Genetic and Functional Diversification of Small RNA Pathways in Plants

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense

Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability

Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development

Genetic and Functional Diversification of Small RNA Pathways in Plants

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense

EIFiso4G augments the synthesis of specific plant proteins involved in normal chloroplast function

Copyright © 2019 American Society of Plant Biologists. All rights reserved. The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 × i4g2, and i4g1 × i4g2 × i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 × i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins

Results and Perspectives from the First Two Years of Neutrino Physics at the LHC by the SND@LHC Experiment

After rapid approval and installation, the SND@LHC Collaboration was able to gather data successfully in 2022 and 2023. Neutrino interactions from νμs originating at the LHC IP1 were observed. Since muons constitute the major background for neutrino interactions, the muon flux entering the acceptance was also measured. To improve the rejection power of the detector and to increase the fiducial volume, a third Veto plane was recently installed. The energy resolution of the calorimeter system was measured in a test beam. This will help with the identification of νe interactions that can be used to probe charm production in the pseudo-rapidity range of SND@LHC (7.2 < η < 8.4). Events with three outgoing muons have been observed and are being studied. With no vertex in the target, these events are very likely from muon trident production in the rock before the detector. Events with a vertex in the detector could be from trident production, photon conversion, or positron annihilation. To enhance SND@LHC’s physics case, an upgrade is planned for HL-LHC that will increase the statistics and reduce the systematics. The installation of a magnet will allow the separation of νμ from ν¯μWe acknowledge the support for the construction and operation of the SND@LHC detector provided by the following funding agencies: CERN; the Bulgarian Ministry of Education and Science within the National Roadmap for Research Infrastructures 2020–2027 (object CERN); ANID—Millennium Program—ICN2019_044 (Chile); the Deutsche Forschungsgemeinschaft (DFG, ID 496466340); the Italian National Institute for Nuclear Physics (INFN); JSPS, MEXT, the Global COE program of Nagoya University, the Promotion and Mutual Aid Corporation for Private Schools of Japan for Japan; the National Research Foundation of Korea with grant numbers 2021R1A2C2011003, 2020R1A2C1099546, 2021R1F1A1061717, and 2022R1A2C100505; Fundação para a Ciência e a Tecnologia, FCT (Portugal), CERN/FIS-INS/0028/2021; the Swiss National Science Foundation (SNSF); TENMAK for Turkey (Grant No. 2022TENMAK(CERN) A5.H3.F2-1). M. Climesu, H. Lacker and R. Wanke are funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Project 496466340. We acknowledge the funding of individuals by Fundação para a Ciência e a Tecnologia, FCT (Portugal) with grant numbers CEECIND/01334/2018, CEECINST/00032/2021 and PRT/BD/153351/2021.CERNBulgarian Ministry of Education and ScienceANID—Millennium ProgramDeutsche ForschungsgemeinschaftItalian National Institute for Nuclear Physics (INFN)JSPS, MEXT, the Global COE program of Nagoya University, the Promotion and Mutual Aid Corporation for Private Schools of Japan for JapanNational Research Foundation of KoreaFundação para a Ciência e a Tecnologia, FCT (Portugal)Swiss National Science Foundation (SNSF)TENMAK for TurkeyPeer Reviewe

Genetic and functional diversification of small RNA pathways in plants.

Multicellular eukaryotes produce small RNA molecules (approximately 21-24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense

Effects of Mutations on <i>AtSN1</i> and 5S rDNA Chromatin Structure and Gene Expression

<div><p>(A) Analysis of CpG (left), CpNpG (center), and CpHpH (right) methylation in <i>AtSN1</i> by bisulfite sequencing of genomic DNA.</p> <p>(B) Blot analysis of 5S rDNA digested with methylation-sensitive restriction enzymes HpaII (left) and MspI (right). HpaII is sensitive to CpG and CpNpG methylation, whereas MspI is sensitive to only CpNpG methylation. Methylation is indicated by the ascending ladder, which corresponds to 5S rDNA multimers (monomer = approximately 0.5 kb). Duplicate samples from each plant were analyzed.</p> <p>(C) ChIP assays using antibodies against dimethyl-histone H3K9 and dimethyl-histone H3K4. Genomic DNA associated with immunoprecipitated chromatin was analyzed by semiquantitative PCR with primer pairs specific for <i>AtSN1</i>, retrotransposon reverse transcriptase (At4g03800) (internal control for H3K9 methylation), and PFK (At4g04040) (internal control for H3K4 methylation). The PCR products were quantitated and compared against the respective internal controls, and the relative H3K4 and H3K9 methylation levels were expressed relative to that in Col-0 (arbitrarily set to 1.00).</p> <p>(D) Detection of <i>AtSN1</i>-specific transcripts by semiquantitative RT-PCR. Primers specific for PFK transcripts were used as the internal control. A parallel set of reactions without addition of reverse transcriptase (RT) was run as a quality control for genomic DNA contamination. The PCR products were normalized relative to PFK, and the expression levels were calculated relative to that in Col-0 (arbitrarily set to 1.00).</p></div