Three consecutive cytosolic glycolysis enzymes modulate autophagic flux.

Autophagy serves as an important recycling route for the growth and survival of eukaryotic organisms in nutrient-deficient conditions. Since starvation induces massive changes in the metabolic flux that are coordinated by key metabolic enzymes, specific processing steps of autophagy may be linked with metabolic flux-monitoring enzymes. We attempted to identify carbon metabolic genes that modulate autophagy using VIGS screening of 45 glycolysis- and Calvin-Benson cycle-related genes in Arabidopsis (Arabidopsis thaliana). Here, we report that three consecutive triose-phosphate-processing enzymes involved in cytosolic glycolysis, triose-phosphate-isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPC), and phosphoglycerate kinase (PGK), designated TGP, negatively regulate autophagy. Depletion of TGP enzymes causes spontaneous autophagy induction and increases AUTOPHAGY-RELATED 1 (ATG1) kinase activity. TGP enzymes interact with ATG101, a regulatory component of the ATG1 kinase complex. Spontaneous autophagy induction and abnormal growth under insufficient sugar in TGP mutants are suppressed by crossing with the atg101 mutant. Considering that triose-phosphates are photosynthates transported to the cytosol from active chloroplasts, the TGP enzymes would be strategically positioned to monitor the flow of photosynthetic sugars and modulate autophagy accordingly. Collectively, these results suggest that TGP enzymes negatively control autophagy acting upstream of the ATG1 complex, which is critical for seedling development.

The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis.

Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription-PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2-ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.

MRF Family Genes Are Involved in Translation Control, Especially under Energy-Deficient Conditions, and Their Expression and Functions Are Modulated by the TOR Signaling Pathway.

Dynamic control of protein translation in response to the environment is essential for the survival of plant cells. Target of rapamycin (TOR) coordinates protein synthesis with cellular energy/nutrient availability through transcriptional modulation and phosphorylation of the translation machinery. However, mechanisms of TOR-mediated translation control are poorly understood in plants. Here, we report that Arabidopsis thaliana MRF (MA3 DOMAIN-CONTAINING TRANSLATION REGULATORY FACTOR) family genes encode translation regulatory factors under TOR control, and their functions are particularly important in energy-deficient conditions. Four MRF family genes (MRF1-MRF4) are transcriptionally induced by dark and starvation (DS). Silencing of multiple MRFs increases susceptibility to DS and treatment with a TOR inhibitor, while MRF1 overexpression decreases susceptibility. MRF proteins interact with eIF4A and cofractionate with ribosomes. MRF silencing decreases translation activity, while MRF1 overexpression increases it, accompanied by altered ribosome patterns, particularly in DS. Furthermore, MRF deficiency in DS causes altered distribution of mRNAs in sucrose gradient fractions and accelerates rRNA degradation. MRF1 is phosphorylated in vivo and phosphorylated by S6 kinases in vitro. MRF expression and MRF1 ribosome association and phosphorylation are modulated by cellular energy status and TOR activity. We discuss possible mechanisms of the function of MRF family proteins under normal and energy-deficient conditions and their functional link with the TOR pathway.

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.

Characterization of Maf1 in Arabidopsis: function under stress conditions and regulation by the TOR signaling pathway.

MAIN CONCLUSION: Maf1 repressor activity is critical for plant survival during environmental stresses, and is regulated by its phosphorylation/dephosphorylation through the activity of TOR and PP4/PP2A phosphatases. Maf1 is a global repressor of RNA polymerase III (Pol III), and is conserved in eukaryotes. Pol III synthesizes small RNAs, 5S rRNA, and tRNAs that are essential for protein translation and cell growth. Maf1 is a phosphoprotein and dephosphorylation of Maf1 promotes its repressor activity in yeast and mammals. Plant Maf1 was identified in citrus plants as a canker elicitor-binding protein, and citrus Maf1 represses cell growth associated with canker development. However, functions of plant Maf1 under diverse stress conditions and its regulation by the target of rapamycin (TOR) signaling components are poorly understood. In this study, the Arabidopsis maf1 mutants were more susceptible to diverse stresses and treatment with the TOR inhibitor Torin-1 than wild-type plants. The maf1 mutants expressed higher levels of Maf1 target RNAs, including 5S rRNA and pre-tRNAs in leaf cells, supporting Pol III repressor activity of Arabidopsis Maf1. Cellular stresses and Torin-1 treatment induced dephosphorylation of Maf1, suggesting Maf1 activation under diverse stress conditions. TOR silencing also stimulated Maf1 dephosphorylation, while silencing of catalytic subunit genes of PP4 and PP2A repressed it. Thus, TOR kinase and PP4/PP2A phosphatases appeared to oppositely modulate the Maf1 phosphorylation status. TOR silencing decreased the abundance of the target RNAs, while silencing of the PP4 and PP2A subunit genes increased it, supporting the positive correlation between Maf1 dephosphorylation and its repressor activity. Taken together, these results suggest that repressor activity of Maf1, regulated by the TOR signaling pathway, is critical for plant cell survival during environmental stresses.

Functional characterization of chaperonin containing T-complex polypeptide-1 and its conserved and novel substrates in Arabidopsis.

Chaperonin containing T-complex polypeptide-1 (CCT) is an evolutionarily conserved chaperonin multi-subunit complex that mediates protein folding in eukaryotes. It is essential for cell growth and survival in yeast and mammals, with diverse substrate proteins. However, only a few studies on plant CCT have been reported to date, due to the essentiality of CCT subunit genes and the large size of the complex. Here, we have investigated the structure and function of the Arabidopsis CCT complex in detail. The plant CCT consisted of eight subunits that assemble to form a high-molecular-mass protein complex, shown by diverse methods. CCT-deficient cells exhibited depletion of cortical microtubules, accompanied by a reduction in cellular α- and ß-tubulin levels due to protein degradation. Cycloheximide-chase assays suggested that CCT is involved in the folding of tubulins in plants. Furthermore, CCT interacted with PPX1, the catalytic subunit of protein phosphatase 4, and may participate in the folding of PPX1 as its substrate. CCT also interacted with Tap46, a regulatory subunit of PP2A family phosphatases, but Tap46 appeared to function in PPX1 stabilization, rather than as a CCT substrate. Collectively, our findings reveal the essential functions of CCT chaperonin in plants and its conserved and novel substrates.

The subfamily II catalytic subunits of protein phosphatase 2A (PP2A) are involved in cortical microtubule organization.

MAIN CONCLUSION: The subfamily II catalytic subunits of protein phosphatase 2A (PP2A) regulate the cortical microtubule dynamics in Arabidopsis, through interaction with TONNEAU2 (TON2)/FASS and modulation of α-tubulin dephosphorylation. Protein phosphatase 2A is a major protein phosphatase in eukaryotes that dephosphorylates many different substrates to regulate their function. PP2A is assembled into a heterotrimeric complex of scaffolding A subunit, regulatory B subunit, and catalytic C subunit. Plant PP2A catalytic C subunit (PP2AC) isoforms are classified into two subfamilies. In this study, we investigated the cellular functions of the Arabidopsis PP2AC subfamily II genes PP2AC-3 and PP2AC-4, particularly regarding the cortical microtubule (MT) organization. PP2AC-3 and PP2AC-4 strongly interacted with the B'' regulatory subunit TON2. Simultaneous silencing of PP2AC-3 and PP2AC-4 by virus-induced gene silencing (PP2AC-3,4 VIGS) significantly altered plant morphology in Arabidopsis, increasing cell numbers in leaves and stems. The leaf epidermis of PP2AC-3,4 VIGS plants largely lost its jigsaw-puzzle shape and exhibited reduced trichome branch numbers. VIGS of PP2AC-3,4 in Arabidopsis transgenic plants that expressed GFP-fused ß-tubulin 6 isoform (GFP-TUB6) for the visualization of MTs caused a reduction in the cortical MT array density in the pavement cells. VIGS of TON2 also led to similar cellular phenotypes and cortical MT patterns compared with those after VIGS of PP2AC-3,4, suggesting that PP2AC-3,4 and their interaction partner TON2 play a role in cortical MT organization in leaf epidermal cells. Furthermore, silencing of PP2AC-3,4 did not affect salt-induced phosphorylation of α-tubulin but delayed its dephosphorylation after salt removal. The reappearance of cortical MT arrays after salt removal was impaired in PP2AC-3,4 VIGS plants. These results suggest an involvement of PP2AC subfamily II in the regulation of cortical MT dynamics under normal and salt-stress conditions in Arabidopsis.

InsP6-sensitive variants of the Gle1 mRNA export factor rescue growth and fertility defects of the ipk1 low-phytic-acid mutation in Arabidopsis.

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP(6)), also known as phytic acid, accumulates in large quantities in plant seeds, serving as a phosphorus reservoir, but is an animal antinutrient and an important source of water pollution. Here, we report that Gle1 (GLFG lethal 1) in conjunction with InsP(6) functions as an activator of the ATPase/RNA helicase LOS4 (low expression of osmotically responsive genes 4), which is involved in mRNA export in plants, supporting the Gle1-InsP(6)-Dbp5 (LOS4 homolog) paradigm proposed in yeast. Interestingly, plant Gle1 proteins have modifications in several key residues of the InsP(6) binding pocket, which reduce the basicity of the surface charge. Arabidopsis thaliana Gle1 variants containing mutations that increase the basic charge of the InsP(6) binding surface show increased sensitivity to InsP(6) concentrations for the stimulation of LOS4 ATPase activity in vitro. Expression of the Gle1 variants with enhanced InsP(6) sensitivity rescues the mRNA export defect of the ipk1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) InsP(6)-deficient mutant and, furthermore, significantly improves vegetative growth, seed yield, and seed performance of the mutant. These results suggest that Gle1 is an important factor responsible for mediating InsP(6) functions in plant growth and reproduction and that Gle1 variants with increased InsP(6) sensitivity may be useful for engineering high-yielding low-phytate crops.

Functional characterization of chloroplast-targeted RbgA GTPase in higher plants.

KEY MESSAGE: Plant RbgA GTPase is targeted to chloroplasts and co-fractionated with chloroplast ribosomes, and plays a role in chloroplast rRNA processing and/or ribosome biogenesis. Ribosome Biogenesis GTPase A (RbgA) homologs are evolutionarily conserved GTPases that are widely distributed in both prokaryotes and eukaryotes. In this study, we investigated functions of chloroplast-targeted RbgA. Nicotiana benthamiana RbgA (NbRbgA) and Arabidopsis thaliana RbgA (AtRbgA) contained a conserved GTP-binding domain and a plant-specific C-terminal domain. NbRbgA and AtRbgA were mainly localized in chloroplasts, and possessed GTPase activity. Since Arabidopsis rbgA null mutants exhibited an embryonic lethal phenotype, virus-induced gene silencing (VIGS) of NbRbgA was performed in N. benthamiana. NbRbgA VIGS resulted in a leaf-yellowing phenotype caused by disrupted chloroplast development. NbRbgA was mainly co-fractionated with 50S/70S ribosomes and interacted with the chloroplast ribosomal proteins cpRPL6 and cpRPL35. NbRbgA deficiency lowered the levels of mature 23S and 16S rRNAs in chloroplasts and caused processing defects. Sucrose density gradient sedimentation revealed that NbRbgA-deficient chloroplasts contained reduced levels of mature 23S and 16S rRNAs and diverse plastid-encoded mRNAs in the polysomal fractions, suggesting decreased protein translation activity in the chloroplasts. Interestingly, NbRbgA protein was highly unstable under high light stress, suggesting its possible involvement in the control of chloroplast ribosome biogenesis under environmental stresses. Collectively, these results suggest a role for RbgA GTPase in chloroplast rRNA processing/ribosome biogenesis, affecting chloroplast protein translation in higher plants.

Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation.

UNLABELLED: The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg(2+) concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE: In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits.

A nuclear-encoded chloroplast-targeted S1 RNA-binding domain protein affects chloroplast rRNA processing and is crucial for the normal growth of Arabidopsis thaliana.

Despite the fact that a variety of nuclear-encoded RNA-binding proteins (RBPs) are targeted to the chloroplast and play essential roles during post-transcriptional RNA metabolism in the chloroplast, the physiological roles of the majority of chloroplast-targeted RBPs remain elusive. Here, we investigated the functional role of a nuclear-encoded S1 domain-containing RBP, designated SDP, in the growth and development of Arabidopsis thaliana. Confocal analysis of the SDP-green fluorescent protein revealed that SDP was localized to the chloroplast. The loss-of-function sdp mutant displayed retarded seed germination and pale-green phenotypes, and grew smaller than the wild-type plants. Chlorophyll a content and photosynthetic activity of the sdp mutant were much lower than those of wild-type plants, and the structures of the chloroplast and the prolamellar body were abnormal in the sdp mutant. The processing of rRNAs in the chloroplast was defective in the sdp mutant, and SDP was able to bind chloroplast 23S, 16S, 5S and 4.5S rRNAs. Notably, SDP possesses RNA chaperone activity. Transcript levels of the nuclear genes involved in chlorophyll biosynthesis were altered in the sdp mutant. Collectively, these results suggest that chloroplast-targeted SDP harboring RNA chaperone activity affects rRNA processing, chloroplast biogenesis and photosynthetic activity, which is crucial for normal growth of Arabidopsis.

Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis.

The nucleolar protein pescadillo (PES) controls biogenesis of the 60S ribosomal subunit through functional interactions with Block of Proliferation 1 (BOP1) and WD Repeat Domain 12 (WDR12) in plants. In this study, we determined protein characteristics and in planta functions of BOP1 and WDR12, and characterized defects in plant cell growth and proliferation caused by a deficiency of PeBoW (PES-BOP1-WDR12) proteins. Dexamethasone-inducible RNAi of BOP1 and WDR12 caused developmental arrest and premature senescence in Arabidopsis, similar to the phenotype of PES RNAi. Both the N-terminal domain and WD40 repeats of BOP1 and WDR12 were critical for specific associations with 60S/80S ribosomes. In response to nucleolar stress or DNA damage, PeBoW proteins moved from the nucleolus to the nucleoplasm. Kinematic analyses of leaf growth revealed that depletion of PeBoW proteins led to dramatically suppressed cell proliferation, cell expansion, and epidermal pavement cell differentiation. A deficiency in PeBoW proteins resulted in reduced cyclin-dependent kinase Type A activity, causing reduced phosphorylation of histone H1 and retinoblastoma-related (RBR) protein. PeBoW silencing caused rapid transcriptional modulation of cell-cycle genes, including reduction of E2Fa and Cyclin D family genes, and induction of several KRP genes, accompanied by down-regulation of auxin-related genes and up-regulation of jasmonic acid-related genes. Taken together, these results suggest that the PeBoW proteins involved in ribosome biogenesis play a critical role in plant cell growth and survival, and their depletion leads to inhibition of cell-cycle progression, possibly modulated by phytohormone signaling.

Cell growth defect factor1/chaperone-like protein of POR1 plays a role in stabilization of light-dependent protochlorophyllide oxidoreductase in Nicotiana benthamiana and Arabidopsis.

Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as chaperone-like protein of POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR.

Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

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.

Pescadillo plays an essential role in plant cell growth and survival by modulating ribosome biogenesis.

Pescadillo (PES) is involved in diverse cellular processes such as embryonic development, ribosomal biogenesis, cell proliferation, and gene transcription in yeast and metazoans. In this study, we characterized cellular functions of plant PES in Nicotiana benthamiana, Arabidopsis, and tobacco BY-2 cells. A GFP fusion protein of PES is predominantly localized in the nucleolus, where its localization requires the N-terminal domain of PES. Silencing of plant PES led to growth arrest and acute cell death. PES interacts with plant homologs of BOP1 and WDR12 in the nucleolus, which are also nucleolar proteins involved in ribosome biogenesis of yeast and mammals. PES, BOP1, and WDR12 cofractionated with ribosome subunits. Depletion of any of these proteins led to defective biogenesis of the 60S ribosome large subunits and disruption of nucleolar morphology. PES-deficient plant cells also exhibited delayed maturation of 25S ribosomal RNA and suppressed global translation. During mitosis in tobacco BY-2 cells, PES is associated with the mitotic microtubules, including spindles and phragmoplasts, and PES deficiency disrupted spindle organization and chromosome arrangement. Collectively, these results suggest that plant PES has an essential role in cell growth and survival through its regulation of ribosome biogenesis and mitotic progression.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

BACKGROUND: Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. RESULTS: Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. CONCLUSIONS: These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

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.

The PP2A regulatory subunit Tap46, a component of the TOR signaling pathway, modulates growth and metabolism in plants.

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway.

Silencing of Nicotiana benthamiana Neuroblastoma-Amplified Gene causes ER stress and cell death.

BACKGROUND: Neuroblastoma Amplified Gene (NAG) was identified as a gene co-amplified with the N-myc gene, whose genomic amplification correlates with poor prognosis of neuroblastoma. Later it was found that NAG is localized in endoplasmic reticulum (ER) and is a component of the syntaxin 18 complex that is involved in Golgi-to-ER retrograde transport in human cells. Homologous sequences of NAG are found in plant databases, but its function in plant cells remains unknown. RESULTS: Nicotiana benthamania Neuroblastoma-Amplified Gene (NbNAG) encodes a protein of 2,409 amino acids that contains the secretory pathway Sec39 domain and is mainly localized in the ER. Silencing of NbNAG by virus-induced gene silencing resulted in growth arrest and acute plant death with morphological markers of programmed cell death (PCD), which include chromatin fragmentation and modification of mitochondrial membrane potential. NbNAG deficiency caused induction of ER stress genes, disruption of the ER network, and relocation of bZIP28 transcription factor from the ER membrane to the nucleus, similar to the phenotypes of tunicamycin-induced ER stress in a plant cell. NbNAG silencing caused defects in intracellular transport of diverse cargo proteins, suggesting that a blocked secretion pathway by NbNAG deficiency causes ER stress and programmed cell death. CONCLUSIONS: These results suggest that NAG, a conserved protein from yeast to mammals, plays an essential role in plant growth and development by modulating protein transport pathway, ER stress response and PCD.