GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17.

KEY MESSAGE: A U-box E3 ubiquitin ligase GhPUB17 is inhibited by GhCyP3 with antifungal activity and acts as a negative regulator involved in cotton resistance to Verticillium dahliae. E3 ubiquitin ligases, the key component enzymes of the ubiquitin-proteasome system, which contains the most diverse structural and functional members involved in the determination of target specificity and the regulation of metabolism, have been well documented in previous studies. Here, we identify GhPUB17, a U-box E3 ligase in cotton that has ubiquitination activity and is involved in the cotton immune response to Verticillium dahliae. The expression level of GhPUB17 is downregulated in the ssn mutant with a constitutively activated immune response (Sun et al., Nat Commun 5:5372, 2014). Infection with V. dahliae or exogenous hormone treatment, including jasmonic acid and salicylic acid, significantly upregulated GhPUB17 in cotton roots, which suggested a possible role for this E3 ligase in the plant immune response to pathogens. Moreover, GhPUB17-knockdown cotton plants are more resistant to V. dahliae, whereas GhPUB17-overexpressing plants are more susceptible to the pathogen, which indicated that GhPUB17 is a negative regulator of cotton resistance to V. dahliae. A yeast two-hybrid (Y2H) assay identified GhCyP3 as a protein that interacts with GhPUB17, and this finding was confirmed by further protein interaction assays. The downregulation of GhCyP3 in cotton seedlings attenuated the plants' resistance to V. dahliae. In addition, GhCyP3 showed antifungal activity against V. dahliae, and the E3 ligase activity of GhPUB17 was repressed by GhCyP3 in vitro. These results suggest that GhPUB17 negatively regulates cotton immunity to V. dahliae and that the antifungal protein GhCyP3 likely interacts with and inhibits the ligase activity of GhPUB17 and plays an important role in the cotton-Verticillium interaction.

GbSOBIR1 confers Verticillium wilt resistance by phosphorylating the transcriptional factor GbbHLH171 in Gossypium barbadense.

Receptor-like kinases (RLKs) are important components of plant innate immunity. Although recent studies have revealed that the RLK suppressor of BIR1-1 (SOBIR1) can interact with multiple receptor-like proteins and is required for resistance against fungal pathogens, how the signal is transduced and triggers immune responses remains enigmatic. In this study, we identified a defence-related RLK from Gossypium barbadense (designated GbSOBIR1) and investigated its functional mechanism. Expression of the GbSOBIR1 gene is ubiquitous in cotton plants and is induced by Verticillium dahliae inoculation. Knock-down of GbSOBIR1 by virus-induced gene silencing resulted in attenuated resistance of cotton plants to V. dahliae, while heterologous overexpression of GbSOBIR1 in Arabidopsis improves resistance. We also found that the kinase region of GbSOBIR1 interacts with a basic helix-loop-helix (bHLH) transcription factor identified as GbbHLH171 in a yeast-two-hybrid screen. GbbHLH171 could interact with and be phosphorylated by GbSOBIR1 in vitro and in vivo and contributes positively to the resistance of cotton against V. dahliae. Furthermore, we found that this phosphorylation is essential to the transcriptional activity and functional role of GbbHLH171. We also show by spectrometric analysis and site-directed mutagenesis that Ser413 is the GbSOBIR1-mediated phosphorylation site of GbbHLH171. These results demonstrate that GbSOBIR1 interacts with GbbHLH171 and plays a critical role in cotton resistance to V. dahliae.

Transcriptome analysis reveals new microRNAs-mediated pathway involved in anther development in male sterile wheat.

BACKGROUND: 337S is a novel bi-pole-photo-thermo-sensitive genic male sterile line in wheat, and sensitive to both long day length/high temperature and short day length/low temperature condition. Although the regulatory function of MicroRNAs (miRNAs) in reproductive development has been increasingly studied, their roles in pre-meiotic and meiotic cells formation of plants have not been clearly explored. Here, we explored the roles of miRNAs in regulating male sterility of 337S at short day length/low temperature condition. RESULTS: Small RNA sequencing and degradome analyses were employed to identify miRNAs and their targets in the 337S whose meiotic cells collapsed rapidly during male meiotic prophase, resulting in failure of meiosis at SL condition. A total of 102 unique miRNAs were detected. Noticeably, the largest miRNA family was MiR1122. The target CCR4-associated factor 1 (CAF1) of miR2275, a subunit of the Carbon Catabolite Repressed 4-Negative on TATA-less (CCR4-NOT) complex, contributes to the process of early meiosis, and was first identified here. Further studies showed that the expression of several pivotal anther-related miRNAs was altered in 337S at SL condition, especially tae-miR1127a, which may be related to male sterility of 337S. Here, we also identified a new member of SWI/SNF factors SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3-like 3 (SMARCA3L3) targeted by tae-miR1127a, whose function might be involved in faithful progression of meiosis in male reproductive cells. CONCLUSION: The miRNA-target interactions of tae-miR2275-CAF1 and tae-miR1127a-SMARCA3L3 might be involved in regulating male fertility in 337S. Our results also implied that multiple roles for SMARCA3L3 and CAF1 in DNA repair and transcriptional regulation jointly orchestrated a tight and orderly system for maintaining chromatin and genome integrity during meiosis.

Proteomic and virus-induced gene silencing (VIGS) Analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae.

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

Functional characterization of cotton genes responsive to Verticillium dahliae through bioinformatics and reverse genetics strategies.

Verticillium wilt causes dramatic cotton yield loss in China. Although some genes or biological processes involved in the interaction between cotton and Verticillium dahliae have been identified, the molecular mechanism of cotton resistance to this disease is still poorly understood. The basic innate immune response for defence is somewhat conserved among plant species to defend themselves in complex environments, which makes it possible to characterize genes involved in cotton immunity based on information from model plants. With the availability of Arabidopsis databases, a data-mining strategy accompanied by virus-induced gene silencing (VIGS) and heterologous expression were adopted in cotton and tobacco, respectively, for global screening and gene function characterization. A total of 232 Arabidopsis genes putatively involved in basic innate immunity were screened as candidate genes, and bioinformatic analysis suggested a role of these genes in the immune response. In total, 38 homologous genes from cotton were singled out to characterize their response to V. dahliae and methyl jasmonate treatment through quantitative real-time PCR. The results revealed that 24 genes were differentially regulated by pathogen inoculation, and most of these genes responded to both Verticillium infection and jasmonic acid stimuli. Furthermore, the efficiency of the strategy was illustrated by the functional identification of six candidate genes via heterologous expression in tobacco or a knock-down approach using VIGS in cotton. Functional categorization of these 24 differentially expressed genes as well as functional analysis suggest that reactive oxygen species, salicylic acid- and jasmonic acid-signalling pathways are involved in the cotton disease resistance response to V. dahliae. Our data demonstrate how information from model plants can allow the rapid translation of information into non-model species without complete genome sequencing, via high-throughput screening and functional identification of target genes based on data-mining and VIGS.

Widely Targeted Metabolomics Provides New Insights into Nutritional Profiling and Reveals the Flavonoid Pathway of Pea (Pisum sativum L.).

To learn more about the nutritional composition and health benefits for human consumers of peas, we used a widely targeted metabolomics-based approach to reveal the metabolite components from three main varieties, and a total of 1095 metabolites were identified. A comparison of 487 differentially accumulated metabolites shared among three varieties of fresh and dried peas found most of the amino acids and derivatives were downregulated and most of the lipids and flavonoids were upregulated in dried peas. Furthermore, comparing the main nutrient profiles exclusively showed that there were few differences in free fatty acids, sugars, vitamins, and alkaloids between dried and fresh peas. Peas are especially enriched with B-group vitamins. Through detailed identification and classification, the flavonoid pathway of peas was revealed; a variety of glycosylated derivatives from kaempferol, quercetin, and luteolin were confirmed to be abundant in peas. It was also found that isoflavones are richer in peas than in many other plants, and putatively the isoflavone synthesis pathway originates from liquiritigenin and naringenin. Our study not only offers guidance for understanding the nutritional components of peas, but also provides the basis for healthy diet analysis of the edible value and health benefits of peas.

BioLadder: A bioinformatic platform primarily focused on proteomic data analysis.

BioLadder (https://www.bioladder.cn/) is an online data analysis platform designed for proteomics research, which includes three classes of experimental data analysis modules and four classes of common data analysis modules. It allows for a variety of proteomics analyses to be conducted easily and efficiently. Additionally, most modules can also be utilized for the analysis of other omics data. To facilitate user experience, we have carefully designed four different kinds of functions for customers to quickly and accurately utilize the relevant analysis modules.

A hybrid XGBoost-ISSA-LSTM model for accurate short-term and long-term dissolved oxygen prediction in ponds.

Dissolved oxygen (DO) is one of the most critical factors to measure the water quality in ponds, which greatly impacts on healthy growth of aquatic organisms. To improve the prediction accuracy of DO and grasp its changing trends, a novel hybrid DO prediction model based on the long short-term memory network (LSTM) optimized by an improved sparrow search algorithm (ISSA) is proposed. Firstly, to discard redundant information and improve the calculation speed of the model, the key factors that have a greater correlation with DO are selected as the input parameters by extreme gradient boosting (XGBoost). Secondly, towards expanding the searching range of sparrows and balancing the global and local search, we introduce an adaptive factor exponential declining strategy for producers, and an arcsine decreasing strategy for scouters, which nonlinearly decreases with the increase of iterations. Besides, we also improve the position updating of scouters, making the sparrows gradually move to the best position. Finally, LSTM is optimized by ISSA to get the best initial weights and thresholds to construct an XGBoost-ISSA-LSTM DO prediction model. Specifically, we first analyze the method for water quality prediction, which can make short-term prediction (including about 1 h, 2 h) and long-term prediction (including about 12 h, 24 h) of DO. In 1-h prediction, the root mean square error (RMSE) of the model is 0.5571, the mean absolute error (MAE) is 0.2572, and the R2 is 0.9276. In 24 h prediction, RMSE of the model is 0.6310, MAE is 0.4562, and R2 is 0.9082. The experimental results show that the proposed model has better generalization performance and higher prediction accuracy compared with other common models. Therefore, the presented model based on XGBoost-ISSA-LSTM is more effective and could meet the actual demand of accurate prediction of DO.

New insights into the evolution of CAF1 family and utilization of TaCAF1Ia1 specificity to reveal the origin of the maternal progenitor for common wheat.

INTRODUCTION: Until now, the most likely direct maternal progenitor (AABB) for common wheat (AABBDD) has yet to be identified. Here, we try to solve this particular problem with the specificity of a novel gene family in wheat and by using large population of rare germplasm resources. OBJECTIVES: Dissect the novelty of TaCAF1Ia subfamily in wheat. Exploit the conservative and specific characteristics of TaCAF1Ia1 to reveal the origin of the maternal progenitor for common wheat. METHODS: Phylogenetic and collinear analysis of TaCAF1 genes were performed to identify the evolutionary specificity of TaCAF1Ia subfamily. The large-scale expression patterns and interaction patterns analysis of CCR4-NOT complex were used to clarify the expressed and structural specificity of TaCAF1Ia subfamily in wheat. The population resequencing and phylogeny analysis of the TaCAF1Ia1 were utilized for the traceability analysis to understand gene-pool exchanges during the transferring and subsequent development from tetraploid to hexaploidy wheat. RESULTS: TaCAF1Ia is a novel non-typical CAF1 subfamily without DEDD (Asp-Glu-Asp-Asp) domain, whose members were extensively duplicated in wheat genome. The replication events had started and constantly evolved from ancestor species. Specifically, it was found that a key member CAF1Ia1 was highly specialized and only existed in the subB genome and S genome. Unlike CAF1s reported in other plants, TaCAF1Ia genes may be new factors for anther development. These atypical TaCAF1s could also form CCR4-NOT complex in wheat but with new interaction sites. Utilizing the particular but conserved characteristics of the TaCAF1Ia1 gene, the comparative analysis of haplotypes composition for TaCAF1Ia1 were identified among wheat populations with different ploidy levels. Based on this, the dual-lineages origin model of maternal progenitor for common wheat and potential three-lineages domestication model for cultivated tetraploid wheat were proposed. CONCLUSION: This study brings fresh insights for revealing the origin of wheat and the function of CAF1 in plants.

iTRAQ based protein profile analysis revealed key proteins involved in regulation of drought-tolerance during seed germination in Adzuki bean.

Adzuki bean is an important legume crop due to its high-quality protein, fiber, vitamins, minerals as well as rich bioactive substances. However, it is vulnerable to drought at the germination stage. However, little information is available about the genetic control of drought tolerance during seed germination in adzuki bean. In this study, some differential expression proteins (DEPs) were identified during seed germination between the drought-tolerant variety 17235 and drought-sensitive variety 17033 in adzuki bean using iTRAQ method. A total of 2834 proteins were identified in the germinating seeds of these two adzuki beans. Compared with the variety 17033, 87 and 80 DEPs were increased and decreased accumulation in variety 17235 under drought, respectively. Meanwhile, in the control group, a few DEPs, including 9 up-regulated and 21 down-regulated proteins, were detected in variety 17235, respectively. GO, KEGG, and PPI analysis revealed that the DEPs related to carbohydrate metabolism and energy production were significantly increased in response to drought stresses. To validate the proteomic function, the ectopic overexpression of V-ATPase in tobacco was performed and the result showed that V-ATPase upregulation could enhance the drought tolerance of tobacco. The results provide valuable insights into genetic response to drought stress in adzuki bean, and the DEPs could be applied to develop biomarkers related to drought tolerant in adzuki bean breeding projects.

SNP-based association study of kernel architecture in a worldwide collection of durum wheat germplasm.

Durum wheat, genetic resource with favorable alleles is considered as natural gene pool for wheat breeding. Kernel size and weight are important factors affecting grain yield in crops. Here, association analysis was performed to dissect the genetic constitution of kernel-related traits in 150 lines collected from 46 countries and regions using a set of EST-derived and genome-wide SNP markers with five consecutive years of data. Total 109 significant associations for eight kernel-related traits were detected under a mix linear model, generating 54 unique SNP markers distributed on 13 of 14 chromosomes. Of which, 19 marker-trait associations were identified in two or more environments, including one stable and pleiotropic SNP BE500291_5_A_37 on chromosome 5A correlated with six kernel traits. Although most of our SNP loci were overlapped with the previously known kernel weight QTLs, several novel loci for kernel traits in durum were reported. Correlation analysis implied that the moderate climatic variables during growth and development of durum are needed for the large grain size and high grain weight. Combined with our previous studies, we found that chromosome 5A might play an important role in durum growth and development.

Genome-Wide Identification of Papain-Like Cysteine Proteases in Gossypium hirsutum and Functional Characterization in Response to Verticillium dahliae.

Cotton, a natural fiber producing crop of huge importance, is often prone to attack of Verticillium dahliae. Papain-like cysteine proteases (PLCPs) constitute a large family in plants and were proposed to involve in plant defense against pathogen attack in a number of studies. However, there is no detailed characterization of PLCP genes in cotton against infection of V. dahliae. In this study, we carried out a genome-wide analysis in cotton and identified seventy-eight PLCPs, which were divided into nine subfamilies based on their evolution phylogeny: RD21 (responsive to desiccation 21), CEP (cysteine endopeptidase), XCP (xylem cysteine peptidase), XBCP3 (xylem bark cysteine peptidase 3), THI, SAG12 (senescence-associated gene 12), RD19 (responsive to desiccation 19), ALP (aleurain-like protease) and CTB (cathepsin B-like). Genes in each subfamily exhibit a similar structure and motif composition. The expression patterns of these genes in different organs were examined, and subfamily RD21 was the most abundant in these families. Expression profiles under abiotic stress showed that thirty-five PLCP genes were induced by multiple stresses. Further transcriptome analysis showed that sixteen PLCP genes were up-regulated in response to V. dahliae in cotton. Among those, GhRD21-7 showed a higher transcription level than most other PLCP genes. Additionally, over-expression of GhRD21-7 led to enhanced resistance and RNAi lines were more susceptible to V. dahliae in cotton. Our results provide valuable information for future functional genomic studies of PLCP gene family in cotton.

Associations of canopy leaf traits with SNP markers in durum wheat (Triticum turgidum L. durum (Desf.)).

The canopy leaves including the top three, i.e., the flag, the 2nd and 3rd from the top, are important for photosynthesis and grain yield of wheat. Molecular markers associated with traits of these leaves should be helpful for the high-yielding breeding. In this study, 1366 single nucleotide polymorphisms (SNP) markers covering the whole genome of durum wheat were used to genotype 150 cultivars collected from 46 countries and regions in the world. Leaf length, leaf width and chlorophyll content of the top three leaves were measured, respectively, in three consecutive years. Association analyses were performed on the leaf traits and SNP markers. A total of 120 SNP marker associations were detected on 13 of the 14 chromosomes. Among these markers, 83 were associated with the canopy leaf traits, 10 with 1000-grain weight, and 29 with kernel number per spike. This study is helpful for better understanding the potential and genetic basis of functional leaves, and facilitates pyramiding of the favorable alleles using marker assisted selection for ideal plant-type and high photosynthesis efficiency in durum wheat breeding.

Cotton cytochrome P450 CYP82D regulates systemic cell death by modulating the octadecanoid pathway.

Plant oxylipins are derived from unsaturated fatty acids and play roles in plant growth and development as well as defence. Although recent studies have revealed that fatty acid metabolism is involved in systemic acquired resistance, the precise function of oxylipins in plant defence remains unknown. Here we report a cotton P450 gene SILENCE-INDUCED STEM NECROSIS (SSN), RNAi suppression of which causes a lesion mimic phenotype. SSN is also involved in jasmonate metabolism and the response to wounding. Fatty acid and oxylipin metabolite analysis showed that SSN overexpression causes hyperaccumulation of hydroxide and ketodiene fatty acids and reduced levels of 18:2 fatty acids, whereas silencing causes an imbalance in LOX (lipoxygenase) expression and excessive hydroperoxide fatty acid accumulation. We also show that an unknown oxylipin-derived factor is a putative mobile signal required for systemic cell death and hypothesize that SSN acts as a valve to regulate HR on pathogen infection.