Arabidopsis APPAN is a Brix family protein that is homologous to yeast Ssf1/Ssf2 and PPan in higher eukaryotes. A previous study, mostly based on physiological experiments, revealed that APPAN plays ...an essential role in female gametogenesis in plants. Here, we investigated cellular functions of APPAN, which could be the molecular basis for developmental defects in snail1/appan mutants. Virus-induced gene silencing (VIGS) of APPAN in Arabidopsis resulted in abnormal shoot apices, leading to defective inflorescences and malformed flowers and leaves. APPAN is localized in the nucleolus and co-sedimented mainly with 60 S ribosome subunit. RNA gel blot analyses showed overaccumulation of processing intermediates, particularly 35 S and P-A3, and the sequences were confirmed by circular RT-PCR. These results suggested that silencing of APPAN causes defective pre-rRNA processing. Metabolic rRNA labeling showed that APPAN depletion mainly reduced 25 S rRNA synthesis. Consistently, based on the ribosome profiling, the levels of 60 S/80 S ribosomes were significantly reduced. Finally, APPAN deficiency caused nucleolar stress with abnormal nucleolar morphology and translocation of nucleolar proteins into the nucleoplasm. Collectively, these results suggest that APPAN plays a crucial role in plant rRNA processing and ribosome biogenesis, and its depletion disrupts plant growth and development.
•Arabidopsis SNAIL1/APPAN is a Brix family protein involved in ribosome biogenesis.•APPAN silencing causes abnormal shoot apices, leading to defective inflorescences.•APPAN deficiency causes defective pre-rRNA processing, mainly reducing 25 S rRNA levels.•APPAN deficiency causes nucleolar stress with abnormal nucleolar morphology.
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.
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.
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 DE-ETIOLATION IN THE DARK AND YELLOWING 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.
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.
The target of rapamycin complex (TORC) plays a key role in plant cell growth and survival by regulating the gene expression and metabolism according to environmental information. TORC activates ...transcription, mRNA translation, and anabolic processes under favorable conditions, thereby promoting plant growth and development. Tomato fruit ripening is a complex developmental process promoted by ethylene and specific transcription factors. TORC is known to modulate leaf senescence in tomato. In this study, we investigated the function of TORC in tomato fruit ripening using virus-induced gene silencing (VIGS) of the TORC genes,
, lethal with SEC13 protein 8 (
), and regulatory-associated protein of TOR (
). Quantitative reverse transcription-polymerase chain reaction showed that the expression levels of tomato TORC genes were the highest in the orange stage during fruit development in Micro-Tom tomato. VIGS of these TORC genes using stage 2 tomato accelerated fruit ripening with premature orange/red coloring and decreased fruit growth, when control tobacco rattle virus 2 (TRV2)-myc fruits reached the mature green stage. TORC-deficient fruits showed early accumulation of carotenoid lycopene and reduced cellulose deposition in pericarp cell walls. The early ripening fruits had higher levels of transcripts related to fruit ripening transcription factors, ethylene biosynthesis, carotenoid synthesis, and cell wall modification. Finally, the early ripening phenotype in Micro-Tom tomato was reproduced in the commercial cultivar Moneymaker tomato by VIGS of the TORC genes. Collectively, these results demonstrate that TORC plays an important role in tomato fruit ripening by modulating the transcription of various ripening-related genes.
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.
The precise regulation of microRNA (miRNA) biogenesis is crucial for plant development, which requires core microprocessors and many fine tuners to coordinate their miRNA processing ...activity/specificity in fluctuating cellular environments. During de-etiolation, light triggers a dramatic accumulation of core microprocessors and primary miRNAs (pri-miRNAs) but decreases pri-miRNA processing activity, resulting in relatively constant miRNA levels. The mechanisms underlying these seemingly contradictory regulatory changes remain unclear. In this study, we identified forkhead-associated domain 2 (FHA2) as a light-stabilized suppressor of miRNA biogenesis. We found that FHA2 deficiency increased the level of mature miRNAs, accompanied by a reduction in pri-miRNAs and target mRNAs. Biochemical assays showed that FHA2 associates with the core microprocessors DCL1, HYL1, and SE, forming a complex to suppress their pri-miRNA processing activity. Further analyses revealed that FHA2 promotes HYL1 binding but inhibits the binding of DCL1-PAZ-RNase-RNA-binding domains (DCL1-PRR) to miRNAs, whereas FHA2 does not directly bind to these RNAs. Interestingly, we found that FHA2 protein is unstable in the dark but stabilized by light during de-etiolation. Consistently, disruption of FHA led to defects in light-triggered changes in miRNA expression and reduced the survival rate of de-etiolated seedlings after prolonged light deprivation. Collectively, these data suggest that FHA2 is a novel light-stabilized suppressor of miRNA biogenesis and plays a role in fine-tuning miRNA processing during de-etiolation.
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 ).
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.
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.
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