Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

Abstract Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family

Genome-wide expression and network analyses of mutants in key brassinosteroid signaling genes

ADDITIONAL FILE 1. List of differentially expressed genes as compared to WS2 in each line and group membership.ADDITIONAL FILE 2. List of marker genes being differentially expressed upon addition of external BRs or in line with a gain of mutation in BR signaling genes at least in 5 studies.ADDITIONAL FILE 3. GO enrichment for genes exclusively differentially expressed in each suppressor.ADDITIONAL FILE: Fig. S1. The T-DNA insertion site for bri1–1D (A), and microscopic images of 7-day old hypocotyl cells for WS2 (B), bri1–5 (C), bri1–5/bak1–1D (D), bri1–5/bri1–1D (E), bri1–5/brs1–1D (F). Fig. S2. PCA plot for assessing the reproducibility of the gene expression dataset. Samples taken from the same genotype are represented in the same color. The plot indicates high consistency between replicate samples as they are located close to each other when plotted on the first and second principal components. Fig. S3. RT-qPCR results for relative expression of selected genes and their corresponding values from microarray analysis. The values represent the log2 of relative expression (sample1/sample2). Rows indicate gene names and columns show the comparison between the indicated lines. Columns with pink header represent the RT-qPCR values, and columns with yellow header are microarray measurements. The red color on the heatmap indicates that the gene has been up-regulated in sample1 as compared to sample2, while blue indicates down-regulation. Fig. S4. Comparing genome-wide expression impact between bri1–5 suppressor lines. Fig. S5. Heatmap of expression of the marker genes that up/down regulation of their expression was confirmed by at least 5 independent references and also affected in the bri1–5 line of our study. For each line, the row-scaled normalized expression data of the 3 biological replicates are shown as adjacent columns. In each row the gradient red color indicates the higher expression for the gene compared to other samples while blue indicates the lower expression. Fig. S6. Pathway analysis (MapMan metabolism) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S7. Pathway analysis (MapMan: large enzyme families) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S8. Pathway analysis (MapMan: gene regulation) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S9. Expression pattern in each mutant line of genes related to ABA signaling, Glutathione metabolism, and ion related hemostasis as discussed in the main text. Mutant lines are represented in the x-axis. The y-axis indicates the log2 normalized expression value of the gene. Table S1. RT-qPCR test of log-fold change (log-FC) of the genes that are overexpressed by activation-tagging in the suppressors at the 7 days seedling stage. Table S2. Summary of the most significant results obtained by MapMan pathway analysis (metabolism, regulation and, large-enzyme families overview). Left column: enriched pathways; entries provide for each line the degree to which the pathway is enriched. P-values are FDR corrected using Benjamini-Hochberg). Table S3. Designed primers for RT-qPCR.BACKGROUND: Brassinosteroid (BR) signaling regulates plant growth and development in concert with other signaling pathways. Although many genes have been identified that play a role in BR signaling, the biological and functional consequences of disrupting those key BR genes still require detailed investigation. RESULTS: Here we performed phenotypic and transcriptomic comparisons of A. thaliana lines carrying a loss-of-function mutation in BRI1 gene, bri1–5, that exhibits a dwarf phenotype and its three activation-tag suppressor lines that were able to partially revert the bri1–5 mutant phenotype to a WS2 phenotype, namely bri1–5/bri1–1D, bri1–5/brs1–1D, and bri1–5/bak1–1D. From the three investigated bri1–5 suppressors, bri1–5/bak1–1D was the most effective suppressor at the transcriptional level. All three bri1–5 suppressors showed altered expression of the genes in the abscisic acid (ABA signaling) pathway, indicating that ABA likely contributes to the partial recovery of the wild-type phenotype in these bri1–5 suppressors. Network analysis revealed crosstalk between BR and other phytohormone signaling pathways, suggesting that interference with one hormone signaling pathway affects other hormone signaling pathways. In addition, differential expression analysis suggested the existence of a strong negative feedback from BR signaling on BR biosynthesis and also predicted that BRS1, rather than being directly involved in signaling, might be responsible for providing an optimal environment for the interaction between BRI1 and its ligand. CONCLUSIONS: Our study provides insights into the molecular mechanisms and functions of key brassinosteroid (BR) signaling genes, especially BRS1.The National Basic Research Program of China, Youth Innovation Promotion Association of the Chinese Academy of Sciences, the Ministry of Science, Research and Technology, Iran, the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO) and UGent Bijzonder onderzoeksfonds.http://www.biomedcentral.com/bmcgenomicspm2021BiochemistryGeneticsMicrobiology and Plant Patholog

A genetic study and meta-analysis of the genetic predisposition of prostate cancer in a Chinese population.

Prostate cancer predisposition has been extensively investigated in European populations, but there have been few studies of other ethnic groups. To investigate prostate cancer susceptibility in the under-investigated Chinese population, we performed single-nucleotide polymorphism (SNP) array analysis on a cohort of Chinese cases and controls and then meta-analysis with data from the existing Chinese prostate cancer genome-wide association study (GWAS). Genotyping 211,155 SNPs in 495 cases and 640 controls of Chinese ancestry identified several new suggestive Chinese prostate cancer predisposition loci. However, none of them reached genome-wide significance level either by meta-analysis or replication study. The meta-analysis with the Chinese GWAS data revealed that four 8q24 loci are the main contributors to Chinese prostate cancer risk and the risk alleles from three of them exist at much higher frequencies in Chinese than European populations. We also found that several predisposition loci reported in Western populations have different effect on Chinese men. Therefore, this first extensive single-nucleotide polymorphism study of Chinese prostate cancer in comparison with European population indicates that four loci on 8q24 contribute to a great risk of prostate cancer in a considerable large proportion of Chinese men. Based on those four loci, the top 10% of the population have six- or two-fold prostate cancer risk compared with men of the bottom 10% or median risk respectively, which may facilitate the design of prostate cancer genetic risk screening and prevention in Chinese men. These findings also provide additional insights into the etiology and pathogenesis of prostate cancer.This work was conducted on behalf of the CHIPGECS and The PRACTICAL consortia (see Supplementary Consortia). We acknowledge the contribution of doctors, nurses and postgraduate research students at the CHIPGENCS sample collecting centers. We thank Orchid and Rosetrees for funding support. This work was also supported by National Natural Science foundation of China for funding support to H Zhang (Grant No: 30671793 and 81072377), N Feng (Grant No: 81272831), X Zhang (Grant No: 30572139, 30872924 and 81072095), S Zhao (Grant No: 81072092 and 81328017), Y Yu (Grant No: 81172448) and Program for New Century Excellent Talents in University from Department of Education of China (NCET-08-0223) and the National High Technology Research and Development Program of China (863 Program 2012AA021101) to X Zhang.This is the final version of the article. It first appeared from Impact Journals via http://dx.doi.org/10.18632/oncotarget.725

Biodegradable Thermosensitive Hydrogel for SAHA and DDP Delivery: Therapeutic Effects on Oral Squamous Cell Carcinoma Xenografts

Background: OSCC is one of the most common malignancies and numerous clinical agents currently applied in combinative chemotherapy. Here we reported a novel therapeutic strategy, SAHA and DDP-loaded PECE (SAHA-DDP/PECE), can improve the therapeutic effects of intratumorally chemotherapy on OSCC cell xenografts. Objective/Purpose: The objective of this study was to evaluate the therapeutic efficacy of the SAHA-DDP/PECE in situ controlled drug delivery system on OSCC cell xenografts. Methods: A biodegradable and thermosensitive hydrogel was successfully developed to load SAHA and DDP. Tumorbeared mice were intratumorally administered with SAHA-DDP/PECE at 50 mg/kg (SAHA) +2 mg/kg (DDP) in 100 ul PECE hydrogel every two weeks, SAHA-DDP at 50 mg/kg(SAHA) +2 mg/kg(DDP) in NS, 2 mg/kg DDP solution, 50 mg/kg SAHA solution, equal volume of PECE hydrogel, or equal volume of NS on the same schedule, respectively. The antineoplastic actions of SAHA and DDP alone and in combination were evaluated using the determination of tumor volume, immunohistochemistry, western blot, and TUNEL analysis. Results: The hydrogel system was a free-flowing sol at 10uC, become gel at body temperature, and could sustain more than 14 days in situ. SAHA-DDP/PECE was subsequently injected into tumor OSCC tumor-beared mice. The results demonstrated that such a strategy as this allows the carrier system to show a sustained release of SAHA and DDP in vivo, and coul

Genetic Evidence for an Indispensable Role of Somatic Embryogenesis Receptor Kinases in Brassinosteroid Signaling

The authors are grateful to the Arabidopsis Biological Resource Center for providing the T-DNA insertion lines discussed in this work. We thank Dr. Yanhai Yin (Iowa State University) for providing anti-BES1 antibody, Dr. Jiayang Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for bri1-301 seeds, and Dr. Xing-wang Deng (Yale University) for cop1-4 and cop1-6 seeds as controls.Author Summary Brassinosteroids (BRs) are a group of plant hormones critical for plant growth and development. BRs are perceived by a cell-surface receptor complex including two distinctive receptor kinases, BRI1 and BAK1. Whereas BRI1 is a true BR-binding receptor, BAK1 does not appear to have BR-binding activity. Therefore, BAK1 is likely a co-receptor in BR signal transduction. The genetic significance of BAK1 was not clearly demonstrated in previous studies largely due to functional redundancy of BAK1 and its closely related homologues. It was not clear whether BAK1 plays an essential role or only an enhancing role in BR signaling. In this study, we identified all possible BAK1 redundant genes in the Arabidopsis thaliana genome and generated single, double, triple, and quadruple mutants. Detailed analysis indicated that, without BAK1 and its functionally redundant proteins, BR signaling is completely disrupted, largely because BRI1 has lost its ability to activate downstream components. These studies provide the first piece of loss-of-functional genetic evidence that BAK1 is indispensable to the early events of the BR signaling pathway.Yeshttp://www.plosgenetics.org/static/editorial#pee

The First Line of Defense: Receptor-like Protein Kinase-Mediated Stomatal Immunity

Stomata regulate gas and water exchange between the plant and external atmosphere, which are vital for photosynthesis and transpiration. Stomata are also the natural entrance for pathogens invading into the apoplast. Therefore, stomata play an important role in plants against pathogens. The pattern recognition receptors (PRRs) locate in guard cells to perceive pathogen/microbe-associated molecular patterns (PAMPs) and trigger a series of plant innate immune responses, including rapid closure of stomata to limit bacterial invasion, which is termed stomatal immunity. Many PRRs involved in stomatal immunity are plasma membrane-located receptor-like protein kinases (RLKs). This review focuses on the current research progress of RLK-mediated signaling pathways involved in stomatal immunity, and discusses questions that need to be addressed in future research

Male gamete biology in flowering plants

Abstract Flowering plant reproduction is characterized by double fertilization, in which two diminutive brother sperm cells initiate embryo and endosperm. The role of the male gamete, although studied structurally for over a century at various levels, is still being explored on a molecular and cellular level. The potential of the male to influence development has been historically underestimated and the reasons for this are obvious: limitations provided by maternal imprinting, the much greater cellular volume of female gametes and the general paucity of paternal effects. However, as more is known about molecular expression of chromatinmodifying proteins, ubiquitin pathway proteins and transcription factors in sperm cells, as well as their ability to achieve effect by intaglio expression, passing transcripts directly into translation, the role of the male is likely to expand. Much of the expression in the male germline that appears to be distinct from patterns of pollen vegetative cell expression may be the result of chromosomal level regulation of transcription. Structure, timing and physiology in flowering plant reproduction In flowering plants, double fertilization launches two dramatically distinct developmental programmes. One of the two sperm cells in a successful pollen tube fuses with the egg cell to form the canonical zygote and embryo; a second brother sperm cell fuses with the central cell to form the male portion of the unique and precocious nutritive endosperm. This unique complement is part of a temporal choreography that is conducted most conspicuously in floral parts that are paradoxically sporophytic and asexual, but also govern the timing of gametophytic events The diversity of structural organization of gametophytes is well described in previous review