from the male and female gametophytes have been comprehensively reviewed recently. Here, we focus on key advances in understanding the mechanistic underpinnings of how these interactions overcome barriers at various pollen-pistil interfaces along the pollen tube growth pathway to facilitate fertilization by desirable mates.

Genome-wide identification and expression analysis of BrAGC genes in Brassica rapa reveal their potential roles in sexual reproduction and abiotic stress tolerance.

AGC protein kinases play important roles in regulating plant growth, immunity, and cell death. However, the function of AGC in Brassica rapa has not yet been clarified. In this study, 62 BrAGC genes were identified, and these genes were distributed on 10 chromosomes and divided into six subfamilies. Analysis of gene structure and conserved motifs showed that the activation segment of BrAGC genes was highly conserved, and genes of the same subfamily showed higher sequence and structural similarity. Collinearity analysis revealed that BrAGCs were more closely related to AtAGCs than to OsAGCs. Expression profile analysis revealed that BrAGCs were preferentially expressed in flowers and BrAGC26, BrAGC33, and BrAGC04 were preferentially expressed in the stigma; the expression of these genes was significantly upregulated after self-incompatibility pollination, and the expression of BrAGC13 and BrAGC32 was significantly upregulated after cross-pollination. In addition, several typical cis-elements involved in the stress response were identified in BrAGC promoters. The expression levels of BrAGC37 and BrAGC44 significantly varied under different types of abiotic stress. Collectively, we identified that BrAGC26, BrAGC33, and BrAGC44 have the greatest potential in regulating pollen-pistil interaction and abiotic stress tolerance, respectively. Our findings will aid future functional investigations of BrAGCs in B. rapa.