Identification of Increased Grain Length 1 (IGL1), a novel gene encoded by a major QTL for modulating grain length in rice.

KEY MESSAGE: A novel quantitative trait locus qIGL1, which performed a positive function in regulating grain length in rice, was cloned by the map-based cloning approach; further studies revealed that it corresponded to LOC_Os03g30530, and the IGL1 appeared to contribute to lengthening and widening of the cells on the surface of grain hulls. Grain length is a prominent determinant for grain weight and appearance quality of rice. In this study, we conducted quantitative trait locus mapping to determine a genomic interval responsible for a long-grain phenotype observed in a japonica cultivar HD385. This led to the identification of a novel QTL for grain length on chromosome 3, named qIGL1 (for Increased Grain Length 1); the HD385 (Handao 385)-derived allele showed enhancement effects on grain length, and such an allele as well as NIP (Nipponbare)-derived allele was designated qigl1 HD385 and qIGL1NIP, respectively. Genetic analysis revealed that the qigl1HD385 allele displayed semidominant effects on grain length. Fine mapping further narrowed down the qIGL1 to an ~ 70.8-kb region containing 9 open reading frames (ORFs). A comprehensive analysis indicated that LOC_Os03g30530, which corresponded to ORF6 and carried base substitutions and deletions in HD385 relative to NIP, thereby causing changes or losses of amino-acid residues, was the true gene for qIGL1. Comparison of grain traits between a pair of near-isogenic lines (NILs), termed NIL-igl1HD385 and NIL-IGL1NIP, discovered that introduction of the igl1HD385 into the NIP background significantly resulted in the elevations of grain length and 1000-grain weight. Closer inspection of grain surfaces revealed that the cell length and width in the longitudinal direction were significantly longer and greater, respectively, in NIL-igl1HD385 line compared with in NIL-IGL1NIP line. Hence, our studies identified a new semidominant natural allele contributing to the increase of grain length and further shed light on the regulatory mechanisms of grain length.

RLL5, an F-box-containing protein, involved in preventing transgene silencing and in maintaining global DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation pathways are antagonistic processes that dynamically regulate site-specific methylation. In this study, we obtained a mutant with reduced luciferase (LUC) luminescence by genetic screening, which was named rll5-1 (for reduced LUC luminescence 5-1). The rll5-1 mutant showed narrower, frizzled and curly leaves, and the low-LUC-luminescence phenotype in the rll5-1 mutant can be largely restored by DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Map-based cloning coupled with genome resequencing data revealed that a nucleotide substitution of G to A was found at the 124th bp of ORF of At4G10190, leading to an aspartate-to-asparagine change at position 42 in such a protein. Bisulfite sequencing data indicated that DNA methylation of 3' region of the double 35S promoter that drives the LUC expression was appreciably increased. Further analysis revealed that there were 4747 hypo-DMRs and 936 hyper-DMRs found in the rll5-1 genome, and the hypo-DMRs was predominantly distributed on TEs, which appeared to stem from the downregulation of a few RdDM pathway genes and DNA methyltransferase genes. Closer inspection demonstrated that there were 1229 hypo-DMRs commonly shared among rll5-1, nrpd1-3 and nrpe1-11, and a total of 1349 hypo-DMRs were common to rll5-1 and cmt2 mutants. Thus, these studies demonstrate the roles of RLL5 in preventing transgene silencing and in maintaining genome-wide DNA methylation in a direct/indirect or locus-specific manner.

Roles of MEM1 in safeguarding Arabidopsis genome against DNA damage, inhibiting ATM/SOG1-mediated DNA damage response, and antagonizing global DNA hypermethylation.

Arabidopsis methylation elevated mutant 1 (mem1) mutants have elevated levels of global DNA methylation. In this study, such mutant alleles showed increased sensitivity to methyl methanesulfonate (MMS). In mem1 mutants, an assortment of genes engaged in DNA damage response (DDR), especially DNA-repair-associated genes, were largely upregulated without MMS treatment, suggestive of activation of the DDR pathway in them. Following MMS treatment, expression levels of multiple DNA-repair-associated genes in mem1 mutants were generally lower than in Col-0 plants, which accounted for the MMS-sensitive phenotype of the mem1 mutants. A group of DNA methylation pathway genes were upregulated in mem1 mutants under non-MMS-treated conditions, causing elevated global DNA methylation, especially in RNA-directed DNA methylation (RdDM)-targeted regions. Moreover, MEM1 seemed to help ATAXIA-TELANGIECTASIA MUTATED (ATM) and/or SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) to fully activate/suppress transcription of a subset of genes regulated simultaneously by MEM1 and ATM and/or SOG1, because expression of such genes decreased/increased consistently in mem1 and atm and/or sog1 mutants, but the decreases/increases in the mem1 mutants were not as dramatic as in the atm and/or sog1 mutants. Thus, our studies reveals roles of MEM1 in safeguarding genome, and interrelationships among DNA damage, activation of DDR, DNA methylation/demethylation, and DNA repair.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Roles of IDM3 and SDJ1/2/3 in Establishment and/or Maintenance of DNA Methylation in Arabidopsis.

Previous studies had demonstrated that in Arabidopsis, IDM3 is involved in ROS1-mediated DNA demethylation pathway, and SUVH-SDJ complex functions as a DNA methylation reader complex for enhancing gene transcription, which presumably recruits ROS1 to the promoters of target genes for DNA demethylation. Here, our analyses, however, showed that the IDM3 and SDJ1/2/3, the components of the SUVH-SDJ complex, are implicated in establishing and/or maintaining DNA methylation as well through DDR (DRD1-DMS3-RDM1) complex. idm3-3 or sdj1/2/3 mutations led to genome-wide DNA hypomethylation, and both mutants shared a large number of common hypo-DMRs (Differentially Methylated Regions) with rdm1-4 and dms3-4, suggesting that IDM3 and SDJ1/2/3 help establish and/or maintain DNA methylation, mediated by RdDM pathway, at a subset of genomic regions largely through DDR complex. IDM3 is able to strongly interact with RDM1 and DMS3, but weakly with SDJ1 and SDJ3; SDJ1 and SDJ3 is capable of interacting separately with RDM1 and DMS3. Furthermore, comparisons of DNA methylation features in idm3-3 and sdj1/2/3 indicated that idm3-3 and sdj1/2/3 mutations make differential impacts on DNA methylation levels and patterns on a genome-wide scale, indicating that they are targeted to quite distinct genomic regions for aiding in DNA methylation. Further analyses on ChIP-seq data demonstrated that RDM1, DMS3 and NRPE1 are enriched in IDM3- and SDJ1/2/3-targted regions. Altogether, our results provide clear demonstration that IDM3 and SDJ1/2/3 play a part in establishing and/or maintaining DNA methylation of a group of genomic regions, through the DDR complex.

Increased Drought Resistance 1 Mutation Increases Drought Tolerance of Upland Rice by Altering Physiological and Morphological Traits and Limiting ROS Levels.

To discover new mutants conferring enhanced tolerance to drought stress, we screened a mutagenized upland rice (Oryza sativa) population (cv. IAPAR9) and identified a mutant, named idr1-1 (increased drought resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1 mutant possessed a significantly enhanced ability to tolerate high-drought stresses. Map-based cloning revealed that the gene LOC_Os05g26890, residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant. IDR1 encodes the Gα subunit of the heterotrimeric G protein (also known as RGA1), and this protein was localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the significantly increased drought tolerance in idr1-1 mutants stemmed from a range of physiological and morphological changes, including greater leaf potentials, increased proline contents, heightened leaf thickness and upregulation of antioxidant-synthesizing and drought-induced genes, under drought-stressed conditions. Especially, reactive oxygen species (ROS) production might be remarkably impaired, while ROS-scavenging ability appeared to be markedly enhanced due to significantly elevated expression of ROS-scavenging enzyme genes in idr1-1 mutants under drought-stressed conditions. In addition, idr1-1 mutants showed reduced expression of OsBRD1. Altogether, these results suggest that mutation of IDR1 leads to alterations in multiple layers of regulations, which ultimately leads to changes in the physiological and morphological traits and limiting of ROS levels, and thereby confers obviously increased drought tolerance to the idr1-1 mutant.

Involvement of MEM1 in DNA demethylation in Arabidopsis.

KEY MESSAGE: MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway.mem1mutation leads to large numbers of hyper-DMRs inArabidopsisgenome. In higher plants, DNA methylation performs important functions in silencing transcribed genes and transposable elements (TEs). Active DNA demethylation mediated by REPRESSOR OF SILENCING 1 (ROS1) is able to antagonize the action of DNA methylation caused by RNA-directed DNA methylation (RdDM) pathway, which plays critical roles in keeping DNA methylation at a proper level. In this study, a new mutant named mem1 (for methylation elevated mutant 1) was isolated from a genetic screen of T-DNA insertional mutant population for lines with elevated DNA methylation at a particular locus through Chop-PCR method. MEM1 possesses a Zf-C3HC domain, and is localized in nucleus as well as highly expressed in cotyledons. Whole-genome bisulfite sequencing data showed that knockout mutation of MEM1 leads to 4519 CG, 1793 CHG and 12739 CHH hyper-DMRs (for differentially methylated regions). Further analysis indicated that there are 2751, 2216 and 2042 overlapped CG hyper-DMRs between mem1-1and three mutants, i.e. ros1-4, rdd and ros3-2, respectively; 797, 2514, and 6766 overlapped CHH hyper-DMRs were observed between mem1-1 and three such mutants, respectively; mem1 nrpd1-3 and mem1 rdm1 double mutants showed nearly complete or partial loss of hypermethylation at 4 tested loci, suggesting that MEM1 performs similar functions as DNA glycosylase/lyases in counteracting excessive DNA methylation, and MEM1 plays important roles as REPRESSOR OF SILENCING 3 (ROS3) in erasing CHH methylation caused by the RdDM pathway. Together, these data demonstrate the involvement of MEM1 in ROS1-mediated DNA demethylation pathway and functional connections between MEM1 and ROS3.

Characterization and fine mapping of Arabidopsis RLL3, a locus required for DNA demethylation pathway.

DNA methylation pattern is found to be established by the combined actions of DNA methylation and demethylation. Compared to the DNA methylation pathway, DNA demethylation pathway, however, remains largely unknown. To better understand the DNA demethylation pathway, we performed genetic screening for Arabidopsis mutants with increased genomic DNA methylation levels through a 2 × 35S:LUC (LUC, luciferase) reporter system. A mutant with reduced LUC luminescence was identified by such a system, therefore named rll3-1 (for reduced LUC luminescence 3-1). The rll3-1 mutant exhibited pleiotropic developmental defects, such as delayed bolting as well as flowering, more branches, etc. By map-based cloning approach, rll3 locus that contains a single nuclear recessive mutation as revealed by the genetic analysis was mapped to a region between molecular markers CL102_B1 M1 and CL102_B3M1, which are located in bacterial artificial chromosome (BAC) clones F9P14 and F12K11, respectively, on chromosome 1. Chop-PCR analysis indicated that a total of seven tested loci displayed elevated DNA methylation levels. Whole-genome bisulfite sequencing further revealed 1536 loci exhibiting increased DNA methylation levels relative to Col-LUC control, among which there are 507 such loci overlapping between the rll3-1 and ros1-7 mutants, suggestive of a functional association between RLL3 and REPRESSOR OF SILENCING 1 (ROS1). Further investigations demonstrated that the expression levels of a few genes (like ROS1, IDM1, etc.), which are involved in DNA demethylation pathway, remained unchanged in the rll3-1 mutant, indicating that the increased DNA methylation levels in rll3-1 mutant are not attributable to downregulation of such genes. Taken together, our studies provide a demonstration of the involvement of RLL3 in the DNA demethylation pathway.

SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis.

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes.

Analysis of the DNA methylation patterns and transcriptional regulation of the NB-LRR-encoding gene family in Arabidopsis thaliana.

KEY MESSAGE: The relationships between transcription and methylation were revealed in Arabidopsis thaliana NB-LRR-encoding genes in wild type (Col-0) and different mutants. Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins constitute a large family that plays predominant roles in disease resistance. However, the regulation of NB-LRR-encoding genes at the transcriptional level is still poorly understood. Recently, DNA cytosine methylation in eukaryotes has been described as serving an important function in regulating gene expression. Here, we analysed the DNA methylation patterns of NB-LRR-encoding genes in Arabidopsis thaliana in samples from a wild type (Col-0) and ago4, met1, cmt3, drm1/2, and ddm1 mutants. Our results revealed that the vast majority of the NB-LRR-encoding genes in Col-0 were methylated, and the DNA methylation occurred predominantly in the CG sequence context. Moreover, DNA methylation was widely distributed in both the promoters and the bodies of most NB-LRR-encoding genes. Our results also showed that the loss of AGO4, MET1, CMT3, DRM1/2 or DDM1 functions generally led to decreased cytosine methylation in the NB-LRR-encoding genes. Analysis of the available transcriptome data from the wild type and the met1, cmt3, drm1/2 and ddm1 mutants revealed that differences in the transcription levels between the wild type and mutants were statistically significant for 63 of the NB-LRR-encoding genes. Of these genes, 38 were significantly upregulated, and the other 25 were significantly downregulated. Some NB-LRR-encoding genes with differential expression levels, which were revealed by the mRNA-Seq data, were confirmed to be significantly upregulated or downregulated in the mutants compared to the wild type by using quantitative RT-PCR. These data suggest that some Arabidopsis NB-LRR-encoding genes are likely to be regulated by altered DNA methylation patterns.

Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OPEN STOMATA 1.

Arabidopsis Senescence-Associated Subtilisin Protease (SASP) has previously been reported to participate in leaf senescence and in the development of inflorescences and siliques. Here, we describe a new role of SASP in the regulation of abscisic acid (ABA) signaling. SASP encodes a subtilase and its expression was considerably induced by darkness, ABA, and ethylene treatments. sasp knockout mutants displayed obvious developmental phenotypes such as early flowering and smaller leaves. In particular, the sasp mutants exhibited enhanced ABA sensitivity during seed germination and seedling growth, heightened ABA-mediated leaf senescence, and increased production of reactive oxygen species (ROS). Importantly, the sasp mutants also showed remarkably increased tolerance to drought, with expression of six ABA signaling-related genes being either up- or down-regulated following ABA treatment. Interaction assays demonstrated that SASP physically interacts with OPEN STOMATA 1 (OST1) at the cell periphery. Co-expression of SASP and OST1 led to degradation of OST1, whereas this degradation was impaired in sasp-1 protoplasts. ROS attenuation assays demonstrated that in sasp-1 mutant guard cells the attenuation rate markedly decreased. Taken together, the results demonstrate that SASP plays an important role in regulating ABA signaling and drought tolerance through interaction with OST1.

Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns.

DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

Sulfamethazine suppresses epigenetic silencing in Arabidopsis by impairing folate synthesis.

DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic regulation is very limited. Using the Arabidopsis thaliana repressor of silencing1 mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation.

A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice.

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

Critical Extracellular Ca2+ Dependence of the Binding between PTH1R and a G-Protein Peptide Revealed by MD Simulations.

The parathyroid hormone type 1 receptor (PTH1R), a canonical class B GPCR, is regulated by a positive allosteric modulator, extracellular Ca2+. Calcium ions prolong the residence time of PTH on the PTH1R, leading to increased receptor activation and duration of cAMP signaling. But the essential mechanism of the allosteric behavior of PTH1R is not fully understood. Here, extensive molecular dynamics (MD) simulations are performed for the PTH1R-G-protein combinations with and without Ca2+ to describe how calcium ions allosterically engage receptor-G-protein coupling. We find that the binding of Ca2+ stabilizes the conformation of the PTH1R-PTH-spep (the α5 helix of Gs protein) complex, especially the extracellular loop 1 (ECL1). Moreover, the MM-GBSA result indicates that Ca2+ allosterically promotes the interaction between PTH1R and spep, consistent with the observation of steered molecular dynamics (SMD) simulations. We further illuminate the possible allosteric signaling pathway from the stable Ca2+-coupling site to the intracellular G-protein binding site. These results unveil structural determinants for Ca2+ allosterism in the PTH1R-PTH-spep complex and give insights into pluridimensional GPCR signaling regulated by calcium ions.

Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.

DNA methylation patterns in plants are dynamically shaped by the antagonistic actions of DNA methylation and demethylation pathways. Although the DNA methylation pathway has been well studied, the DNA demethylation pathway, however, are not fully understood so far. To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screening for proteins that were involved in preventing epigenetic gene silencing, and then the ones, which were also implicated in DNA demethylation pathway, were used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6-1 (rll6-1). Map-based cloning revealed that rll6-1 mutation was located on chromosome 4, and there were a total of 10 candidate genes residing within such a region. Analyses of genome-wide methylation patterns of rll6-1 mutant showed that mutation of RLL6 locus led to 3,863 hyper-DMRs (DMRs for differentially methylated regions) throughout five Arabidopsis chromosomes, and elevated DNA methylation level of 2 × 35S promoter, which was similar to that found in the ros1 (repressor of silencing 1) mutant. Further analysis demonstrated that there were 1,456 common hyper-DMRs shared by rll6-1 and ros1-7 mutants, suggesting that both proteins acted together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that mutation of RLL6 locus did not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrate that RLL6 locus-encoded protein not only participates in transcriptional anti-silencing of a transgene, but is also involved in DNA demethylation pathway.

Mechanism of enhanced sensitivity of mutated ß-adrenergic-like octopamine receptor to amitraz in honeybee Apis mellifera: An insight from MD simulations.

BACKGROUND: Amitraz is one of the critical acaricides/insecticides for effective control of pest infestation of Varroa destructor mite, a devastating parasite of Apis mellifera, because of its low toxicity to honeybees. Previous assays verified that a typical G protein-coupled receptor, ß-adrenergic-like octopamine receptor (Octß2R), is the unique target of amitraz, but the honeybee Octß2R resists to amitraz. However, the underlying molecular mechanism of the enhanced sensitivity or toxicity of amitraz to mutated honeybee Octß2RE208V/I335T/I350V is not fully understood. Here, molecular dynamics simulations are employed to explore the implied mechanism of the enhanced sensitivity to amitraz in mutant honeybee Octß2R. RESULTS: We found that amitraz binding stabilized the structure of Octß2R, particularly the intracellular loop 3 associated with the Octß2R signaling. Then, it was further demonstrated that both mutations and ligand binding resulted in a more rigid and compact amitraz binding site, as well as the outward movement of the transmembrane helix 6, which was a prerequisite for G protein coupling and activation. Moreover, mutations were found to promote the binding between Octß2R and amitraz. Finally, community analysis illuminated that mutations and amitraz strengthened the residue-residue communication within the transmembrane domain, which might facilitate the allosteric signal propagation and activation of Octß2R. CONCLUSION: Our results unveiled structural determinants of improved sensitivity in the Octß2R-amitraz complex and may contribute to further structure-based drug design for safer and less toxic selective insecticides. © 2022 Society of Chemical Industry.

Identification and regulatory network analysis of SPL family transcription factors in Populus euphratica Oliv. heteromorphic leaves.

The SQUAMOSA promoter-binding protein-like (SPL) family play a key role in guiding the switch of plant growth from juvenile to adult phases. Populus euphratica Oliv. exhibit typical heterophylly, and is therefore an ideal model for studying leaf shape development. To investigate the role and regulated networks of SPLs in the morphogenesis of P. euphratica heteromorphic leaves. In this study, 33 P. euphratica SPL (PeuSPL) genes were identified from P. euphratica genome and transcriptome data. Phylogenetic analysis depicted the classification of these SPL genes into two subgroups. The expression profiles and regulatory networks of P. euphratica SPL genes analysis displayed that major P. euphratica SPL family members gradually increases from linear to broad-ovate leaves, and they were involved in the morphogenesis regulation, stress response, transition from vegetative to reproductive growth, photoperiod, and photosynthesis etc. 14 circRNAs, and 33 lncRNAs can promote the expression of 12 of the P. euphratica SPLs by co-decoying miR156 in heteromorphic leaf morphogenesis. However, it was found that the effect of PeuSPL2-4 and PeuSPL9 in leaf shape development was contrasting to their homologous genes of Arabidopsis. Therefore, it was suggested that the SPL family were evolutionarily conserved for regulation growth, but were varies in different plant for regulation of the organ development.

Impact of DNA Demethylases on the DNA Methylation and Transcription of Arabidopsis NLR Genes.

Active DNA demethylation is an important epigenetic process that plays a key role in maintaining normal gene expression. In plants, active DNA demethylation is mediated by DNA demethylases, including ROS1, DME, DML2, and DML3. In this study, the available bisulfite sequencing and mRNA sequencing data from ros1 and rdd mutants were analyzed to reveal how the active DNA demethylation process shapes the DNA methylation patterns of Arabidopsis nucleotide-binding leucine-rich repeat (NLR) genes, a class of important plant disease resistance genes. We demonstrate that the CG methylation levels of three NLR genes (AT5G49140, AT5G35450, and AT5G36930) are increased in the ros1 mutants relative to the wild-type plants, whereas the CG methylation level of AT2G17050 is decreased. We also observed increased CG methylation levels of AT4G11170 and AT5G47260 and decreased CG methylation levels of AT5G38350 in rdd mutants. We further found that the expression of three NLR genes (AT1G12280, AT1G61180, and AT4G19520) was activated in both ros1 and rdd mutants, whereas the expression of another three NLR genes (AT1G58602, AT1G59620, and AT1G62630) was repressed in these two mutants. Quantitative reverse transcriptase-polymerase chain reaction detection showed that the expression levels of AT1G58602.1, AT4G19520.3, AT4G19520.4, and AT4G19520.5 were decreased in the ros1 mutant; AT3G50950.1 and AT3G50950.2 in the rdd mutant were also decreased in expression compared to Col-0, whereas AT1G57630.1, AT1G58602.2, and AT5G45510.1 were upregulated in the rdd mutant relative to Col-0. These results indicate that some NLR genes are regulated by DNA demethylases. Our study demonstrates that each DNA demethylase (ROS1, DML2, and DML3) exerts a specific effect on the DNA methylation of the NLR genes, and active DNA demethylation is part of the regulation of DNA methylation and transcriptional activity of some Arabidopsis NLR genes.