Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specifi c Ca 2 + signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca 2 + transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through 1 O 2 -mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.

Obg is a ribosome-associated GTPase essential for bacterial viability and is conserved in most organisms, from bacteria to eukaryotes. Obg is also expressed in plants, which predicts an important role for this molecule in plant viability; however, the functions of the plant Obg homologs have not been reported. Here, we first identified Arabidopsis AtObgC as a plant chloroplast-targeting Obg and elucidated its molecular biological and physiological properties. AtObgC encodes a plant-specific Obg GTPase that contains an N-terminal region for chloroplast targeting and has intrinsic GTP hydrolysis activity. A targeting assay using a few AtObgC N-terminal truncation mutants revealed that AtObgC localizes to chloroplasts and its transit peptide consists of more than 50 amino acid residues. Interestingly, GFP-fused full-length AtObgC exhibited a punctate staining pattern in chloroplasts of Arabidopsis protoplasts, which suggests a dimerization or multimerization of AtObgC. Moreover, its Obg fold was indispensable for the generation of the punctate staining pattern, and thus, was supposed to be important for such oligomerization of AtObgC by mediating the protein-protein interaction. In addition, the T-DNA insertion AtObgC null mutant exhibited an embryonic lethal phenotype that disturbed the early stage of embryogenesis. Altogether, our results provide a significant implication that AtObgC as a chloroplast targeting GTPase plays an important role at the early embryogenesis by exerting its function in chloroplast protein synthesis.

ABSTRACT. SNAREs ('Soluble N-ethyl-maleimide sensitive factor attachment protein receptors') play a critical role in the membrane fusion step of the vesicular transport system in eukaryotes. The number of the genes encoding SNARE proteins is estimated to be 64 in Arabidopsis thaliana. This number is much larger than those in other eukaryotes, suggesting a complex membrane trafficking in plants. The Arabidopsis SNAREs, the SYP7 group proteins, SYP71, SYP72, and SYP73, form a plant-specific SNARE subfamily with not-yet-identified functions. We have previously reported that the SYP7 subfamily proteins are predominantly localized to the endoplasmic reticulum in the Arabidopsis suspension cultured cells under transient expression condition. However, several proteomic analyzes indicated the plasma membrane localizations of one of SYP7 subfamily proteins, SYP71. In order to confirm the expression patterns and subcellular localization of SYP7, we performed combination analyses including promoter GUS analysis, a sucrose density gradient centrifugation analysis, as well as an observation on transgenic Arabidopsis plants expressing GFP-fused SYP71 under control of its native promoter. From these analyses, we concluded that one of the SYP7 subfamily proteins, SYP71, is predominantly expressed in all vegetative tissues and mainly localized to the plasma membrane. We also found that SYP71 is localized to the endoplasmic reticulum in the dividing cells of various types of tissues.

The genomes of free-living bacteria frequently exchange genes via lateral gene transfer (LGT), which has played a major role in bacterial evolution. LGT also played a significant role in the acquisition of genes from non-cyanobacterial bacteria to the lineage of “primary” algae and land plants. Small GTPases are widely distributed among prokaryotes and eukaryotes. In this study, we inferred the evolutionary history of organelle-targeted small GTPases in plants. Arabidopsis thaliana contains at least one ortholog in seven subfamilies of OBG-HflX-like and TrmE-Era-EngA-YihA-Septin-like GTPase superfamilies (together referred to as Era-like GTPases). Subcellular localization analysis of all Era-like GTPases in Arabidopsis revealed that all 30 eubacteria-related GTPases are localized to chloroplasts and/or mitochondria, whereas archaea-related DRG and NOG1 are localized to the cytoplasm and nucleus, respectively, suggesting that chloroplast- and mitochondrion-localized GTPases are derived from the ancestral cyanobacterium and α-proteobacterium, respectively, through endosymbiotic gene transfer (EGT). However, phylogenetic analyses revealed that plant organelle GTPase evolution is rather complex. Among the eubacterium-related GTPases, only four localized to chloroplasts (including one dual targeting GTPase) and two localized to mitochondria were derived from cyanobacteria and α-proteobacteria, respectively. Three other chloroplast-targeted GTPases were related to α-proteobacterial proteins, rather than to cyanobacterial GTPases. Furthermore, we found that four other GTPases showed neither cyanobacterial nor α-proteobacterial affiliation. Instead, these GTPases were closely related to clades from other eubacteria, such as Bacteroides (Era1, EngB-1, and EngB-2) and green non-sulfur bacteria (HflX). This study thus provides novel evidence that LGT significantly contributed to the evolution of organelle-targeted Era-like GTPases in plants.

Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes.

A rice glutaredoxin isoform (OsGrxC2;2) with antioxidant capacity is expressed abundantly in seed tissues and is localized to storage vacuoles in aleurone layers in developing and mature seeds. Seed tissues undergo drastic water loss at the late stage of seed development, and thus need to tolerate oxidative injuries associated with desiccation. We previously found a rice glutaredoxin isoform, OsGrxC2;2, as a gene expressed abundantly in developing seeds. Since glutaredoxin is involved in antioxidant defense, in the present study we investigated the subcellular localization and expression profile of OsGrxC2;2 and whether OsGrxC2;2 has a role in the defense against reactive oxygen species. Western blotting and immunohistochemistry revealed that the OsGrxC2;2 protein accumulated at a high level in the embryo and aleurone layers of developing and mature seeds. The OsGrxC2;2 in developing seeds was particularly localized to aleurone grains, which are storage organelles derived from vacuoles. Overexpression of OsGrxC2;2 resulted in an enhanced tolerance to menadione in yeast and methyl viologen in green leaves of transgenic rice plants. These results suggest that OsGrxC2;2 participates in the defense against oxidative stress in developing and mature seeds.

The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k cat values 0.12 ؎ 0.007 min ؊1 (n ‫؍‬ 6) and 0.087 ؎ 0.002 min ؊1 (n ‫؍‬ 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S prerRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono-and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.In eukaryotes, ribosome biogenesis is a complex and coordinated process that involves the synthesis and processing of rRNA, and its assembly with ribosomal proteins (1, 2). Ribosome biogenesis begins with transcription of rDNA repeats by RNA polymerase I and III, followed by processing and assembly of pre-rRNA intermediates, which are exported to the cytoplasm from the nucleolus and nucleus (3-9). These processes require various ribosome biogenesis factors such as assembly factors, as well as non-ribosomal factors including snoRNPs, endo-and exonucleases, pseudouridine synthases, methyltransferases, RNA helicases, RNA chaperones, ATPases and GTPases (3,4,[6][7][8]10).Several GTPases are essential for the biogenesis and assembly of ribosomal subunits (1, 10 -13). In Escherichia coli, the process of the 50S ribosomal subunit assembly requires Obg, EngA, and HflX (14 -16), whereas Bacillus subtilis needs . Interestingly, most GTPases involved in ribosomal biogenesis belong to the YlqF/YawG family, which is characterized by a circularly permuted order of GTPase motifs within the GTP-binding domain (1,11,20). In E. coli, YjeQ binds to the 30S ribosomal subunit, whereas YloQ and YqeH show similar activities in B. subtilis (21-23). YlqF (RbgA) participates in late stage assembly of the 50...

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