Nitric Oxide Regulates Protein Methylation during Stress Responses in Plants.

Methylation and nitric oxide (NO)-based S-nitrosylation are highly conserved protein posttranslational modifications that regulate diverse biological processes. In higher eukaryotes, PRMT5 catalyzes Arg symmetric dimethylation, including key components of the spliceosome. The Arabidopsis prmt5 mutant shows severe developmental defects and impaired stress responses. However, little is known about the mechanisms regulating the PRMT5 activity. Here, we report that NO positively regulates the PRMT5 activity through S-nitrosylation at Cys-125 during stress responses. In prmt5-1 plants, a PRMT5C125S transgene, carrying a non-nitrosylatable mutation at Cys-125, fully rescues the developmental defects, but not the stress hypersensitive phenotype and the responsiveness to NO during stress responses. Moreover, the salt-induced Arg symmetric dimethylation is abolished in PRMT5C125S/prmt5-1 plants, correlated to aberrant splicing of pre-mRNA derived from a stress-related gene. These findings define a mechanism by which plants transduce stress-triggered NO signal to protein methylation machinery through S-nitrosylation of PRMT5 in response to environmental alterations.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output.

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

The 3' processing of antisense RNAs physically links to chromatin-based transcriptional control.

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. At Arabidopsis thaliana FLC, antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3' processing factors, reduces FLC transcription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over the FLC gene body. SDG26 interacts with the RNA 3' processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repress FLC Overall, our work provides insights into RNA-mediated chromatin silencing.

Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5.

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is involved in a multitude of biological processes in eukaryotes. Symmetric arginine dimethylation mediated by PRMT5 modulates constitutive and alternative pre-mRNA splicing of diverse genes to regulate normal growth and development in multiple species; however, the underlying molecular mechanism remains largely unknown. A genetic screen for suppressors of an Arabidopsis symmetric arginine dimethyltransferase mutant, atprmt5, identified two gain-of-function alleles of pre-mRNA processing factor 8 gene (prp8-8 and prp8-9), the highly conserved core component of the U5 small nuclear ribonucleoprotein (snRNP) and the spliceosome. These two atprmt5 prp8 double mutants showed suppression of the developmental and splicing alterations of atprmt5 mutants. In atprmt5 mutants, the NineTeen complex failed to be assembled into the U5 snRNP to form an activated spliceosome; this phenotype was restored in the atprmt5 prp8-8 double mutants. We also found that loss of symmetric arginine dimethylation of Sm proteins prevents recruitment of the NineTeen complex and initiation of spliceosome activation. Together, our findings demonstrate that symmetric arginine dimethylation has important functions in spliceosome assembly and activation, and uncover a key molecular mechanism for arginine methylation in pre-mRNA splicing that impacts diverse developmental processes.

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity.

In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting.

A systematic proteomic analysis of NaCl-stressed germinating maize seeds.

Salt (NaCl) is a common physiological stressor of plants. To better understand how germinating seeds respond to salt stress, we examined the changes that occurred in the proteome of maize seeds during NaCl-treated germination. Phenotypically, salt concentrations less than 0.2 M appear to delay germination, while higher concentrations disrupt development completely, leading to seed death. The identities of 96 proteins with expression levels altered by NaCl-incubation were established using 2-DE-MALDI-TOF-MS and 2-DE-MALDI-TOF-MS/MS. Of these 96 proteins, 79 were altered greater than twofold when incubated with a 0.2 M salt solution, while 51 were altered when incubated with a 0.1 M salt solution. According to their functional annotations in the Swiss-Prot protein-sequence databases, these proteins are mainly involved in seed storage, energy metabolism, stress response, and protein metabolism. Notably, the expression of proteins that respond to abscisic acid signals increased in response to salt stress. The results of this study provide important clues as to how NaCl stresses the physiology of germinating maize seeds.

Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing.

Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator flowering locus KH domain (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor flowering locus C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper pre-mRNA splicing that impacts diverse developmental processes.

VAL1 acts as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC.

Polycomb (PcG) silencing is crucial for development, but how targets are specified remains incompletely understood. The cold-induced Polycomb Repressive Complex 2 (PRC2) silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate PcG regulation. Association of the DNA binding protein VAL1 to FLC PcG nucleation regionis an important step. VAL1 co-immunoprecipitates APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and PRC1. Here, we show that ASAP and PRC1 are necessary for co-transcriptional repression and chromatin regulation at FLC. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulation increases at the FLC nucleation region during cold, but unlike the PRC2-delivered H3K27me3, does not spread across the locus. H2Aub thus involved in the transition to epigenetic silencing at FLC, facilitating H3K27me3 accumulation and long-term epigenetic memory. Overall, our work highlights the importance of VAL1 as an assembly platform co-ordinating activities necessary for epigenetic silencing at FLC.

Large-scale analysis of phosphorylated proteins in maize leaf.

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.

Comparative temporal analyses of the Pinus sylvestris L. var. mongolica litv. apical bud proteome from dormancy to growth.

Bud dormancy in perennial plants adapts to environmental and seasonal changes. Bud dormancy is of ecological interest because it affects forest population growth characteristics and is of economical interest because it impacts wood production levels. To understand Pinus sylvestris L. var. mongolica litv. bud-dormancy and bud-burst mechanisms, we characterized the proteomes of their apical buds at the four critical stages that occur during the dormancy-to-growth transition. Ninety-six proteins with altered expression patterns were identified using NanoLC-ESI-MS/MS. The majority of these proteins (57%) are involved in metabolic and other cellular processes. For 28% of the proteins, a function could not be assigned. However, because their expression levels changed, they may be potential candidate bud development- or dormancy-related proteins. Of the 75 non-redundant bud proteins identified, ascorbate peroxidase, pathogenesis-related protein PR-10, and heat shock proteins dramatically increased during August and November, suggesting that they may involved in the initiation of bud dormancy. Conversely, S-adenosylmethionine synthetase, abscisic acid/stress-induced proteins, superoxide dismutase (SOD), caffeoyl-CoA O-methyltransferase, actin, and type IIIa membrane protein cp-wap13 had greater expression levels during April, suggesting that they may be involved in the initiation of bud dormancy-release. Cell division cycle protein 48 and eukaryotic initiation factors 4A-15 and 4A had greater expression levels during May, suggesting that they may regulate cell proliferate and differentiation in the shoot apical meristem. These observations provide insights into the molecular mechanisms that induce or break bud dormancy.

Phosphoproteomic identification and phylogenetic analysis of ribosomal P-proteins in Populus dormant terminal buds.

To better understand the role that reversible phosphorylation plays in woody plant ribosomal P-protein function, we initiated a phosphoproteomic investigation of P-proteins from Populus dormant terminal buds. Using gel-free (in-solution) protein digestion and phosphopeptide enrichment combined with a nanoUPLC-ESI-MS/MS strategy, we identified six phosphorylation sites on eight P-proteins from Populus dormant terminal buds. Among these, six Ser sites and one Thr site were identified in the highly conserved C-terminal region of eight P-proteins of various P-protein subfamilies, including two P0, two P1, three P2 and one P3 protein. Among these, the Thr site was shown to be novel and has not been identified in any other organisms. Sequence analysis indicated that the phosphothreonine sites identified in the C-terminus of Ptr RPP2A exclusively occurred in woody species of Populus, etc. The identified phosphopeptides shared a common phosphorylation motif of (S/T)XX(D/E) and may be phosphorylated in vivo by casein kinase 2 as suggested by using Scansite analysis. Furthermore, phylogenetic analysis suggested that divergence of P2 also occurred in Populus, including type I and type II. To the best of our knowledge, this is the first systematic phosphoproteomic and phylogenetic analysis of P-proteins in woody plants, the results of which will provide a wealth of resources for future understanding and unraveling of the regulatory mechanisms of Populus P-protein phosphorylation during the maintenance of dormancy.

Large-scale Proteomic and Phosphoproteomic Analyses of Maize Seedling Leaves During De-etiolation.

De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light. During this process light, an important environmental signal, triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts, and etiolated leaves transition from the "sink" stage to the "source" stage. De-etiolation has been extensively studied in maize (Zea mays L.). However, little is known about how this transition is regulated. In this study, we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize. We identified 16,420 proteins in proteome, among which 14,168 proteins were quantified. In addition, 8746 phosphorylation sites within 3110 proteins were identified. From the combined proteomic and phosphoproteomic data, we identified a total of 17,436 proteins. Only 7.0% (998/14,168) of proteins significantly changed in abundance during de-etiolation. In contrast, 26.6% of phosphorylated proteins exhibited significant changes in phosphorylation level; these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions. Based on phosphoproteomic analysis, 34.0% (1057/3110) of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites, and 37 proteins contained more than 16 phosphorylation sites, indicating that multi-phosphorylation is ubiquitous during the de-etiolation process. Our results suggest that plants might preferentially regulate the level of posttranslational modifications (PTMs) rather than protein abundance for adapting to changing environments. The study of PTMs could thus better reveal the regulation of de-etiolation.

Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3.

New breeding technologies accelerate germplasm improvement and reduce the cost of goods in seed production1-3. Many such technologies could use in vivo paternal haploid induction (HI), which occurs when double fertilization precedes maternal (egg cell) genome loss. Engineering of the essential CENTROMERIC HISTONE (CENH3) gene induces paternal HI in Arabidopsis4-6. Despite conservation of CENH3 function across crops, CENH3-based HI has not been successful outside of the Arabidopsis model system7. Here we report a commercially operable paternal HI line in wheat with a ~7% HI rate, identified by screening genome-edited TaCENH3α-heteroallelic combinations. Unlike in Arabidopsis, edited alleles exhibited reduced transmission in female gametophytes, and heterozygous genotypes triggered higher HI rates than homozygous combinations. These developments might pave the way for the deployment of CENH3 HI technology in diverse crops.

Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation.

The ubiquitin system is crucial for the development and fitness of higher plants. De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L. (exposed to light for 1, 6, or 12 h) achieved through immunoprecipitation-based high-resolution mass spectrometry (MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites (fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones. Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.

A shotgun phosphoproteomics analysis of embryos in germinated maize seeds.

To better understand the role that reversible protein phosphorylation plays in seed germination, we initiated a phosphoproteomic investigation of embryos of germinated maize seeds. A total of 776 proteins including 39 kinases, 16 phosphatases, and 33 phosphoproteins containing 36 precise in vivo phosphorylation sites were identified. All the phosphorylation sites identified, with the exception of the phosphorylation site on HSP22, have not been reported previously (Lund et al. in J Biol Chem, 276, 29924-29929, 2001). Assayed with QRT-PCR, the transcripts of ten kinase genes were found to be dramatically up-regulated during seed germination and those of four phosphatase genes were up-regulated after germination, which indicated that reversible protein phosphorylation occurred and complex regulating networks were activated during this period. At least one-third of these phosphoproteins are key components involved in biological processes which relate to seed germination, such as DNA repair, gene transcription, RNA splicing and protein translation, suggesting that protein phosphorylation plays an important role in seed germination. As far as we know, this is the first phosphoproteomic study on a monocot and it will lay a solid foundation for further study of the molecular mechanisms of seed germination and seedling development.