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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.

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.

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 .

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.

Abstract Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that D E-ETIOLATION IN THE DARK A ND Y ELLOWING IN THE LIGHT (DAY) , a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), 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 InsP6 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-InsP6-Dbp5 (LOS4 homolog) paradigm proposed in yeast. Interestingly, plant Gle1 proteins have modifications in several key residues of the InsP6 binding pocket, which reduce the basicity of the surface charge. Arabidopsis thaliana Gle1 variants containing mutations that increase the basic charge of the InsP6 binding surface show increased sensitivity to InsP6 concentrations for the stimulation of LOS4 ATPase activity in vitro. Expression of the Gle1 variants with enhanced InsP6 sensitivity rescues the mRNA export defect of the ipk1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) InsP6-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 InsP6 functions in plant growth and reproduction and that Gle1 variants with increased InsP6 sensitivity may be useful for engineering high-yielding low-phytate crops.

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.

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.

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

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.