Pollen Number and Ribosome Gene Expression Altered in a Genome-Editing Mutant of REDUCED POLLEN NUMBER1 Gene

The number of pollen grains varies within and between species. However, little is known about the molecular basis of this quantitative trait, in contrast with the many studies available on cell differentiation in the stamen. Recently, the first gene responsible for pollen number variation, REDUCED POLLEN NUMBER1 (RDP1), was isolated by genome-wide association studies of Arabidopsis thaliana and exhibited the signature of natural selection. This gene encodes a homolog of yeast Mrt4 (mRNA turnover4), which is an assembly factor of the large ribosomal subunit. However, no further data were available to link ribosome function to pollen development. Here, we characterized the RDP1 gene using the standard A. thaliana accession Col-0. The frameshift mutant, rdp1-3 generated by CRISPR/Cas9 revealed the pleiotropic effect of RDP1 in flowering, thus demonstrating that this gene is required for a broad range of processes other than pollen development. We found that the natural Col-0 allele conferred a reduced pollen number against the Bor-4 allele, as assessed using the quantitative complementation test, which is more sensitive than transgenic experiments. Together with a historical recombination event in Col-0, which was identified by sequence alignment, these results suggest that the coding sequence of RDP1 is the candidate region responsible for the natural phenotypic variation. To elucidate the biological processes in which RDP1 is involved, we conducted a transcriptome analysis. We found that genes responsible for ribosomal large subunit assembly/biogenesis were enriched among the differentially regulated genes, which supported the hypothesis that ribosome biogenesis is disturbed in the rdp1-3 mutant. Among the pollen-development genes, three key genes encoding basic helix-loop-helix (bHLH) transcription factors (ABORTED MICROSPORES (AMS), bHLH010, and bHLH089), as well as direct downstream genes of AMS, were downregulated in the rdp1-3 mutant. In summary, our results suggest a specialized function of ribosomes in pollen development through RDP1, which harbors natural variants under selection.

Long noncoding RNA TINCR facilitates hepatocellular carcinoma progression and dampens chemosensitivity to oxaliplatin by regulating the miR-195-3p/ST6GAL1/NF-κB pathway

Background Long non-coding RNAs (lncRNA) have an essential role in progression and chemoresistance of hepatocellular carcinoma (HCC). In-depth study of specific regulatory mechanisms is of great value in providing potential therapeutic targets. The present study aimed to explore the regulatory functions and mechanisms of lncRNA TINCR in HCC progression and oxaliplatin response. Methods The expression of TINCR in HCC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation, migration, invasion, and chemosensitivity were evaluated by cell counting kit 8 (CCK8), colony formation, transwell, and apoptosis assays. Luciferase reporter assays and RNA pulldown were used to identify the interaction between TINCR and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) via miR-195-3p. The corresponding functions were verified in the complementation test and in vivo animal experiment. Results TINCR was upregulated in HCC and associated with poor patient prognosis. Silencing TINCR inhibited HCC proliferation, migration, invasion, and oxaliplatin resistance while overexpressing TINCR showed opposite above-mentioned functions. Mechanistically, TINCR acted as a competing endogenous (ceRNA) to sponge miR-195-3p, relieving its repression on ST6GAL1, and activated nuclear factor kappa B (NF-κB) signaling. The mouse xenograft experiment further verified that knockdown TINCR attenuated tumor progression and oxaliplatin resistance in vivo. Conclusions Our finding indicated that there existed a TINCR/miR-195-3p/ST6GAL1/NF-κB signaling regulatory axis that regulated tumor progression and oxaliplatin resistance, which might be exploited for anticancer therapy in HCC.

Genome-Wide Identification, Primary Functional Characterization of the NHX Gene Family in Canavalia rosea, and Their Possible Roles for Adaptation to Tropical Coral Reefs

Canavalia rosea, distributed in the coastal areas of tropical and subtropical regions, is an extremophile halophyte with good adaptability to high salinity/alkaline and drought tolerance. Plant sodium/hydrogen (Na+/H+) exchanger (NHX) genes encode membrane transporters involved in sodium ion (Na+), potassium ion (K+), and lithium ion (Li+) transport and pH homeostasis, thereby playing key roles in salinity tolerance. However, the NHX family has not been reported in this leguminous halophyte. In the present study, a genome-wide comprehensive analysis was conducted and finally eight CrNHXs were identified in C. rosea genome. Based on the bioinformatics analysis about the chromosomal location, protein domain, motif organization, and phylogenetic relationships of CrNHXs and their coding proteins, as well as the comparison with plant NHXs from other species, the CrNHXs were grouped into three major subfamilies (Vac-, Endo-, and PM-NHX). Promoter analyses of cis-regulatory elements indicated that the expression of different CrNHXs was affected by a series of stress challenges. Six CrNHXs showed high expression levels in five tested tissues of C. rosea in different levels, while CrNHX1 and CrNHX3 were expressed at extremely low levels, indicating that CrNHXs might be involved in regulating the development of C. rosea plant. The expression analysis based on RNA-seq showed that the transcripts of most CrNHXs were obviously decreased in mature leaves of C. rosea plant growing on tropical coral reefs, which suggested their involvement in this species’ adaptation to reefs and specialized islands habitats. Furthermore, in the single-factor stress treatments mimicking the extreme environments of tropical coral reefs, the RNA-seq data also implied CrNHXs holding possible gene-specific regulatory roles in the environmental adaptation. The qRT-PCR based expression profiling exhibited that CrNHXs responded to different stresses to varying degrees, which further confirmed the specificity of CrNHXs’ in responding to abiotic stresses. Moreover, the yeast functional complementation test proved that some CrNHXs could partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. This study provides comprehensive bio-information and primary functional identification of NHXs in C. rosea, which could help improve the salt/alkaline tolerance of genetically modified plants for further studies. This research also contributes to our understanding of the possible molecular mechanism whereby NHXs maintain the ion balance in the natural ecological adaptability of C. rosea to tropical coral islands and reefs.

A tonoplast-localized magnesium transporter is involved in stomatal opening in Arabidopsis

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing are still not completely understood. Here, through large-scale screening, we identified an Arabidopsis mutant (cst2 for closed stomata2) defective in stomatal opening under light condition. A map-based cloning combined with complementation test revealed that the mutant phenotype was caused by a nucleotide substitution of a gene, which domains show similarity to human Mg efflux transporter ACDP/CNNM. Functional analysis showed that CST2 encodes a tonoplast-localized transporter for Mg. This protein is constitutively and highly expressed in the guard cells. Furthermore, CST2 is phosphorylated by calcineurin B-like protein (CBL)-interacting protein kinases 26 (CIPK26) in vitro, which is probably required for its activation. Knockout of this gene resulted in stomatal closing and growth retardation under high Mg concentration conditions, while over-expression of this gene increased tolerance to high Mg. Our results indicate that CST2 plays an important role in maintaining Mg homeostasis in plant cells through sequestering Mg into vacuoles especially in guard cells and that this homeostasis is required for stomatal opening, which provide a novel insight into mechanism of stomatal opening in plants.

A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiae's DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panel's 'Mosaic beer' clade and share an enrichment for C>A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C>A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C>A mutations if left unrepaired.

TRIM22 inhibits the proliferation of gastric cancer cells through the Smad2 protein

TRIM22 is involved in tumorigenesis and development, but its mechanism is not clear. In this study, we investigated the expression and biological role of TRIM22 in gastric cancer. We found that TRIM22 mRNA and protein expression was abnormally low in gastric cancer tissues and cells and correlated with tumor size and depth of invasion. Overexpression of TRIM22 significantly inhibited the proliferation, colony formation, and migration of gastric cancer cells and downregulated the expression of HSPA6. However, the HSPA6-siRNA complementation test showed that TRIM22 did not regulate cell proliferation through HSPA6. Furthermore, overexpression of TRIM22 downregulated the phosphorylation of Smad2 and Smad3. In addition, TRIM22 directly binds to Smad2, and overexpression of Smad2 can reverse the inhibition of cell proliferation and migration induced by TRIM22. In vivo, overexpression of TRIM22 significantly inhibited the growth of subcutaneous xenografts in nude mice. Our study indicates that TRIM22 has an important role in the development of gastric cancer and may inhibit the proliferation of gastric cancer cells through Smad2.

Candidate Gene Test for Quantitative Trait Loci Conferring Hypercholesterolemia on Mouse Chromosome 1

Objectives The TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a subcongenic mouse with TH donor segment, ∼25 Mb, on chromosome (Chr) 1 in a C57BL/6J (B6) background that harbors quantitative trait loci (QTL) conferring hypercholesterolemia, named Tchol1 (Tallyho Associated Cholesterol 1). The subcongenic mouse developed hypercholesterolemia compared to B6 mice demonstrating that distal segment of Chr 1 from TH genome is necessary to cause the hypercholesterolemia. In this study, we tested the candidacy of the apolipoprotein A2 (Apoa2) gene for Tachol1 by the quantitative complementation test. Apoa2, known regulator of cholesterol metabolism, maps to the Tchol1 locus. Methods To carry out the quantitative complementation test, both TH-homozygous Tachol1 subcongenic and B6-homozygous (B6) mice were mated to the Apoa2 knockout heterozygous [wild-type (wt)/null] mice to produce four types of animals; TH/wt, TH/null, B6/wt, and B6/null. Both male and female mice were weaned onto standard rodent chow and maintained. Blood was collected when animals were euthanized at 16 weeks of age. Total plasma cholesterol levels were determined using colorimetric assays. A two-way ANOVA was used to evaluate Apoa2 (null vs. wt) and Tachol1 (TH vs. B6) interaction effects for dependent variables, followed by the multiple comparison post test with Tukey correction using GraphPad Prism 8. Results Total plasma cholesterol levels were: 137 ± 5 (TH/wt), 119 ± 8 (TH/null), 103 ± 8 (B6/wt), and 80 ± 4 (B6/null) for males, and 149 ± 8 (TH/wt), 130 ± 9 (TH/null), 98 ± 3 (B6/wt), and 103 ± 6 (B6/null) for females [mean ± s.e.m; mg/dl]. Two-way ANOVA revealed no significant interaction between Tchol1 and Apoa2 knockout alleles for total plasma cholesterol levels in both males and females. However, there were significant main effects of Tchol1 and Apoa2 knockout alleles on total plasma cholesterol levels in males, while significant main effects of Tchol1 on them in females. Conclusions No significant interaction effect between knockout and QTL alleles is interpreted as evidence that the knockout locus is not equal to the QTL. Our results suggest that the Apoa2 gene is not identical to the Tchol1 QTL. Funding Sources AHA 18AIREA33960437, NIH 1 R15 DK113604-01A1, the WV-INBRE grant (P20GM103434), and the COBRE ACCORD grant (1P20GM121299).

GRAS transcription factor LOSS OF AXILLARY MERISTEMS is essential for stamen and runner formation in wild strawberry

Axillary bud development is a major factor that impacts plant architecture. A runner is an elongated shoot that develops from axillary buds and is frequently used for clonal propagation of strawberry. However, the genetic control underlying runner production is largely unknown. Here, we identified and characterized loss of axillary meristems (lam), an EMS-induced mutant of the diploid woodland strawberry (Fragaria vesca) that lacked stamens in flowers and had reduced numbers of branch crowns and runners. The reduced branch crown and runner phenotypes were caused by a failure of axillary meristem initiation. The causative mutation of lam was located in FvH4_3g41310, which encodes a GRAS transcription factor, and was validated by a complementation test. lamCR mutants generated by CRISPR/Cas9 produced flowers without stamens and had fewer runners than the wild type. LAM was broadly expressed in meristematic tissues. Gibberellic acid (GA) application induced runner outgrowth from the remaining buds in lam, but failed to do so at the empty axils of lam. In contrast, treatment with the GA biosynthesis inhibitor paclobutrazol (PBZ) converted the runners into branch crowns. Moreover, genetic studies indicated that lam is epistatic to suppressor of runnerless (srl), a mutant of FveRGA1 in the gibberellic acid pathway, during runner formation. Our results demonstrate that LAM is required for stamen and runner formation and acts sequentially with GA from bud initiation to runner outgrowth, providing insights into the molecular regulation of these economically important organs in strawberry.

Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

Genetic variability and vegetative compatibility in Aspergillus niger isolated from various food substances in Benin City Nigeria

In this investigation, Aspergillus niger isolated from eight food substances, have been classified based on the absence of heterokaryon formation. The size of their sporangia were differentiated, the wild and mutant strains were subjected to vegetative compatibility tests in order to group them into different vegetative compatibility groups (VCGs) which include VCG-1, VCG-2, VCG-3 and VCG-4. The strains were further tested for the possible formation of a stable heterokaryon using nit mutants generated on potato dextrose agar (PDA) containing 2.5% chlorate (KClO3), represented as PDC. Based on the vegetative compatibility groups, nit mutants were paired on a minimal medium (MM) for complementation test. Interestingly, there was compatibility with mycelia showing anastomoses but without the formation of heterokaryon. The vegetative compatibility groups suggested four genotypes and polymorphism in the het loci. A population study for detailed genotyping is suggested in order to unravel the genetic recombination in A. niger.