N6-methyladenosine RNA methylation modulates liquid‒liquid phase separation in plants.

Membraneless biomolecular condensates form distinct subcellular compartments that enable a cell to orchestrate numerous biochemical reactions in a spatiotemporal-specific and dynamic manner. Liquid‒liquid phase separation (LLPS) facilitates the formation of membraneless biomolecular condensates, which are crucial for many cellular processes in plants, including embryogenesis, the floral transition, photosynthesis, pathogen defense, and stress responses. The main component required for LLPS is a protein harboring key characteristic features, such as intrinsically disordered regions, low-complexity sequence domains, and prion-like domains. RNA is an additional component involved in LLPS. Increasing evidence indicates that modifications in proteins and RNAs play pivotal roles in LLPS. In particular, recent studies have indicated that N6-methyladenosine (m6A) modification of messenger RNA is crucial for LLPS in plants and animals. In this review, we provide an overview of recent developments in the role of mRNA methylation in LLPS in plant cells. Moreover, we highlight the major challenges in understanding the pivotal roles of RNA modifications and elucidating how m6A marks are interpreted by RNA-binding proteins crucial for LLPS.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

FIONA1-mediated mRNA m6 A methylation regulates the response of Arabidopsis to salt stress.

N6 -methyladenosine (m6 A) is an mRNA modification widely found in eukaryotes and plays a crucial role in plant development and stress responses. FIONA1 (FIO1) is a recently identified m6 A methyltransferase that regulates Arabidopsis (Arabidopsis thaliana) floral transition; however, its role in stress response remains unknown. In this study, we demonstrate that FIO1-mediated m6 A methylation plays a vital role in salt stress response in Arabidopsis. The loss-of-function fio1 mutant was sensitive to salt stress. Importantly, the complementation lines expressing the wild-type FIO1 exhibited the wild-type phenotype, whereas the complementation lines expressing the mutant FIO1m , in which two critical amino acid residues essential for methyltransferase activity were mutated, did not recover the wild-type phenotype under salt stress, indicating that the salt sensitivity is associated with FIO1 methyltransferase activity. Furthermore, FIO1-mediated m6 A methylation regulated ROS production and affected the transcript level of several salt stress-responsive genes via modulating their mRNA stability in an m6 A-dependent manner in response to salt stress. Importantly, FIO1 is associated with salt stress response by specifically targeting and differentially modulating several salt stress-responsive genes compared with other m6 A writer. Collectively, our findings highlight the molecular mechanism of FIO1-mediated m6 A methylation in the salt stress adaptation.

Unique features of mRNA m6A methylomes during expansion of tomato (Solanum lycopersicum) fruits.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA. Although the role of m6A has been demonstrated in many biological processes, including embryonic development, flowering time control, microspore generation, fruit ripening, and stress responses, its contribution to other aspects of plant development still needs to be explored. Herein, we show the potential link between m6A deposition and the expansion of tomato (Solanum lycopersicum) fruits through parallel m6A-immunoprecipitation-sequencing (m6A-seq) and RNA-seq analyses. We found that global m6A levels increased during tomato fruit expansion from immature green to mature green stage. m6A-seq revealed that thousands of protein-coding genes are m6A-modified mainly in the 3'-untranslated regions. m6A-seq and RNA-seq analyses showed a positive association between m6A methylation and mRNA abundance. In particular, a large number of fruit expansion-related genes involved in hormone responses and endoreduplication were m6A modified and expressed more actively than the non-m6A-modified genes, suggesting a potential role of m6A modification in tomato fruit expansion. Importantly, altering m6A levels by direct injection of 3-deazaneplanocin A (DA; m6A writer inhibitor) or meclofenamic acid (MA; m6A eraser inhibitor) into tomato fruits suppressed fruit expansion; however, injection of exogenous DA or MA accelerated or delayed fruit ripening, respectively. Collectively, these results suggest a dynamic role of m6A methylation in the expansion and ripening of tomato fruits.

Arabidopsis N6-methyladenosine methyltransferase FIONA1 regulates floral transition by affecting the splicing of FLC and the stability of floral activators SPL3 and SEP3.

N 6-methyladenosine (m6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m6A methyltransferase that regulates the floral transition in Arabidopsis through influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC). In this study, we confirmed that FIONA1 is an m6A methyltransferase that installs m6A marks in a small group of mRNAs. Furthermore, we show that, in addition to its role in influencing the stability of CO, SOC1, and FLC, FIONA1-mediated m6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which together play a vital role in floral transition in Arabidopsis. Our study confirms the function of FIONA1 as an m6A methyltransferase and suggests a close molecular link between FIONA1-mediated m6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time 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.

Epitranscriptomic mRNA modifications governing plant stress responses: underlying mechanism and potential application.

Plants inevitably encounter environmental adversities, including abiotic and biotic stresses, which significantly impede plant growth and reduce crop yield. Thus, fine-tuning the fate and function of stress-responsive RNAs is indispensable for plant survival under such adverse conditions. Recently, post-transcriptional RNA modifications have been studied as a potent route to regulate plant gene expression under stress. Among over 160 mRNA modifications identified to date, N6 -methyladenosine (m6 A) in mRNAs is notable because of its multifaceted roles in plant development and stress response. Recent transcriptome-wide mapping has revealed the distribution and patterns of m6 A in diverse stress-responsive mRNAs in plants, building a foundation for elucidating the molecular link between m6 A and stress response. Moreover, the identification and characterization of m6 A writers, readers and erasers in Arabidopsis and other model crops have offered insights into the biological roles of m6 A in plant abiotic stress responses. Here, we review the recent progress of research on mRNA modifications, particularly m6 A, and their dynamics, distribution, regulation and biological functions in plant stress responses. Further, we posit potential strategies for breeding stress-tolerant crops by engineering mRNA modifications and propose the future direction of research on RNA modifications to gain a much deeper understanding of plant stress biology.

ALKBH9C, a potential RNA m6 A demethylase, regulates the response of Arabidopsis to abiotic stresses and abscisic acid.

Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases (referred to as 'erasers'), biological functions of only a few ALKBH proteins have been characterized to date. In this study, we determined the function of ALKBH9C (At4g36090) in seed germination and seedling growth of Arabidopsis thaliana in response to abiotic stress and abscisic acid (ABA). Seed germination of the alkbh9c mutant was delayed in response to salt, drought, cold and ABA. Moreover, seedling growth of the mutant was repressed under salt stress or ABA but enhanced under drought conditions. Notably, the stress-responsive phenotypes were associated with the altered expression of several m6 A-modified transcripts related to salt, drought or ABA response. Global m6 A levels were increased in the alkbh9c mutant, and ALKBH9C bound to m6 A-modified RNAs and had in vitro m6 A demethylase activity, suggesting its potential role as an m6 A eraser. The m6 A levels in several stress-responsive genes were increased in the alkbh9c mutant, and the stability of m6 A-modified transcripts was altered in the mutant. Collectively, our results suggest that m6 A eraser ALKBH9C is crucial for seed germination and seedling growth of Arabidopsis in response to abiotic stresses or ABA via affecting the stability of stress-responsive transcripts.

RsmD, a Chloroplast rRNA m2G Methyltransferase, Plays a Role in Cold Stress Tolerance by Possibly Affecting Chloroplast Translation in Arabidopsis.

Ribosomal RNA (rRNA) methylation is a pivotal process in the assembly and activity of ribosomes, which in turn play vital roles in the growth, development and stress responses of plants. Although few methyltransferases responsible for rRNA methylation have been identified in plant chloroplasts, the nature and function of these enzymes in chloroplasts remain largely unknown. In this study, we characterized ArabidopsisRsmD (At3g28460), an ortholog of the methyltransferase responsible for N2-methylguanosine (m2G) modification of 16S rRNA in Escherichia coli. Confocal microscopic analysis of an RsmD- green fluorescent protein fusion protein revealed that RsmD is localized to chloroplasts. Primer extension analysis indicated that RsmD is responsible for m2G methylation at position 915 in the 16S rRNA of Arabidopsis chloroplasts. Under cold stress, rsmd mutant plants exhibited retarded growth, i.e. had shorter roots, lower fresh weight and pale-green leaves, compared with wild-type (WT) plants. However, these phenotypes were not detected in response to drought or salt stress. Notably, the rsmd mutant was hypersensitive to erythromycin or lincomycin and accumulated fewer chloroplast proteins compared with the WT, suggesting that RsmD influences translation in chloroplasts. Complementation lines expressing RsmD in the rsmd mutant background recovered WT phenotypes. Importantly, RsmD harbored RNA methyltransferase activity. Collectively, the findings of this study indicate that RsmD is a chloroplast 16S rRNA methyltransferase responsible for m2G915 modification that plays a role in the adaptation of Arabidopsisto cold stress.

Alpha-ketoglutarate-dependent dioxygenase homolog 10B, an N6 -methyladenosine mRNA demethylase, plays a role in salt stress and abscisic acid responses in Arabidopsis thaliana.

N6 -methyladenosine (m6 A) is an abundant methylation mark in eukaryotic mRNAs. It is installed and removed by methyltransferases ("writers") and demethylases ("erasers"), respectively. A recent study has demonstrated that alpha-ketoglutarate-dependent dioxygenase homolog 10B (ALKBH10B) is an mRNA m6 A eraser affecting floral transition in Arabidopsis thaliana. However, the roles of m6 A eraser proteins, including ALKHB10B, in plant adaptation to abiotic stresses are largely unknown. In this study, we aimed to determine the role of ALKBH10B in the response of A. thaliana to abiotic stresses and abscisic acid (ABA). The m6 A level increased in response to salt stress, and m6 A levels in alkbh10b mutants were higher than those in the wild-type under both normal and salt stress conditions. Germination of alkbh10b mutant seeds was markedly delayed under salt stress but not under dehydration, cold, or ABA conditions. Seedling growth and survival rate of alkbh10b mutants were enhanced under salt stress. Notably, salt-tolerant phenotypes of alkbh10b mutants were correlated with decreased levels of several m6 A-modified genes, including ATAF1, BGLU22, and MYB73, which are negative effectors of salt stress tolerance. In response to ABA, both seedling and root growth of alkbh10b mutants were inhibited via upregulating ABA signaling-related genes, including ABI3 and ABI4. Collectively, these findings indicate that ALKBH10B-mediated m6 A demethylation affects the transcript levels of stress-responsive genes, which are important for seed germination, seedling growth, and survival of Arabidopsis thaliana in response to salt stress or ABA.

RNA methylation in chloroplasts or mitochondria in plants.

Recent advances in our understanding of epitranscriptomic RNA methylation have expanded the complexity of gene expression regulation beyond epigenetic regulation involving DNA methylation and histone modifications. The instalment, removal, and interpretation of methylation marks on RNAs are carried out by writers (methyltransferases), erasers (demethylases), and readers (RNA-binding proteins), respectively. Contrary to an emerging body of evidence demonstrating the importance of RNA methylation in the diverse fates of RNA molecules, including splicing, export, translation, and decay in the nucleus and cytoplasm, their roles in plant organelles remain largely unclear and are only now being discovered. In particular, extremely high levels of methylation marks in chloroplast and mitochondrial RNAs suggest that RNA methylation plays essential roles in organellar biogenesis and functions in plants that are crucial for plant development and responses to environmental stimuli. Thus, unveiling the cellular components involved in RNA methylation in cell organelles is essential to better understand plant biology.

N4-methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function, development, and abscisic acid response in Arabidopsis.

Although the essential role of messenger RNA methylation in the nucleus is increasingly understood, the nature of ribosomal RNA (rRNA) methyltransferases and the role of rRNA methylation in chloroplasts remain largely unknown. A recent study revealed that CMAL (for Chloroplast mr aW- Like) is a chloroplast-localized rRNA methyltransferase that is responsible for N4-methylcytidine (m4 C) in 16S chloroplast rRNA in Arabidopsis thaliana. In this study, we further examined the role of CMAL in chloroplast biogenesis and function, development, and hormone response. The cmal mutant showed reduced chlorophyll biosynthesis, photosynthetic activity, and growth-defect phenotypes, including severely stunted stems, fewer siliques, and lower seed yield. The cmal mutant was hypersensitive to chloroplast translation inhibitors, such as lincomycin and erythromycin, indicating that the m4 C-methylation defect in the 16S rRNA leads to a reduced translational activity in chloroplasts. Importantly, the stunted stem of the cmal mutant was partially rescued by exogenous gibberellic acid or auxin. The cmal mutant grew poorer than wild type, whereas the CMAL-overexpressing transgenic Arabidopsis plants grew better than wild type in the presence of abscisic acid. Altogether, these results indicate that CMAL is an indispensable rRNA methyltransferase in chloroplasts and is crucial for chloroplast biogenesis and function, photosynthesis, and hormone response during plant growth and development.

The coordinated action of PPR4 and EMB2654 on each intron half mediates trans-splicing of rps12 transcripts in plant chloroplasts.

The pentatricopeptide repeat proteins PPR4 and EMB2654 have been shown to be required for the trans-splicing of plastid rps12 transcripts in Zea mays (maize) and Arabidopsis, respectively, but their roles in this process are not well understood. We investigated the functions of the Arabidopsis and Oryza sativa (rice) orthologs of PPR4, designated AtPPR4 (At5g04810) and OsPPR4 (Os4g58780). Arabidopsis atppr4 and rice osppr4 mutants are embryo-lethal and seedling-lethal 3 weeks after germination, respectively, showing that PPR4 is essential in the development of both dicot and monocot plants. Artificial microRNA-mediated mutants of AtPPR4 displayed a specific defect in rps12 trans-splicing, with pale-green, yellowish or albino phenotypes, according to the degree of knock-down of AtPPR4 expression. Comparison of RNA footprints in atppr4 and emb2654 mutants showed a similar concordant loss of extensive footprints at the 3' end of intron 1a and at the 5' end of intron 1b in both cases. EMB2654 is known to bind within the footprint region in intron 1a and we show that AtPPR4 binds to the footprint region in intron 1b, via its PPR motifs. Binding of both PPR4 and EMB2654 is essential to juxtapose the two intron halves and to maintain the RNAs in a splicing-competent structure for the efficient trans-splicing of rps12 intron 1, which is crucial for chloroplast biogenesis and plant development. The similarity of EMB2654 and PPR4 orthologs and their respective binding sites across land plant phylogeny indicates that their coordinate function in rps12 trans-splicing has probably been conserved for 500 million years.

High-throughput deep sequencing reveals the important role that microRNAs play in the salt response in sweet potato (Ipomoea batatas L.).

BACKGROUND: MicroRNAs (miRNAs), a class of small regulatory RNAs, have been proven to play important roles in plant growth, development and stress responses. Sweet potato (Ipomoea batatas L.) is an important food and industrial crop that ranks seventh in staple food production. However, the regulatory mechanism of miRNA-mediated abiotic stress response in sweet potato remains unclear. RESULTS: In this study, we employed deep sequencing to identify both conserved and novel miRNAs from salinity-exposed sweet potato cultivars and its untreated control. Twelve small non-coding RNA libraries from NaCl-free (CK) and NaCl-treated (Na150) sweet potato leaves and roots were constructed for salt-responsive miRNA identification in sweet potatoes. A total of 475 known miRNAs (belonging to 66 miRNA families) and 175 novel miRNAs were identified. Among them, 51 (22 known miRNAs and 29 novel miRNAs) were significantly up-regulated and 76 (61 known miRNAs and 15 novel miRNAs) were significantly down-regulated by salinity stress in sweet potato leaves; 13 (12 known miRNAs and 1 novel miRNAs) were significantly up-regulated and 9 (7 known miRNAs and 2 novel miRNAs) were significantly down-regulated in sweet potato roots. Furthermore, 636 target genes of 314 miRNAs were validated by degradome sequencing. Deep sequencing results confirmed by qRT-PCR experiments indicated that the expression of most miRNAs exhibit a negative correlation with the expression of their targets under salt stress. CONCLUSIONS: This study provides insights into the regulatory mechanism of miRNA-mediated salt response and molecular breeding of sweet potatoes though miRNA manipulation.

Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses.

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

Functional Characterization of a Putative RNA Demethylase ALKBH6 in Arabidopsis Growth and Abiotic Stress Responses.

RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases, the function of most ALKBHs is yet to be determined. The Arabidopsis thaliana genome contains thirteen genes encoding ALKBH proteins, the functions of which are largely unknown. In this study, we characterized the function of a potential eraser protein, ALKBH6 (At4g20350), during seed germination and seedling growth in Arabidopsis under abiotic stresses. The seeds of T-DNA insertion alkbh6 knockdown mutants germinated faster than the wild-type seeds under cold, salt, or abscisic acid (ABA) treatment conditions but not under dehydration stress conditions. Although no differences in seedling and root growth were observed between the alkbh6 mutant and wild-type under normal conditions, the alkbh6 mutant showed a much lower survival rate than the wild-type under salt, drought, or heat stress. Cotyledon greening of the alkbh6 mutants was much higher than that of the wild-type upon ABA application. Moreover, the transcript levels of ABA signaling-related genes, including ABI3 and ABI4, were down-regulated in the alkbh6 mutant compared to wild-type plants. Importantly, the ALKBH6 protein had an ability to bind to both m6A-labeled and m5C-labeled RNAs. Collectively, these results indicate that the potential eraser ALKBH6 plays important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses.

TAF15b, involved in the autonomous pathway for flowering, represses transcription of FLOWERING LOCUS C.

TATA-binding protein-associated factors (TAFs) are general transcription factors within the transcription factor IID (TFIID) complex, which recognizes the core promoter of genes. In addition to their biochemical function, it is known that several TAFs are involved in the regulation of developmental processes. In this study, we found that TAF15b affects flowering time, especially through the autonomous pathway (AP) in Arabidopsis. The mutant taf15b shows late flowering compared with the wild type plant during both long and short days, and vernalization accelerates the flowering time of taf15b. In addition, taf15b shows strong upregulation of FLOWERING LOCUS C (FLC), a flowering repressor in Arabidopsis, and the flc taf15b double mutant completely offsets the late flowering of taf15b, indicating that TAF15b is a typical AP gene. The taf15b mutant also shows increased transcript levels of COOLAIR, an antisense transcript of FLC. Consistently, chromatin immunoprecipitation (ChIP) analyses showed that the TAF15b protein is enriched around both sense and antisense transcription start sites of the FLC locus. In addition, co-immunoprecipitation showed that TAF15b interacts with RNA polymerase II (Pol II), while ChIP showed increased enrichment of the phosphorylated forms, both serine 2 (Ser2) and Ser5, of the C-terminal domain of Pol II at the FLC locus, which is indicative of transcriptional elongation. Finally, taf15b showed higher enrichment of the active histone marker, H3K4me3, on FLC chromatin. Taken together, our results suggest that TAF15b affects flowering time through transcriptional repression of FLC in Arabidopsis.

CFM9, a Mitochondrial CRM Protein, Is Crucial for Mitochondrial Intron Splicing, Mitochondria Function and Arabidopsis Growth and Stress Responses.

Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.

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.

A chloroplast-targeted pentatricopeptide repeat protein PPR287 is crucial for chloroplast function and Arabidopsis development.

BACKGROUND: Even though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis ( http://ppr.plantenergy.uwa.edu.au ). RESULTS: The homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants. CONCLUSIONS: These results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.