Glycine-Rich RNA-Binding Protein AtGRP7 Functions in Nickel and Lead Tolerance in Arabidopsis.

Plant glycine-rich RNA-binding proteins (GRPs) play crucial roles in the response to environmental stresses. However, the functions of AtGRP7 in plants under heavy metal stress remain unclear. In the present study, in Arabidopsis, the transcript level of AtGRP7 was markedly increased by Ni but was decreased by Pb. AtGRP7-overexpressing plants improved Ni tolerance, whereas the knockout mutant (grp7) was more susceptible than the wild type to Ni. In addition, grp7 showed greatly enhanced Pb tolerance, whereas overexpression lines showed high Pb sensitivity. Ni accumulation was reduced in overexpression lines but increased in grp7, whereas Pb accumulation in grp7 was lower than that in overexpression lines. Ni induced glutathione synthase genes GS1 and GS2 in overexpression lines, whereas Pb increased metallothionein genes MT4a and MT4b and phytochelatin synthase genes PCS1 and PCS2 in grp7. Furthermore, Ni increased CuSOD1 and GR1 in grp7, whereas Pb significantly induced FeSOD1 and FeSOD2 in overexpression lines. The mRNA stability of GS2 and PCS1 was directly regulated by AtGRP7 under Ni and Pb, respectively. Collectively, these results indicate that AtGRP7 plays a crucial role in Ni and Pb tolerance by reducing Ni and Pb accumulation and the direct or indirect post-transcriptional regulation of genes related to heavy metal chelators and antioxidant enzymes.

Phylogenetic and functional analyses of N6-methyladenosine RNA methylation factors in the wheat scab fungus Fusarium graminearum.

In eukaryotes, N6-methyladenosine (m6A) RNA modification plays a crucial role in governing the fate of RNA molecules and has been linked to various developmental processes. However, the phyletic distribution and functions of genetic factors responsible for m6A modification remain largely unexplored in fungi. To get insights into the evolution of m6A machineries, we reconstructed global phylogenies of potential m6A writers, readers, and erasers in fungi. Substantial copy number variations were observed, ranging from up to five m6A writers in early-diverging fungi to a single copy in the subphylum Pezizomycotina, which primarily comprises filamentous fungi. To characterize m6A factors in a phytopathogenic fungus Fusarium graminearum, we generated knockout mutants lacking potential m6A factors including the sole m6A writer MTA1. However, the resulting knockouts did not exhibit any noticeable phenotypic changes during vegetative and sexual growth stages. As obtaining a homozygous knockout lacking MTA1 was likely hindered by its essential role, we generated MTA1-overexpressing strains (MTA1-OE). The MTA1-OE5 strain showed delayed conidial germination and reduced hyphal branching, suggesting its involvement during vegetative growth. Consistent with these findings, the expression levels of MTA1 and a potential m6A reader YTH1 were dramatically induced in germinating conidia, followed by the expression of potential m6A erasers at later vegetative stages. Several genes including transcription factors, transporters, and various enzymes were found to be significantly upregulated and downregulated in the MTA1-OE5 strain. Overall, our study highlights the functional importance of the m6A methylation during conidial germination in F. graminearum and provides a foundation for future investigations into m6A modification sites in filamentous fungi.IMPORTANCEN6-methyladenosine (m6A) RNA methylation is a reversible posttranscriptional modification that regulates RNA function and plays a crucial role in diverse developmental processes. This study addresses the knowledge gap regarding phyletic distribution and functions of m6A factors in fungi. The identification of copy number variations among fungal groups enriches our knowledge regarding the evolution of m6A machinery in fungi. Functional characterization of m6A factors in a phytopathogenic filamentous fungus Fusarium graminearum provides insights into the essential role of the m6A writer MTA1 in conidial germination and hyphal branching. The observed effects of overexpressing MTA1 on fungal growth and gene expression patterns of m6A factors throughout the life cycle of F. graminearum further underscore the importance of m6A modification in conidial germination. Overall, this study significantly advances our understanding of m6A modification in fungi, paving the way for future research into its roles in filamentous growth and potential applications in disease control.

N6 -Methyladenosine mRNA methylation is important for salt stress tolerance in Arabidopsis.

As the most abundant internal modification of mRNA, N6 -methyladenosine (m6 A) methylation of RNA is emerging as a new layer of epitranscriptomic gene regulation in cellular processes, including embryo development, flowering-time control, microspore generation and fruit ripening, in plants. However, the cellular role of m6 A in plant responses to environmental stimuli remains largely unexplored. In this study, we show that m6 A methylation plays an important role in salt stress tolerance in Arabidopsis. All mutants of m6 A writer components, including MTA, MTB, VIRILIZER (VIR) and HAKAI, displayed salt-sensitive phenotypes in an m6 A-dependent manner. The vir mutant, in which the level of m6 A was most highly reduced, exhibited salt-hypersensitive phenotypes. Analysis of the m6 A methylome in the vir mutant revealed a transcriptome-wide loss of m6 A modification in the 3' untranslated region (3'-UTR). We demonstrated further that VIR-mediated m6 A methylation modulates reactive oxygen species homeostasis by negatively regulating the mRNA stability of several salt stress negative regulators, including ATAF1, GI and GSTU17, through affecting 3'-UTR lengthening linked to alternative polyadenylation. Our results highlight the important role played by epitranscriptomic mRNA methylation in the salt stress response of Arabidopsis and indicate a strong link between m6 A methylation and 3'-UTR length and mRNA stability during stress adaptation.

Overexpression of phytochelatin synthase AtPCS2 enhances salt tolerance in Arabidopsis thaliana.

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.

Rice OsRH58, a chloroplast DEAD-box RNA helicase, improves salt or drought stress tolerance in Arabidopsis by affecting chloroplast translation.

BACKGROUND: Despite increasing characterization of DEAD-box RNA helicases (RHs) in chloroplast gene expression regulation at posttranscriptional levels in plants, their functional roles in growth responses of crops, including rice (Oryza sativa), to abiotic stresses are yet to be characterized. In this study, rice OsRH58 (LOC_Os01g73900), a chloroplast-localized DEAD-box RH, was characterized for its expression patterns upon stress treatment and its functional roles using transgenic Arabidopsis plants under normal and abiotic stress conditions. RESULTS: Chloroplast localization of OsRH58 was confirmed by analyzing the expression of OsRH58-GFP fusion proteins in tobacco leaves. Expression of OsRH58 in rice was up-regulated by salt, drought, or heat stress, whereas its expression was decreased by cold, UV, or ABA treatment. The OsRH58-expressing Arabidopsis plants were taller and had more seeds than the wild type under favorable conditions. The transgenic plants displayed faster seed germination, better seedling growth, and a higher survival rate than the wild type under high salt or drought stress. Importantly, levels of several chloroplast proteins were increased in the transgenic plants under salt or dehydration stress. Notably, OsRH58 harbored RNA chaperone activity. CONCLUSIONS: These findings suggest that the chloroplast-transported OsRH58 possessing RNA chaperone activity confers stress tolerance by increasing translation of chloroplast mRNAs.

Exogenous Glutathione Enhances Mercury Tolerance by Inhibiting Mercury Entry into Plant Cells.

Despite the increasing understanding of the crucial roles of glutathione (GSH) in cellular defense against heavy metal stress as well as oxidative stress, little is known about the functional role of exogenous GSH in mercury (Hg) tolerance in plants. Here, we provide compelling evidence that GSH contributes to Hg tolerance in diverse plants. Exogenous GSH did not mitigate the toxicity of cadmium (Cd), copper (Cu), or zinc (Zn), whereas application of exogenous GSH significantly promoted Hg tolerance during seed germination and seedling growth of Arabidopsis thaliana, tobacco, and pepper. By contrast, addition of buthionine sulfoximine, an inhibitor of GSH biosynthesis, severely retarded seed germination and seedling growth of the plants in the presence of Hg. The effect of exogenous GSH on Hg specific tolerance was also evident in the presence of other heavy metals, such as Cd, Cu, and Zn, together with Hg. GSH treatment significantly decreased H2O2 and O2- levels and lipid peroxidation, but increased chlorophyll content in the presence of Hg. Importantly, GSH treatment resulted in significantly less accumulation of Hg in Arabidopsis plants, and thin layer chromatography and nuclear magnetic resonance analysis revealed that GSH had much stronger binding affinity to Hg than to Cd, Cu, or Zn, suggesting that tight binding of GSH to Hg impedes Hg uptake, leading to low Hg accumulation in plant cells. Collectively, the present findings reveal that GSH is a potent molecule capable of conferring Hg tolerance by inhibiting Hg accumulation in plants.

The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis.

Nucleostemin is a nucleolar GTP-binding protein that is involved in stem cell proliferation, embryonic development, and ribosome biogenesis in mammals. Plant nucleostemin-like 1 (NSN1) plays a role in embryogenesis, and apical and floral meristem development. In this study, a nucleolar function of NSN1 in the regulation of ribosome biogenesis was identified. Green fluorescent protein (GFP)-fused NSN1 localized to the nucleolus, which was primarily determined by its N-terminal domain. Recombinant NSN1 and its N-terminal domain (NSN1-N) bound to RNA in vitro. Recombinant NSN1 expressed GTPase activity in vitro. NSN1 silencing in Arabidopsis thaliana and Nicotiana benthamiana led to growth retardation and premature senescence. NSN1 interacted with Pescadillo and EBNA1 binding protein 2 (EBP2), which are nucleolar proteins involved in ribosome biogenesis, and with several ribosomal proteins. NSN1, NSN1-N, and EBP2 co-fractionated primarily with the 60S ribosomal large subunit in vivo. Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation. NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression. Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis.

DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants.

This study investigated protein characteristics and physiological functions of DER (Double Era-like GTPase) of higher plants. Nicotiana benthamiana DER (NbDER) contained two tandemly repeated GTP-binding domains (GD) and a C-terminal domain (CTD) that was similar to the K-homology domain involved in RNA binding. Both GDs possessed GTPase activity and contributed to the maximum GTPase activity of NbDER. NbDER fused to green fluorescent protein was localized primarily to chloroplast nucleoids. Arabidopsis der null mutants exhibited an embryonic lethal phenotype, indicating an essential function of DER during plant embryogenesis. Virus-induced gene silencing of NbDER resulted in a leaf-yellowing phenotype caused by disrupted chloroplast biogenesis. NbDER was associated primarily with the chloroplast 50S ribosomal subunit in vivo, and both the CTD and the two GD contributed to the association. Recombinant proteins of NbDER and its CTD could bind to 23S and 16S ribosomal RNAs in vitro. Depletion of NbDER impaired processing of plastid-encoded ribosomal RNAs, resulting in accumulation of the precursor rRNAs in the chloroplasts. NbDER-deficient chloroplasts contained significantly reduced levels of mature 23S and 16S rRNAs and diverse mRNAs in the polysomal fractions, suggesting decreased translation in chloroplasts. These results suggest that DER is involved in chloroplast rRNA processing and ribosome biogenesis in higher plants.

Different roles of glycine-rich RNA-binding protein7 in plant defense against Pectobacterium carotovorum, Botrytis cinerea, and tobacco mosaic viruses.

Glycine-rich RNA-binding protein7 (AtGRP7) has previously been demonstrated to confer plant defense against Pseudomonas syringae DC3000. Here, we show that AtGRP7 can play different roles in plant defense against diverse pathogens. AtGRP7 enhances resistance against a necrotrophic bacterium Pectobacterium carotovorum SCC1 or a biotrophic virus tobacco mosaic virus. By contrast, AtGRP7 plays a negative role in defense against a necrotrophic fungus Botrytis cinerea. These results provide evidence that AtGRP7 is a potent regulator in plant defense response to diverse pathogens, and suggest that the regulation of RNA metabolism by RNA-binding proteins is important for plant innate immunity.

S1 domain-containing STF modulates plastid transcription and chloroplast biogenesis in Nicotiana benthamiana.

• In this study, we examined the biochemical and physiological functions of Nicotiana benthamiana S1 domain-containing Transcription-Stimulating Factor (STF) using virus-induced gene silencing (VIGS), cosuppression, and overexpression strategies. • STF : green fluorescent protein (GFP) fusion protein colocalized with sulfite reductase (SiR), a chloroplast nucleoid-associated protein also present in the stroma. Full-length STF and its S1 domain preferentially bound to RNA, probably in a sequence-nonspecific manner. • STF silencing by VIGS or cosuppression resulted in severe leaf yellowing caused by disrupted chloroplast development. STF deficiency significantly perturbed plastid-encoded multimeric RNA polymerase (PEP)-dependent transcript accumulation. Chloroplast transcription run-on assays revealed that the transcription rate of PEP-dependent plastid genes was reduced in the STF-silenced leaves. Conversely, the exogenously added recombinant STF protein increased the transcription rate, suggesting a direct role of STF in plastid transcription. Etiolated seedlings of STF cosuppression lines showed defects in the light-triggered transition from etioplasts to chloroplasts, accompanied by reduced light-induced expression of plastid-encoded genes. • These results suggest that STF plays a critical role as an auxiliary factor of the PEP transcription complex in the regulation of plastid transcription and chloroplast biogenesis in higher plants.

PRBP plays a role in plastid ribosomal RNA maturation and chloroplast biogenesis in Nicotiana benthamiana.

In the present study, we investigated protein characteristics and physiological functions of PRBP (plastid RNA-binding protein) in Nicotiana benthamiana. PRBP fused to green fluorescent protein (GFP) localized to the chloroplasts. Recombinant PRBP proteins bind to single-stranded RNA in vitro, but not to DNA in a double- or a single-stranded form. Virus-induced gene silencing (VIGS) of PRBP resulted in leaf yellowing in N. benthamiana. At the cellular level, PRBP depletion disrupted chloroplast biogenesis: chloroplast number and size were reduced, and the thylakoid membrane was poorly developed. In PRBP-silenced leaves, protein levels of plastid-encoded genes were significantly reduced, whereas their mRNA levels were normal regardless of their promoter types indicating that PRBP deficiency primarily affects translational or post-translational processes. Depletion of PRBP impaired processing of the plastid-encoded 4.5S ribosomal RNA, resulting in accumulation of the larger precursor rRNAs in the chloroplasts. In addition, PRBP-deficient chloroplasts contained significantly reduced levels of mature 4.5S and 5S rRNAs in the polysomal fractions, indicating decreased chloroplast translation. These results suggest that PRBP plays a role in chloroplast rRNA processing and chloroplast development in higher plants.

Transgenic Arabidopsis and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses.

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions.

Molecular cloning of a cDNA encoding a high mobility group protein in Cucumis sativus and its expression by abiotic stress treatments.

A cDNA encoding a high mobility group B (HMGB) protein was isolated from Cucumis sativus and characterized with respect to its sequence, expression and responses to various abiotic stress treatments. The predicted polypeptide of 146 amino acid residues contains characteristic features of HMGB family proteins including the N-terminal basic region, one HMG-box and a stretch of acidic amino acid residues at the C-terminus. In vitro nucleic acid-binding assay revealed that the HMGB protein bound to both single-stranded DNA and double-stranded DNA. DNA gel blot analysis indicated that the HMGB gene is a single copy gene in cucumber genome. RNA gel blot analysis showed that the cucumber HMGB was more abundantly expressed in the roots than in shoots and leaves. Various abiotic stresses, including cold, drought and high salinity, down regulated markedly the expression of the HMGB in cucumber. The present report identifies a novel gene encoding HMGB protein in cucumber that shows a significant response to abiotic stress treatments.

Molecular cloning and characterization of a cDNA encoding a kinase in Cucumis sativus and its expression by abiotic stress treatments.

As a part of an integrated study of stress-related gene expression, a cDNA clone coding for a protein kinase in the root of Cucumis sativus was isolated and characterized with respect to its sequence and the expression patterns upon various abiotic stress treatments. The predicted polypeptide of 352 amino acid residues contains characteristic features of both the serine/threonine and tyrosine kinase families. In vitro kinase assay confirmed that the isolated protein kinase has autophosphorylation activity. Southern blot analysis showed that the kinase gene is a single-copy gene. Northern blot analysis showed that the kinase gene was more abundantly expressed in the roots and shoots than in the leaves. A quantitative real-time reverse-transcription-polymerase chain reaction analysis revealed that, among the abiotic stresses tested, drought treatment markedly decreased the transcript level of the kinase, whereas the expression of the kinase gene significantly increased by cold treatment. High salinity did not influence its expression. The present report identifies a dual specificity protein kinase in cucumber that shows different responses to abiotic stress treatments.

Prokaryotic and eukaryotic translational machineries respond differently to the frameshifting RNA signal from plant or animal virus.

Many mutational and structural analyses of the RNA signals propose a hypothesis that programmed frameshifting occurs by a specific interaction between ribosome and frameshifting signals comprised of a shifty site and a downstream RNA structure, in which the exact nature of the interaction has not yet been proven. To address this question, we analyzed the frameshifting sequence elements from animal or plant virus in yeast and Escherichia coli. Frameshifting efficiencies varied in yeast, but not in E. coli, depending on the specific conformation of mouse mammary tumor virus (MMTV) RNA pseudoknot. Similar changes in frameshifting efficiencies were observed in yeast, but not in E. coli, for the mutations in frameshifting sequence elements from cereal yellow dwarf virus serotype RPV (CYDV-RPV). The differential response of MMTV or CYDV-RPV frameshifting signal to prokaryotic and eukaryotic translational machineries implies that ribosome pausing alone is insufficient to mediate frameshifting, and additional events including specific interaction between ribosome and RNA structural element are required for efficient frameshifting. These results supports the hypothesis that frameshifting occurs by a specific interaction between ribosome and frameshifting signal.