Chloroplast RNA Binding Research Articles

OsCRP1, a Ribonucleoprotein Gene, Regulates Chloroplast mRNA Stability That Confers Drought and Cold Tolerance.

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded and highly abundant proteins that are proposed to function in chloroplast RNA metabolism. However, the molecular mechanisms underlying the regulation of chloroplast RNAs involved in stress tolerance are poorly understood. Here, we demonstrate that CHLOROPLAST RNA-BINDING PROTEIN 1 (OsCRP1), a rice (Oryza sativa) cpRNP gene, is essential for stabilization of RNAs from the NAD(P)H dehydrogenase (NDH) complex, which in turn enhances drought and cold stress tolerance. An RNA-immunoprecipitation assay revealed that OsCRP1 is associated with a set of chloroplast RNAs. Transcript profiling indicated that the mRNA levels of genes from the NDH complex significantly increased in the OsCRP1 overexpressing compared to non-transgenic plants, whereas the pattern in OsCRP1 RNAi plants were opposite. Importantly, the OsCRP1 overexpressing plants showed a higher cyclic electron transport (CET) activity, which is essential for elevated levels of ATP for photosynthesis. Additionally, overexpression of OsCRP1 resulted in significantly enhanced drought and cold stress tolerance with higher ATP levels compared to wild type. Thus, our findings suggest that overexpression of OsCRP1 stabilizes a set of mRNAs from genes of the NDH complex involved in increasing CET activity and production of ATP, which consequently confers enhanced drought and cold tolerance.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

Nitric oxide associated protein 1 is associated with chloroplast perturbation and disease symptoms in Nicotiana benthamiana infected with South African cassava mosaic virus

Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation and response to biotic stress. The association between NOA1 and NO accumulation in Arabidopsis noa1 mutants has been linked to the inability of noa1 mutants to accumulate carbon reserves such as fumarate, leading to chloroplast dysfunction and a pale green leaf phenotype. To understand the role played by NOA1 in response to South African cassava mosaic virus infection in Nicotiana benthamiana, the expression of NbNOA1 and the accumulation of NO in leaf samples was compared between south african cassava mosaic (SACMV)-infected and mock-infected plants at 14 and 28 dpi. Real-time qPCR was used to measure SACMV viral load which increased significantly by 20% from 14 to 28 dpi as chlorosis and symptom severity progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p-value=0.01 and 5-fold lower at 28, p-value=0.00). At 14 dpi, NO accumulation remained unchanged in infected leaf tissue compared to mock inoculated, while at 28 dpi, NO accumulation was 40% lower (p-value=0.01). At 28 dpi, the decrease in NbNOA1 expression and NO accumulation was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaves. Furthermore, the expression of chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation elongation factor Tu, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and plastid ribosome recycling factor) were found to be repressed in infected N. benthamiana. GC–MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection, a decrease in photopigments and carbon stores, accompanied by an increase in glucose and decrease in fumarate, leads to a decline in NbNOA1expression and NO levels. This is manifested by suppressed translation factors and disruption of chloroplast function, thereby contributing to chlorotic disease symptoms.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

CSP41b, a protein identified via FOX hunting using Eutrema salsugineum cDNAs, improves heat and salinity stress tolerance in transgenic Arabidopsis thaliana

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses.

The proteome response ofHordeum spontaneumto salinity stress

Hordeum spontaneum (wild barley) is a good gene source to improve salt tolerance in barley because it rapidly hybridizes and recombines with barley cultivars. Proteomics can assist in identifying proteins associated with a certain environmental or developmental signal. We employed a proteomic approach to understand the mechanisms of plant responses to salinity in a salt tolerant accession of H. spontaneum. At the 4-leaf stage, wild barley plants were exposed to 0 (control treatment) or 300 mM NaCl (salt treatment). The salt treatment lasted 3 weeks. Total proteins of leaf 4 were extracted and separated by two-dimensional gel electrophoresis. More than 500 protein spots were reproducibly detected. Of these, 29 spots showed significant differences between salt treatment and control. Using MALDI-TOF-TOF MS, we identified 29 cellular proteins, which represented 16 different proteins. These were classified into six categories and a group with unknown biological function. The proteins identified were involved in many different cellular functions. Three spots were identified as unknown proteins; searching in the NCBI database revealed that there was a 71% match with clathrin assembly protein putative [Ricinus communis], a 67% match with actin binding protein [Zea mays], and a 66% match with phosphatidylinositol kinase [Arabidopsis thaliana]. Other proteins identified included ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), oxygen-evolving enhancer protein (OEE), photosystem II reaction centerWprotein (Psbw), ribosomal proteins, chloroplast RNA binding protein (ChRBP), superoxide dismutase (SOD), malate dehydrogenase (MDH), thioredoxin h (Trx), nucleoside diphosphate kinase (NDPK), profilin, translationally-controlled tumor protein (TCTP), polyamine oxidase (PAO) and universal stress protein family (USP).

Arabidopsis Chloroplast RNA Binding Proteins CP31A and CP29A Associate with Large Transcript Pools and Confer Cold Stress Tolerance by Influencing Multiple Chloroplast RNA Processing Steps

Chloroplast RNA metabolism is mediated by a multitude of nuclear encoded factors, many of which are highly specific for individual RNA processing events. In addition, a family of chloroplast ribonucleoproteins (cpRNPs) has been suspected to regulate larger sets of chloroplast transcripts. This together with their propensity for posttranslational modifications in response to external cues suggested a potential role of cpRNPs in the signal-dependent coregulation of chloroplast genes. We show here on a transcriptome-wide scale that the Arabidopsis thaliana cpRNPs CP31A and CP29A (for 31 kD and 29 kD chloroplast protein, respectively), associate with large, overlapping sets of chloroplast transcripts. We demonstrate that both proteins are essential for resistance of chloroplast development to cold stress. They are required to guarantee transcript stability of numerous mRNAs at low temperatures and under these conditions also support specific processing steps. Fine mapping of cpRNP-RNA interactions in vivo suggests multiple points of contact between these proteins and their RNA ligands. For CP31A, we demonstrate an essential function in stabilizing sense and antisense transcripts that span the border of the small single copy region and the inverted repeat of the chloroplast genome. CP31A associates with the common 3'-terminus of these RNAs and protects them against 3'-exonucleolytic activity.

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3′-untranslated regions

The mature 3′-end of many chloroplast mRNAs is generated by the processing of the 3′-untranslated region (3′-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3′-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3′-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3′-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3′-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3′-UTRs, finding that each 3′-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3′-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3′-UTR processing activity on ortholog spinach 3′-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3′-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3′-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.

Logical Operation Based Literature Association with Genes and its application, PosMed.

PosMed prioritizes candidate genes for positional cloning by employing our original database search engine GRASE, which uses an inferential process similar to an artificial neural network comprising documental neurons (or 'documentrons') that represent each document contained in databases such as MEDLINE and OMIM (Yoshida, et al. 2009, Makita, et al. 2009). PosMed immediately ranks the candidate genes by connecting phenotypic keywords to the genes through connections representing gene–gene interactions other biological relationships, such as metabolite–gene, mutant mouse–gene, drug–gene, disease–gene, and protein–protein interactions, ortholog data, and gene–literature connections.To make proper relationships between genes and literature, we manually curate queries, which are defined by logical operation rules, against MEDLINE. For example, to detect a set of MEDLINE documents for the AT1G03880 gene in A. thaliana, we applied the following logical query: (‘AT1G03880’ OR ‘CRU2’ OR ‘CRB’ OR ‘CRUCIFERIN 2' OR ‘CRUCIFERIN B’) AND (‘Arabidopsis’) NOT (‘chloroplast RNA binding’). Curators refined these queries in mouse, rice and A. thaliana. For human and rat genes, we use mouse curation results via ortholog genes in PosMed.PosMed is available at "http://omicspace.riken.jp/PosMed":http://omicspace.riken.jp/PosMed

Logical Operation Based Literature Association with Genes and its application, PosMed.

AbstractPosMed prioritizes candidate genes for positional cloning by employing our original database search engine GRASE, which uses an inferential process similar to an artificial neural network comprising documental neurons (or 'documentrons') that represent each document contained in databases such as MEDLINE and OMIM (Yoshida, et al. 2009, Makita, et al. 2009). PosMed immediately ranks the candidate genes by connecting phenotypic keywords to the genes through connections representing gene–gene interactions other biological relationships, such as metabolite–gene, mutant mouse–gene, drug–gene, disease–gene, and protein–protein interactions, ortholog data, and gene–literature connections.To make proper relationships between genes and literature, we manually curate queries, which are defined by logical operation rules, against MEDLINE. For example, to detect a set of MEDLINE documents for the AT1G03880 gene in A. thaliana, we applied the following logical query: (‘AT1G03880’ OR ‘CRU2’ OR ‘CRB’ OR ‘CRUCIFERIN 2' OR ‘CRUCIFERIN B’) AND (‘Arabidopsis’) NOT (‘chloroplast RNA binding’). Curators refined these queries in mouse, rice and A. thaliana. For human and rat genes, we use mouse curation results via ortholog genes in PosMed.PosMed is available at "http://omicspace.riken.jp/PosMed":http://omicspace.riken.jp/PosMed

A proteomic analysis of oligo(dT)-bound mRNP containing oxidative stress-induced Arabidopsis thaliana RNA-binding proteins ATGRP7 and ATGRP8

Plants are highly adapted to respond to a range of environmental stresses commonly by altering their gene expression and metabolism as a result of cell signalling which may be mediated by reactive oxygen species. The glycine-rich RNA-binding proteins ATGRP7 and ATGRP8 were rapidly upregulated in response to peroxide-induced oxidative stress and were amongst the most abundant RNA binding proteins isolated by oligo(dT) chromatography. The oligo(dT)-bound mRNP complexes were analysed proteomically, and were seen to contain potential isoforms of the ATGRP proteins; other proteins that contain an RNA Recognition Motif (RRM); and chloroplast RNA binding proteins. These findings suggest that ATGRP proteins have an evolutionarily conserved function in the regulation of gene expression at the posttranscriptional level in response to environmental stress.

Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.

A Diterpene as an Endogenous Signal for the Activation of Defense Responses to Infection with Tobacco mosaic virus and Wounding in Tobacco

The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing element between residues -52 and -27 that contributes to transcriptional regulation. In this study, we have isolated a silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone. Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus sequence-type RNA binding domains characteristic of heterogeneous nuclear ribonucleoproteins (hnRNPs). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element-dependent manner, confirming the role of SEBF as a transcriptional repressor.

A chloroplast RNA binding protein from stromal thylakoid membranes specifically binds to the 5' untranslated region of the psbA mRNA.

The intrachloroplastic localization of post-transcriptional gene expression steps represents one key determinant for the regulation of chloroplast development. We have characterized an RNA binding protein of 63 kDa (RBP63) from Chlamydomonas reinhardtii chloroplasts, which cofractionates with stromal thylakoid membranes. Solubility properties suggest that RBP63 is a peripheral membrane protein. Among RNA probes from different 5' untranslated regions of chloroplast transcripts, RBP63 preferentially binds to the psbA leader. This binding is dependent on a region comprising seven consecutive A residues, which is required for D1 protein synthesis. A possible role for this newly discovered RNA binding protein in membrane targeting of psbA gene expression is discussed.