Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference–based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

SummaryWhite lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to )P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA i )-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under )P stress. Here, we show that LaMATE had high expression in proteoid roots not only under )P, but also under )Fe, )N, )Mn and þAl stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein.LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA i -based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.

Background: Previous studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome. Results: Genome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable -estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/ absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation.

Colorectal cancer (CRC) is a global healthcare problem. Radioresistance is a huge setback for CRC radiotherapy. In this text, the roles and molecular mechanisms of long non-coding RNA HOTAIR in CRC tumorigenesis and radioresistance were further investigated. ATG12 mRNA, HOTAIR, and microRNA-93 (miR-93) levels were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. Protein levels of LC3 I, LC3 II, p62, ATG12, cleaved caspase 3, Bax, and Bcl-2 were detected by western blotting assay in cells and were examined by immunohistochemistry (IHC) assay in tissues. Cell survival fractions, viability, and apoptotic rates were determined by clonogenic survival assay, CCK-8 assay, and flow cytometry analysis, respectively. The relationships of HOTAIR, miR-93, and ATG12 were tested by bioinformatics analysis and luciferase reporter assay. Mouse xenograft tumor models were established to investigate the influence of HOTAIR knockdown on CRC radioresistance in vivo. We found that HOTAIR expression was markedly upregulated in plasma from CRC patients after radiotherapy and CRC cells after irradiation. HOTAIR knockdown, miR-93 overexpression, or ATG12 silencing weakened cell viability, induced cell apoptosis, inhibited cell autophagy, and enhanced cell radiosensitivity in CRC. HOTAIR exerted its functions by downregulating miR-93. Moreover, HOTAIR functioned as a molecular sponge of miR-93 to regulate ATG12 expression. ATG12 protein expression was markedly upregulated and associated with miR-93 and HOTAIR expression in CRC tissues. Furthermore, HOTAIR knockdown enhanced radiosensitivity of CRC xenograft tumors by regulating miR-93/ATG12 axis. In conclusion, HOTAIR knockdown potentiated radiosensitivity through regulating miR-93/ATG12 axis in CRC, further elucidating the roles and molecular basis of HOTAIR in CRC radioresistance.

Background: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. Results: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. Conclusions: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.

The Scarecrow (SCR) transcription factor plays a crucial role in root cell radial patterning and is required for maintenance of the quiescent centre and differentiation of the endodermis. In response to phosphorus (P) deficiency, white lupin (Lupinus albus L.) root surface area increases some 50-fold to 70-fold due to the development of cluster (proteoid) roots. Previously it was reported that SCR-like expressed sequence tags (ESTs) were expressed during early cluster root development. Here the cloning of two white lupin SCR genes, LaSCR1 and LaSCR2, is reported. The predicted amino acid sequences of both LaSCR gene products are highly similar to AtSCR and contain C-terminal conserved GRAS family domains. LaSCR1 and LaSCR2 transcript accumulation localized to the endodermis of both normal and cluster roots as shown by in situ hybridization and gene promoter::reporter staining. Transcript analysis as evaluated by quantitative real-time-PCR (qRT-PCR) and RNA gel hybridization indicated that the two LaSCR genes are expressed predominantly in roots. Expression of LaSCR genes was not directly responsive to the P status of the plant but was a function of cluster root development. Suppression of LaSCR1 in transformed roots of lupin and Medicago via RNAi (RNA interference) delivered through Agrobacterium rhizogenes resulted in decreased root numbers, reflecting the potential role of LaSCR1 in maintaining root growth in these species. The results suggest that the functional orthologues of AtSCR have been characterized.

Background: Propofol has been identified to perform anti-tumor functions in glioma. However, the molecular mechanisms underlying propofol-induced prevention on migration and invasion of glioma cells remain unclear. Methods: Cell proliferation, invasion and migration were measured by 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di-phenytetrazoliumromide assay and transwell assay, respectively. The expression of microRNA (miR)-206 and Rho-associated coiled coil-containing protein kinase 1 (ROCK1) was detected by quantitative real-time polymerase chain reaction. Western blot was used to measure the activation of the PI3K/AKT pathway. The interaction between miR-206 and ROCK1 was analyzed using the dual-luciferase reporter assay, RNA immunoprecipitation assay, and pull-down assay. Results: Propofol treatment inhibited the migration, invasion, and PI3K/AKT pathway activation in glioma cells. MiR-206 was decreased in glioma tissues and cells, while propofol exposure induced the upregulation of miR-206 in glioma cells. Besides that, we also found overexpressed miR-206 enhanced propofol-mediated inhibition on the migration, invasion, and PI3K/AKT pathway activation of glioma cells. Subsequently, ROCK1 was confirmed to be a target of miR-206. ROCK1 was elevated in glioma tissues and cells, but was reduced by propofol exposure in glioma cells. The rescue assay indicated that the miR-206/ROCK1 axis was involved in propofol-induced inhibition on the migration, invasion, and PI3K/AKT pathway activation in glioma cells. Conclusion: Propofol inhibited the migration and invasion of glioma cells by blocking the PI3K/AKT pathway through the miR-206/ROCK1 axis, suggesting an effective clinical implication for the anesthetic to prevent the metastasis of glioma.

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