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Phosphorylation‐mediated signalling in flowering: prospects and retrospects of phosphoproteomics in crops
Abstract
Protein phosphorylation is a major post-translational modification, regulating protein function, stability, and subcellular localization. To date, annotated phosphorylation data are available mainly for model organisms and humans, despite the economic importance of crop species and their large kinomes. Our understanding of the phospho-regulation of flowering in relation to the biology and interaction between the pollen and pistil is still significantly lagging, limiting our knowledge on kinase signalling and its potential applications to crop production. To address this gap, we bring together relevant literature that were previously disconnected to present an overview of the roles of phosphoproteomic signalling pathways in modulating molecular and cellular regulation within specific tissues at different morphological stages of flowering. This review is intended to stimulate research, with the potential to increase crop productivity by providing a platform for novel molecular tools.
... This highly diverse range of PTMs includes phosphorylation, ubiquitination, SUMOylation, γ-carboxylation, poly(ADPribosyl)ation, acetylation, redox modification, methylation, glycosylation, acylation, alkylation, hydroxylation, nitration and nucleotide addition, among others (Vu et al. 2018, Arefian et al. 2021, Gough and Sadanandom 2021, Péter et al. 2021, Wang et al. 2021c). The UniProtKB/Swiss-Prot databases have identified over 450 different PTMs (Conibear 2020, Zhang and Zeng 2020, Wang et al. 2021b, which demonstrates the complexity of cellular proteomes. ...
The thiol group of cysteine (Cys) residues, often present in the active center of the protein, are of particular importance to protein function which is significantly determined by the redox state of a protein’s environment. Our knowledge of different thiol-based oxidative post-translational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last ten years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation, and S-acylation. The role of each oxiPTM depends on the redox cellular state, which, in turn, depends on cellular homeostasis under either optimal or stressful conditions. Under these conditions, the metabolism of molecules such as glutathione (GSH), NADPH, nitric oxide (NO), hydrogen sulfide (H2S), and hydrogen peroxide (H2O2) can be altered, exacerbated and, consequently, outside the cell´s control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.
Rice is a staple food crop worldwide; however, salinity stress is estimated to reduce its global production by 50%. Knowledge about initial molecular signaling and proteins associated with sensing salinity among crop plants is limited. We characterized early salt effects on the proteome and metabolome of rice tissues. Omics results were validated by western blotting and multiple reaction monitoring assays and integrated with physiological changes. We identified 8160 proteins and 2045 metabolites in rice tissues. Numerous signaling pathways were induced rapidly or partially by salinity. Combined data showed the most susceptible proteins or metabolites in each pathway that likely affected the sensitivity of rice to salinity, such as PLA1, BON3 (involved in sensing stress), SnRK2, pro-resilin, GDT1, G-proteins, calmodulin activators (Ca2+ and abscisic acid signaling), MAPK3/5, MAPKK1/3 (MAPK pathway), SOS1, ABC F/D, PIP2-7, and K+ transporter-23 (transporters), OPR1, JAR1, COL1, ABA2, and MAPKK3 (phytohormones). Additionally, our results expanded the stress-sensing function of receptor-like kinases, phosphatidylinositols, and Na+ sensing proteins (IPUT1). Combined analyses revealed the most sensitive components of signaling pathways causing salt-susceptibility in rice and suggested potential targets for crop improvement.
Rice is a model monocot plant and a major food crop worldwide that is known as the most salt-sensitive cereal. Phosphorylation of proteins regulates the stimulus perception and triggers responses; however, remains largely unknown in crops, particularly in rice under salinity. Here, we report in-depth physiological, proteomic, and phosphoproteomic kinetics of rice shoot and root tissues in response to short-term salt stress. We identified 8160 proteins, 5684 phosphopeptides that assigned to 3142 phosphoproteins. In the root, hypophosphorylation of KAT1, BORs, HAKs, NHX2, and NHX8 transporters, RBOHs and nitrate reductase implied less efficiency in ion, minerals, and ROS haemostasis, thus likely resulting in membrane damages and sensitivity of rice to salinity. Predominantly, the PKA, CDK, MAPK, and PP2A families modulated plant response through interaction with densely interconnected protein modules. We identified 17 phosphomotifs reflecting the identities of the upstream kinases. Furthermore, phylogenetic mapping of 245 identified kinases together with the kinase of known target motifs and function, expanded the biological characters of kinases in rice. We also assigned target motifs for rice kinase families, including PDK1, PKA, CAMKL, CK1, CDK, GSK, and MAPK. This study expands the set of kinase substrates and their signaling networks. Our data also assigns new stress-responsive functions to a large number of novel phosphoproteins, thus will serve as a resource for further studies of salt stress signaling in plants.
Rice is one of the most important crops worldwide. Crop production is constrained markedly, however, by abiotic stresses such as salinity. To elucidate early stress response signaling networks involved in rice, we report in this study an original quantitative proteomic analysis of the rice seedlings subjected to short-term salt stress. We detected 570 differentially regulated proteins (DRPs) in the root sample. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that DRPs of the root were mainly involved in membrane trafficking, kinase activity, and ion toxicity responses. Interactome analysis revealed the central role of root proteins involved in membrane trafficking in the early response to salinity, such as cell surface receptor-like kinases (RLKs), phosphatidylinositols (PIs), calcium-dependent protein kinases 1 and 5, calcineurin B-like protein-interacting proteins, protein phosphatase 2C (PP2C) inhibitors, and abscisic acid receptors (PYL5/10), indicating activation of S-type anion channel. Furthermore, the proteogenomic analysis revealed 128 unique genome search-specific peptides with high-quality mass spectromety (MS/MS) spectra. We identified 38 novel protein-coding genes, refined the annotation of 17 existing gene models, and suggested several novel stress-responsive proteins, such as RLK5, peroxidase 27, and growth-regulating factor 2. Novel peptides had an ortholog match in the curated protein sequence set of other plant species. In conclusion, this study identifies novel stress-responsive proteins and genes of rice, thus warrant future consideration as candidates for molecular breeding of stress-tolerant crop varieties.
Maintaining fertility in a fluctuating environment is key to the reproductive success of flowering plants. Meiosis and pollen formation are particularly sensitive to changes in growing conditions, especially temperature. We have previously identified cyclin-dependent kinase G1 (CDKG1) as a master regulator of temperature-dependent meiosis and this may involve the regulation of alternative splicing (AS), including of its own transcript. CDKG1 mRNA can undergo several AS events, potentially producing two protein variants: CDKG1L and CDKG1S, differing in their N-terminal domain which may be involved in co-factor interaction. In leaves, both isoforms have distinct temperature-dependent functions on target mRNA processing, but their role in pollen development is unknown. In the present study, we characterize the role of CDKG1L and CDKG1S in maintaining Arabidopsis fertility. We show that the long (L) form is necessary and sufficient to rescue the fertility defects of the cdkg1-1 mutant, while the short (S) form is unable to rescue fertility. On the other hand, an extra copy of CDKG1L reduces fertility. In addition, mutation of the ATP binding pocket of the kinase indicates that kinase activity is necessary for the function of CDKG1. Kinase mutants of CDKG1L and CDKG1S correctly localize to the cell nucleus and nucleus and cytoplasm, respectively, but are unable to rescue either the fertility or the splicing defects of the cdkg1-1 mutant. Furthermore, we show that there is partial functional overlap between CDKG1 and its paralog CDKG2 that could in part be explained by overlapping gene expression. © Copyright © 2020 Nibau, Dadarou, Kargios, Mallioura, Fernandez-Fuentes, Cavallari and Doonan.
As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.
Background:
Fruit skin color play important role in commercial value of cucumber, which is mainly determined by the content and composition of chlorophyll and anthocyanins. Therefore, understanding the related genes and metabolomics involved in composition of fruit skin color is essential for cucumber quality and commodity value.
Results:
The results showed that chlorophyll a, chlorophyll b and carotenoid content in fruit skin were higher in Lv (dark green skin) than Bai (light green skin) on fruit skin. Cytological observation showed more chloroplast existed in fruit skin cells of Lv. A total of 162 significantly different metabolites were found between the fruit skin of the two genotypes by metabolome analysis, including 40 flavones, 9 flavanones, 8 flavonols, 6 anthocyanins, and other compounds. Crucial anthocyanins and flavonols for fruit skin color, were detected significantly decreased in fruit skin of Bai compared with Lv. By RNA-seq assay, 4516 differentially expressed genes (DEGs) were identified between two cultivars. Further analyses suggested that low expression level of chlorophyll biosynthetic genes, such as chlM, por and NOL caused less chlorophylls or chloroplast in fruit skin of Bai. Meanwhile, a predicted regulatory network of anthocyanin biosynthesis was established to illustrate involving many DEGs, especially 4CL, CHS and UFGT.
Conclusions:
This study uncovered significant differences between two cucumber genotypes with different fruit color using metabolome and RNA-seq analysis. We lay a foundation to understand molecular regulation mechanism on formation of cucumber skin color, by exploring valuable genes, which is helpful for cucumber breeding and improvement on fruit skin color.
In Arabidopsis, CONSTANS (CO) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS1 (CO1) and CONSTANS2 (CO2). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 (co1 co2) result in a small (3 d) but significant (P<0.0001) acceleration of heading time both in PHOTOPERIOD1 (PPD1) sensitive (Ppd-A1b, functional ancestral allele) and insensitive (Ppd-A1a, functional dominant allele) backgrounds. Under short days (SD), co1 co2 mutants headed 13 d earlier than the wild type (P<0.0001) in the presence of Ppd-A1a. However, in the presence of Ppd-A1b, spikes from both genotypes failed to emerge by 180 d. These results indicate that CO1 and CO2 operate mainly as weak heading time repressors in both LD and SD. By contrast, in ppd1 mutants with loss-of-function mutations in both PPD1 homeologs, the wild type Co1 allele accelerated heading time >60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1, CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1. Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat.
Background:
Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development.
Results:
Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERKs were investigated. Gene duplication and synteny analyses and the calculation of Ka/Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana, B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERKs exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds.
Conclusion:
Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.
Photoperiod is a major environmental cue that determines the floral transition from vegetative to reproductive development in flowering plants. Arabidopsis thaliana responds to photoperiodic signals mainly through a central regulator CONSTANS (CO). Although it has been suggested that phosphorylation of CO contributes to its role in photoperiodic control of flowering, how this is regulated so far remains unknown. Here, we report that a glycogen synthase kinase-3 member, SHAGGY-like kinase 12 (SK12), plays an important role in preventing precocious flowering through phosphorylating CO. Loss of function of SK12 causes early flowering. SK12 expression in seedlings is decreased during the floral transition, and its expression in vascular tissues is required for repressing flowering. SK12 interacts with and phosphorylates CO at threonine 119, thus facilitating CO degradation. Our findings suggest that site-specific phosphorylation of CO by SK12 is critical for modulating the photoperiodic output for the floral induction in Arabidopsis .
Tyrosine phosphorylation constitutes up to 5% of the total phophoproteome. However, only limited studies are available on protein tyrosine kinases (PTKs) that catalyze protein tyrosine phosphorylation in plants. In this study, domain analysis of the 27 annotated PTK genes in rice genome led to the identification of 18 PTKs with tyrosine kinase domain. The kinase domain of rice PTKs shared high homology with that of dual specificity kinase BRASSINOSTEROID- INSENSITIVE 1 (BRI1) of Arabidopsis. In phylogenetic analysis, rice PTKs clustered with receptor-like cytoplasmic kinases-VII (RLCKs-VII) of Arabidopsis. mRNAseq analysis using Genevestigator revealed that rice PTKs except PTK9 and PTK16 express at moderate to high level in most tissues. PTK16 expression was highly abundant in panicle at flowering stage. mRNAseq data analysis led to the identification of drought, heat, salt, and submergence stress regulated PTK genes in rice. PTK14 was upregulated under all stresses. qRT-PCR analysis also showed that all PTKs except PTK10 were significantly upregulated in root under osmotic stress. Tissue specificity and abiotic stress mediated differential regulation of PTKs suggest their potential role in development and stress response of rice. The candidate dual specificity PTKs identified in this study paves way for molecular analysis of tyrosine phosphorylation in rice.
Flowering time is of great significance for crop reproduction, yield, and regional adaptability, which is intricately regulated by various environmental cues and endogenous signals. Genetic approaches in Arabidopsis have revealed the elaborate underlying mechanisms of sensing the dynamic change of photoperiod via a coincidence between light signaling and circadian clock, the cellular time keeping system, to precisely control photoperiodic flowering time, and many other signaling pathways including internal hormones and external temperature cues. Extensive studies in rice (Oryza sativa.), one of the short-day plants (SDP), have uncovered the multiple major genetic components in regulating heading date, and revealed the underlying mechanisms for regulating heading date. Here we summarize the current progresses on the molecular basis for rice heading date control, especially focusing on the integration mechanism between photoperiod and circadian clock, and epigenetic regulation and heading procedures in response to abiotic stresses.
The temporary callose layer surrounding the tetrads of microspores is critical for male gametophyte development in flowering plants, as abnormal callose deposition can lead to microspore abortion. A sophisticated signaling network regulates callose biosynthesis, but these pathways are poorly understood. In this study, we characterized a rice male sterile mutant, oslecrk5, which showed defective callose deposition during meiosis. OsLecRK5 encodes a plasma membrane (PM)-localized lectin receptor like kinase, which can form dimer with itself. Moreover, normal anther development requires the K-phosphorylation site (a conserved residue at the ATP-binding site) of OsLecRK5. In vitro assay showed that OsLecRK5 phosphorylates the callose synthesis enzyme UGP1, enhancing callose biosynthesis during anther development. Together, our results demonstrated that PM-localized OsLecRK5 phosphorylates UGP1 and promotes its activity in callose biosynthesis in rice. This is the first evidence that an RLK positively regulates callose biosynthesis.
Arabidopsis thaliana MUT9-like kinases (MLKs), a family of the plant-specific casein kinase 1 (CK1), have been implicated collectively in multiple biological processes including flowering. Three of the four MLKs (MLK1/2/4) have been characterized, however, little is known about MLK3, the most divergent member of MLKs. Here, we demonstrated that disruption of MLK3 transcript in mlk3 caused early flowering with retarded leaf growth under long-day conditions. In vitro kinase assay showed the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3) and mutation of a conserved residue (K146R) abolished the catalytic activity. Ectopic expression of MLK3 but not MLK3(K146R) rescued the morphological defects of mlk3, indicating that an intact MLK3 is critical for maintaining proper flowering time. Transcriptomic analysis revealed that the floral repressor FLOWERING LOCUS C (FLC) was down-regulated significantly in mlk3, suggesting that MLK3 negatively regulates flowering. Hence, MLK3 plays a role in repressing the transition from vegetative to reproductive phase in A. thaliana. This study sheds light on the delicate control of flowering time by A. thaliana CK1 specific to the plant kingdom.
Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy³. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release⁴. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE1¹, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.
Plants are essential for life and are extremely diverse organisms with unique molecular capabilities¹. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.
Background: In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice.
Results: Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes.
Conclusions: In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.
As an important post-translational modification (PTM), protein phosphorylation is involved in the regulation of almost all of biological processes in eukaryotes. Due to the rapid progress in mass spectrometry-based phosphoproteomics, a large number of phosphorylation sites (p-sites) have been characterized but remain to be curated. Here, we briefly summarized the current progresses in the development of data resources for the collection, curation, integration and annotation of p-sites in eukaryotic proteins. Also, we designed the eukaryotic phosphorylation site database (EPSD), which contained 1 616 804 experimentally identified p-sites in 209 326 phosphoproteins from 68 eukaryotic species. In EPSD, we not only collected 1 451 629 newly identified p-sites from high-throughput (HTP) phosphoproteomic studies, but also integrated known p-sites from 13 additional databases. Moreover, we carefully annotated the phosphoproteins and p-sites of eight model organisms by integrating the knowledge from 100 additional resources that covered 15 aspects, including phosphorylation regulator, genetic variation and mutation, functional annotation, structural annotation, physicochemical property, functional domain, disease-associated information, protein-protein interaction, drug-target relation, orthologous information, biological pathway, transcriptional regulator, mRNA expression, protein expression/proteomics and subcellular localization. We anticipate that the EPSD can serve as a useful resource for further analysis of eukaryotic phosphorylation. With a data volume of 14.1 GB, EPSD is free for all users at http://epsd.biocuckoo.cn/.
The ability to sense environmental temperature and to coordinate growth and development accordingly, is critical to the reproductive success of plants. Flowering time is regulated at the level of gene expression by a complex network of factors that integrate environmental and developmental cues. One of the main players, involved in modulating flowering time in response to changes in ambient temperature is FLOWERING LOCUS M (FLM). FLM transcripts can undergo extensive alternative splicing producing multiple variants, of which FLM-β and FLM-δ are the most representative. While FLM-β codes for the flowering repressor FLM protein, translation of FLM-δ has the opposite effect on flowering. Here we show that the cyclin-dependent kinase G2 (CDKG2), together with its cognate cyclin, CYCLYN L1 (CYCL1) affects the alternative splicing of FLM, balancing the levels of FLM-β and FLM-δ across the ambient temperature range. In the absence of the CDKG2/CYCL1 complex, FLM-β expression is reduced while FLM-δ is increased in a temperature dependent manner and these changes are associated with an early flowering phenotype in the cdkg2 mutant lines. In addition, we found that transcript variants retaining the full FLM intron 1 are sequestered in the cell nucleus. Strikingly, FLM intron 1 splicing is also regulated by CDKG2/CYCL1. Our results provide evidence that temperature and CDKs regulate the alternative splicing of FLM, contributing to flowering time definition.
Control of flowering time has been a major focus of comparative genetic analyses in plant development. This study reports on a forward genetic approach to define previously uncharacterized components of flowering control pathways in the long-day legume, pea (Pisum sativum L.). We isolated two complementation groups of late-flowering mutants in pea that define two uncharacterized loci, LATE BLOOMER 3 (LATE3) and LATE4, and describe their diverse effects on vegetative and reproductive development. A map-based comparative approach was employed to identify the underlying genes for both loci, revealing that that LATE3 and LATE4 are orthologs of CYCLIN DEPENDENT KINASE8 (CDK8) and CYCLIN C1 (CYCC1), components of the CDK8 kinase module of the Mediator complex, which is a deeply conserved regulator of transcription in eukaryotes. We confirm the genetic and physical interaction of LATE3 and LATE4, and show that they contribute to the transcriptional regulation of key flowering genes, including the induction of the florigen gene FTa1 and repression of the floral repressor LF. Our results establish the conserved importance of the CDK8 module in plants and provide evidence for the function of CYCC1 orthologs in the promotion of flowering and the maintenance of normal reproductive development.
Background:
The receptor-like kinase FEROINA (FER) plays a crucial role in controlling plant vegetative growth partially by sensing the rapid alkalinization factor (RALF) peptide. However, the role of RALF1-FER in the vegetative-reproductive growth transition remains unknown. Here, we analyze the mechanism through which FER affects the flowering time in Arabidopsis.
Results:
We found that the FER mRNA levels exhibit an oscillating pattern with a diurnal rhythm and that the clock oscillator CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) up-regulates the expression of FER by associating with its chromatin. In addition, FER expression is regulated by clock genes, and FER also modulates the expression patterns of clock genes. Consistent with its gene expression pattern, FER positively regulates flowering by modulating the transcript accumulation and mRNA alternative splicing of certain flowering-related genes, including FLOWERING LOCUS C (FLC) and its homolog MADS AFFECTING FLOWERING (MAF). However, the RALF1 ligand negatively regulates flowering compared with FER.
Conclusions:
We found that FER, which is up-regulated by CCA1, controls the flowering time by regulating the transcript accumulation and mRNA alternative splicing (AS) of some important flowering genes, and these findings link FER to the floral transition.
Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination. Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture. The mutant gene encodes a novel plasma membrane‐localized legume‐lectin receptor kinase that we named OsLecRK‐S.7. OsLecRK‐S.7 was expressed at different levels in all tested tissues and throughout anther development. In vitro kinase assay showed OsLecRK‐S.7 capable of autophosporylation. Mutation in s13283 (E560K) and mutation of the conserved ATP binding site (K418E) both knocked out the kinase activity. Mass spectrometry showed Thr376, Ser378, Thr386, Thr403, and Thr657 to be the autophosphorylation sites. Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees, but did not abolish the gene function in fertility complementation. oslecrk‐s.7 mutant plant overexpressing OsLecRK‐S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers, and these phenotypes were abolished by E560K or K418E mutation. The results indicated that OsLecRK‐S.7 was a key regulator of pollen development. This article is protected by copyright. All rights reserved.
Verticillium wilt is a plant vascular disease causing severe yield and quality losses in many crops and is caused by the soil-borne plant pathogenic fungus Verticillium dahliae. To investigate the molecular mechanisms of the cotton–V. dahliae interaction, a time-course phosphoproteomic analysis of roots of susceptible and resistant cotton lines in response to V. dahliae was performed. In total, 1716 unique phosphoproteins were identified in the susceptible (S) and resistant (R) cotton lines. Of these, 359 phosphoproteins were significantly different in R1 (1 day after V. dahliae inoculation) vs R0 (mock) group and 287 phosphoproteins in R2 (3 days after V. dahliae inoculation) vs R0 group. Moreover, 263 proteins of V. dahliae-regulated phosphoproteins were significantly changed in S1 (1 day after V. dahliae inoculation) vs S0 (mock) group and 197 proteins in S2 (3 days after V. dahliae inoculation) vs S0 group. Thirty phosphoproteins were significantly changed and common to the resistant and susceptible cotton lines following inoculation with V. dahliae. Specifically, 92 phosphoproteins were shared in both in R1 vs R0 and R2 vs R0 but not in susceptible cotton lines. There were 38 common phosphoproteins shared in both S1 vs S0 and S2 vs S0 but not in resistant cotton lines. GO terms and Kyoto Encyclopedia of Genes and Genomes pathway analyses displayed an abundance of known and novel phosphoproteins related to plant–pathogen interactions, signal transduction, and metabolic processes, which were correlated with resistance against fungal infection. These data provide new perspectives and inspiration for understanding molecular defense mechanisms of cotton roots against V. dahliae infection.
Changes in light quality indicative of competition for this essential resource influence plant growth and developmental transitions; however, little is known about neighbor proximity-induced acceleration of reproduction. Phytochrome B (phyB) senses light cues from plant competitors, ultimately leading to the expression of the floral inducers FLOWERING LOCUS T (FT) and TWIN SISTER of FT (TSF). Here we show that PHYTOCHROME INTERACTING FACTORs 4, 5 and 7 (PIF4, PIF5 and PIF7) mediate neighbor proximity-induced flowering, with PIF7 playing a prominent role. These transcriptional regulators act directly downstream of phyB to promote expression of FT and TSF. Neighbor proximity enhances PIF accumulation towards the end of the day, coinciding with enhanced floral inducer expression. We present evidence supporting direct PIF-regulated TSF expression. The relevance of our findings is illustrated by the prior identification of FT, TSF and PIF4 as loci underlying flowering time regulation in natural conditions. In plants, phytochrome B senses light cues indicative of neighbouring competitors leading to accelerated flowering. Here the authors show that PHYTOCHROME INTERACTING FACTORs 4, 5 and 7 (PIF4, PIF5 and PIF7) mediate neighbor proximity-induced flowering acting downstream of phyB to promote expression of FT and TSF.
Background:
Organic acid secretion is a widespread physiological response of plants to alkalinity. However, the characteristics and underlying mechanism of the alkali-induced secretion of organic acids are poorly understood.
Results:
Oxalate was the main organic acid synthesized and secreted in grapevine (a hybrid of Vitis amurensis, V. berlandieri and V. riparia) roots, while acetate synthesis and malate secretion were also promoted under NaHCO3 stress. NaHCO3 stress enhanced the H+ efflux rate of grapevine roots, which is related to the plasma membrane H+-ATPase activity. Transcriptomic profiling revealed that carbohydrate metabolism was the most significantly altered biological process under NaHCO3 stress; a total of seven genes related to organic acid metabolism were significantly altered, including two phosphoenolpyruvate carboxylases and phosphoenolpyruvate carboxylase kinases. Additionally, the expression levels of five ATP-binding cassette transporters, particularly ATP-binding cassette B19, and two Al-activated malate transporter 2 s were substantially upregulated by NaHCO3 stress. Phosphoproteomic profiling demonstrated that the altered phosphoproteins were primarily related to binding, catalytic activity and transporter activity in the context of their molecular functions. The phosphorylation levels of phosphoenolpyruvate carboxylase 3, two plasma membrane H+-ATPases 4 and ATP-binding cassette B19 and pleiotropic drug resistance 12 were significantly increased. Additionally, the inhibition of ethylene synthesis and perception completely blocked NaHCO3-induced organic acid secretion, while the inhibition of indoleacetic acid synthesis reduced NaHCO3-induced organic acid secretion.
Conclusions:
Our results demonstrated that oxalate was the main organic acid produced under alkali stress and revealed the necessity of ethylene in mediating organic acid secretion. Additionally, we further identified several candidate genes and phosphoproteins responsible for organic acid metabolism and secretion.
Heat stress is a serious and widespread threat to the quality and yield of many crop species, including grape (Vitis vinifera L.), which is cultivated worldwide. Here, we conducted phosphoproteomic and acetylproteomic analyses of leaves of grape plants cultivated under four distinct temperature regimes. The phosphorylation or acetylation of a total of 1011 phosphoproteins with 1828 phosphosites and 96 acetyl proteins with 148 acetyl sites changed when plants were grown at 35 °C, 40 °C, and 45 °C in comparison with the proteome profiles of plants grown at 25 °C. The greatest number of changes was observed at the relatively high temperatures. Functional classification and enrichment analysis indicated that phosphorylation, rather than acetylation, of serine/arginine-rich splicing factors was involved in the response to high temperatures. This finding is congruent with previous observations by which alternative splicing events occurred more frequently in grapevine under high temperature. Changes in acetylation patterns were more common than changes in phosphorylation patterns in photosynthesis-related proteins at high temperatures, while heat-shock proteins were associated more with modifications involving phosphorylation than with those involving acetylation. Nineteen proteins were identified with changes associated with both phosphorylation and acetylation, which is consistent with crosstalk between these posttranslational modification types.
Extensive research over several decades in plant light signaling mediated by photoreceptors has identified the molecular mechanisms for how phytochromes regulate photomorphogenic development, which includes degradation of phytochrome-interacting factors (PIFs) and inactivation of COP1-SPA complexes with the accumulation of master transcription factors for photomorphogenesis, such as HY5. However, the initial biochemical mechanism for the function of phytochromes has not been fully elucidated. Plant phytochromes have long been known as phosphoproteins, and a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified. However, there is no report thus far of a protein kinase that acts on phytochromes. On the other hand, plant phytochromes have been suggested as autophosphorylating serine/threonine protein kinases, proposing that the kinase activity might be important for their functions. Indeed, the autophosphorylation of phytochromes has been reported to play an important role in the regulation of plant light signaling. More recently, evidence that phytochromes function as protein kinases in plant light signaling has been provided using phytochrome mutants displaying reduced kinase activities. In this review, we highlight recent advances in the reversible phosphorylation of phytochromes and their functions as protein kinases in plant light signaling.
Background
Salinity is a major abiotic stress that limits the growth, productivity, and geographical distribution of plants. A comparative proteomics and gene expression analysis was performed to better understand salinity tolerance mechanisms in chickpea.
Results
Ten days of NaCl treatments resulted in the differential expression of 364 reproducible spots in seedlings of two contrasting chickpea genotypes, Flip 97-43c (salt tolerant, T1) and Flip 97-196c (salt susceptible, S1). Notably, after 3 days of salinity, 80% of the identified proteins in T1 were upregulated, while only 41% in S2 had higher expression than the controls. The proteins were classified into eight functional categories, and three groups of co-expression profile. The second co-expressed group of proteins had higher and/or stable expression in T1, relative to S2, suggesting coordinated regulation and the importance of some processes involved in salinity acclimation. This group was mainly enriched in proteins associated with photosynthesis (39%; viz. chlorophyll a-b binding protein, oxygen-evolving enhancer protein, ATP synthase, RuBisCO subunits, carbonic anhydrase, and fructose-bisphosphate aldolase), stress responsiveness (21%; viz. heat shock 70 kDa protein, 20 kDa chaperonin, LEA-2 and ascorbate peroxidase), and protein synthesis and degradation (14%; viz. zinc metalloprotease FTSH 2 and elongation factor Tu). Thus, the levels and/or early and late responses in the activation of targeted proteins explained the variation in salinity tolerance between genotypes. Furthermore, T1 recorded more correlations between the targeted transcripts and their corresponding protein expression profiles than S2.
Conclusions
This study provides insight into the proteomic basis of a salt-tolerance mechanism in chickpea, and offers unexpected and poorly understood molecular resources as reliable starting points for further dissection.
Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants' signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.
In flowering plants, extensive male-female interactions during pollen germination on the stigma, pollen tube growth and guidance in the transmitting tract, and pollen tube reception by the female gametophyte are required for successful double fertilization in which various signaling cascades are involved. Peptide/receptor-like kinase-mediated signaling has been found playing important roles in these male-female interactions. Here, we mainly summarized the progress made on the regulatory roles of peptide/receptor-like kinase-mediated signaling pathways in four critical stages during reproduction in higher plants.
The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.
O-GlcNAcylation and phosphorylation are two posttranslational modifications that antagonistically regulate protein function. However, the regulation of and the cross talk between these two protein modifications are poorly understood in plants. Here we investigated the role of O-GlcNAcylation during vernalization, a process whereby prolonged cold exposure promotes flowering in winter wheat (Triticum aestivum), and analyzed the dynamic profile of O-GlcNAcylated and phosphorylated proteins in response to vernalization. Altering O-GlcNAc signaling by chemical inhibitors affected the vernalization response, modifying the expression of VRN genes and subsequently affecting flowering transition. Over a vernalization time-course, O-GlcNAcylated and phosphorylated peptides were enriched from winter wheat plumules by Lectin weak affinity chromatography and iTRAQ-TiO2, respectively. Subsequent mass spectrometry and gene ontology term enrichment analysis identified 168 O-GlcNAcylated proteins that are mainly involved in responses to abiotic stimulus and hormones, metabolic processing, and gene expression; and 124 differentially expressed phosphorylated proteins that participate in translation, transcription, and metabolic processing. Of note, 31 vernalization-associated proteins were identified that carried both phosphorylation and O-GlcNAcylation modifications, of which the majority (97%) exhibited the coexisting module and the remainder exhibited the potential competitive module. Among these, TaGRP2 was decorated with dynamic O-GlcNAcylation (S87) and phosphorylation (S152) modifications, and the mutation of S87 and S152 affected the binding of TaGRP2 to the RIP3 motif of TaVRN1 in vitro. Our data suggest that a dynamic network of O-GlcNAcylation and phosphorylation at key pathway nodes regulate the vernalization response and mediate flowering in wheat.
The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.
In flowering plants, pollen tubes undergo tip growth to deliver two nonmotile sperm to the ovule where they fuse with an egg and central cell to achieve double fertilization. This extended journey involves rapid growth and changes in gene activity that manage compatible interactions with at least seven different cell types. Nearly half of the genome is expressed in haploid pollen, which facilitates genetic analysis, even of essential genes. These unique attributes make pollen an ideal system with which to study plant cell–cell interactions, tip growth, cell migration, the modulation of cell wall integrity, and gene expression networks. We highlight the signaling systems required for pollen tube navigation and the potential roles of Ca2+ signals. The dynamics of pollen development make sexual reproduction highly sensitive to heat stress. Understanding this vulnerability may generate strategies to improve seed crop yields that are under threat from climate change. Expected final online publication date for the Annual Review of Plant Biology Volume 70 is April 29, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Maize originated as a tropical plant that required short days to transition from vegetative to reproductive development. ZmCCT10 [CO, CONSTANS, CO-LIKE and TIMING OF CAB1 (CCT) transcription factor family] is a regulator of photoperiod response and was identified as a major QTL controlling photoperiod sensitivity in maize. We modulated expression of ZmCCT10 in transgenic maize using two constitutive promoters with different expression levels. Transgenic plants over expressing ZmCCT10 with either promoter were delayed in their transition from vegetative to reproductive development but were not affected in their switch from juvenile-to-adult vegetative growth. Strikingly, transgenic plants containing the stronger expressing construct had a prolonged period of vegetative growth accompanied with dramatic modifications to plant architecture that impacted both vegetative and reproductive traits. These plants did not produce ears, but tassels were heavily branched. In more than half of the transgenic plants, tassels were converted into a branched leafy structure resembling phyllody, often composed of vegetative plantlets. Analysis of expression modules controlling the floral transition and meristem identity linked these networks to photoperiod dependent regulation, whereas phase change modules appeared to be photoperiod independent. Results from this study clarified the influence of the photoperiod pathway on vegetative and reproductive development and allowed for the fine-tuning of the maize flowering time model.
Tobacco mosaic virus (TMV) is a common source of biological stress that significantly affects plant growth and development. It is also useful as a model in studies designed to clarify the mechanisms involved in plant viral disease. Plant responses to abiotic stress were recently reported to be regulated by complex mechanisms at the post-translational modification (PTM) level. Protein phosphorylation is one of the most widespread and major PTMs in organisms. Using immobilized metal ion affinity chromatography (IMAC) enrichment, high-pH C18 chromatography fraction, and high-accuracy mass spectrometry (MS), a set of proteins and phosphopeptides in both TMV-infected tobacco and control tobacco were identified. A total of 4905 proteins and 3998 phosphopeptides with 3063 phosphorylation sites were identified. These 3998 phosphopeptides were assigned to 1311 phosphoproteins, as some proteins carried multiple phosphorylation sites. Among them, 530 proteins and 337 phosphopeptides corresponding to 277 phosphoproteins differed between the two groups. There were 43 upregulated phosphoproteins, including phosphoglycerate kinase, pyruvate phosphate dikinase, protein phosphatase 2C, and serine/threonine protein kinase. To the best of our knowledge, this is the first phosphoproteomic analysis of leaves from a tobacco cultivar, K326. The results of this study advance our understanding of tobacco development and TMV action at the protein phosphorylation level.
Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5,500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. In particular, the activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms.
Plants have evolved specific photoreceptors that capture informational cues from sunlight. The phytochrome, cryptochrome, and UVR8 photoreceptors perceive red/far-red, blue/UV-A, and UV-B light, respectively, and control overlapping photomorphogenic responses important for plant growth and development. A major repressor of such photomorphogenic responses is the E3 ubiquitin ligase formed by CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSOR OF PHYA-105 (SPA) proteins, which acts by regulating the stability of photomorphogenesis-promoting transcription factors. The direct interaction of light-activated photoreceptors with the COP1/SPA complex represses its activity via nuclear exclusion of COP1, disruption of the COP1-SPA interaction, and/or SPA protein degradation. This process enables plants to integrate different light signals at the level of the COP1/SPA complex to enact appropriate photomorphogenic responses according to the light environment.
Non-availability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NP) and phosphoproteins (NPPs) execute crucial cellular functions that form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. Four-week-old seedlings of a grain legume, chickpea, were subjected to gradual dehydration and nuclear proteins were extracted from unstressed control as well as from 72 and 144 h stressed tissues. We identified 4832 NPs and 478 phosphosites, corresponding to 299 unique NPPs involved in multivariate cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases and transcription factors, besides 660 uncharacterised proteins. Spliceosome complex and splicing related proteins were dominant among differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also, regulatory proteins of key processes viz., protein degradation, regulation of flowering time and circadian clock were observed to undergo dehydration-induced dephosphorylation. The characterization of novel regulatory proteins would provide new insights into stress adaptation and enable directed genetic manipulations for developing climate-resilient crops.
Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9‐LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with transcriptional repression of euchromatic and heterochromatic loci. This study demonstratesthat mlk4‐3, aT‐DNA insertion mutant of MLK4, flowered late and overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3 at Thr3in vitro and in vivo, and thecatalytic activity required the conserved lysine residue K175. The mutation K175Rof MLK4failed to restore the H3T3ph level or complement the phenotypic defects of mlk4‐3. The FLC/MAF‐clade genes includingFLC, MAF4 and MAF5 were significantly up‐regulated in mlk4‐3. The double mutant mlk4‐3 flc‐3 flowered earlier than mlk4‐3, suggesting functional FLC is crucial for flowering repression in mlk4‐3. ChIP assay showed that MLK4 bound to FLC/MAF chromatin and the H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. Consistently, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4‐3, but diminished in 35S::MLK4(K175R)/mlk4‐3plants. Moreover, the amount of RNA Pol II deposited at FLC/MAFwas clearly enrichedin mlk4‐3 relative to wild type. Therefore, MLK4‐dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of flowering negative regulatorFLC/MAF. This study sheds light on the delicate control of flowering by the plant‐specific CK1, MLK4,via posttranslational modification of histone H3.
Abiotic stresses are the prime factors that affect the growth of all plants. With current trends in global climate change further exacerbating the situation, its effects on plants, especially crops, could be catastrophic. In this regard, any increment in our understanding as to how plant growth is maintained under unfavorable environments, will be highly desirable and significant. Environmental stimuli are perceived in plant cells through appropriate receptors, which activate downstream signal transduction pathways, eventually leading to an appropriate response that aid the plant's survival or completion of its reproductive cycle. These signal transduction machineries are multigenic, comprising numerous signaling molecules working in an orchestrated manner. The two component system (TCS) is one of the most important signaling machinery deployed by plants to sense the environmental cues. TCS is also involved in other vital cellular processes such as the response to cytokinins and ethylene. TCS is a multi‐step phosphorelay (MSP) system composed of three partners, which are histidine kinases (HKs) working as receptors, histidine containing phosphotransfer proteins (HPTs; serving as shuttle proteins) and response regulators (RRs) functioning as output proteins. Sensory HKs are one of the important classes of protein kinase, which function as a sensor in the signaling cascade. Knowledge gained from the model plant Arabidopsis confirms the role of these protein kinases in stress and hormone signaling. Extensive functional diversity of these HKs has been reported in other plants as well. Furthermore, downstream signal partners, HPTs and RRs also take part in the aforesaid bioprocesses. Altogether, TCS signaling machinery, comprising a complex network of HKs, HPTs, and RRs, play a crucial role in governing various cellular processes under normal as well as stress conditions. In this chapter, we summarize the role of TCS circuitry in plant stress signaling and provide a comparative account of their roles among a set of diverse monocot and dicot plant species.
Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein–protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.
O-Phosphorylation (phosphorylation of the hydroxyl-group of S, T, and Y residues) is among the first described and most thoroughly studied posttranslational modification (PTM). Y-Phosphorylation, catalyzed by Y-kinases, is a key step in both signal transduction and regulation of enzymatic activity in mammalian systems. Canonical Y-kinase sequences are absent from plant genomes/kinomes, often leading to the assumption that plant cells lack O-phospho-l-tyrosine (pY). However, recent improvements in sample preparation, coupled with advances in instrument sensitivity and accessibility, have led to results that unequivocally disproved this assumption. Identification of hundreds of pY-peptides/proteins, followed by validation using genomic, molecular, and biochemical approaches, implies previously unappreciated roles for this "animal PTM" in plants. Herein, we review extant results from studies of pY in plants and propose a strategy for preparation and analysis of pY-peptides that will allow a depth of coverage of the plant pY-proteome comparable to that achieved in mammalian systems.
Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L‐type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell‐specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co‐receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female–male gametophyte interactions during plant fertilisation.
Phosphorylation of proteins is the most dynamic protein modification, and its analysis aids in determining the functional and regulatory principles of important cellular pathways. The legumes constitute the third largest family of higher plants, Fabaceae, comprising about 20,000 species and are second to cereals in agricultural importance on the basis of global production. Therefore, an understanding of the developmental and adaptive processes of legumes demands identification of their regulatory components. The most crucial signature of the legume family is the symbiotic nitrogen fixation, which makes this fascinating and interesting to investigate phosphorylation events. The research on protein phosphorylation in legumes has been focused primarily on two model species, Medicago truncatula and Lotus japonicus. The development of reciprocal research in other species, particularly the crops, is lagging behind which has limited its beneficial uses in agricultural productivity. In this chapter, we outline the titanium dioxide-based enrichment of phosphopeptides for nuclear proteome analysis of a grain legume, chickpea.
Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.
Rice ( Oryza sativa ) is a facultative short‐day ( SD ) plant, flowering early under SD and late under long‐day ( LD ) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD . Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post‐transcriptional level.
We found that constitutive expression of Ghd7 delays flowering in the wild‐type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co‐expressing OsGIGANTEA ( Os GI ) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and Os GI play antagonist roles in regulating Ghd7 protein stability and flowering time.
We show that Os PHYA , Os PHYB and Os GI could directly interact with Ghd7. Interestingly, Os PHYA and Os PHYB could inhibit the interaction between Os GI and Ghd7, thus helping to stabilize Ghd7 protein.
Our results revealed a new level of Ghd7 regulation by phytochromes and Os GI in photoperiodic control of flowering in rice.
In flowering plants, pollen development is a critical step for reproductive success and necessarily involves complex genetic regulatory networks. Calcium-dependent protein kinases (CPKs) are plant-specific calcium sensors involved in the regulation of plant development and adaption to the environment; however, whether they play a role in regulating male reproduction remains elusive. Here, we found that the knockdown of spikelet-specific OsCPK21 causes pollen abortion in OsCPK21-RNAi transgenic plants. Severe defects in pollen development initiated at stage 10 of anther development and simultaneous cell death occurred in the pollen cells of OsCPK21-RNAi plants. Microarray analysis and qRT-PCR revealed that the transcription of OsCPK21 is coordinated with that of MIKC*-type MADS box transcription factors OsMADS62, OsMADS63, and OsMADS68 during rice anther development. We further showed that OsCPK21 indirectly up-regulates the transcription of OsMADS62, OsMADS63, and OsMADS68 through the potential MYB binding site, DRE/CRT element, and/or new ERF binding motif localised in the promoter region of these three MADS genes. These findings suggest that OsCPK21 plays an essential role in pollengenesis, possibly via indirectly regulating the transcription of MIKC*-type MADS box proteins.
EARLY FLOWERING3 (ELF3) functions as a nighttime repressor required for sustaining circadian rhythms and coordinating growth and development in various plant species. The rice genome carries two ELF3 homologs, namely OsELF3-1 and OsELF3-2. Previous studies have suggested that OsELF3-1 has a predominant role in controlling rice photoperiodic flowering, while also contributing to the transcriptional regulation of rice floral regulators expressed in the morning. However, OsELF3-1 has not been functionally characterized. Here, we observed that the oself3-1 mutation suppresses the photoperiod-insensitive early flowering of photoperiod sensitivity5 (se5), which is a chromophore-deficient rice mutant. Detailed analyses of the se5oself3-1 double mutant revealed the recovery of the phytochrome-dependent expression of Grain number, plant height, and heading date7 (Ghd7), a floral repressor, and Light-harvesting chlorophyll a/b binding protein (Lhcb) genes. Although the oself3-1 mutation recovered Ghd7 expression in the se5 background, there was a lack of Ghd7 expression in the phyAphyBphyC triple mutant background. These observations suggest OsELF3-1 represses Ghd7 expression by inhibiting the phytochrome signaling pathway. Comparative genome analyses indicated that OsELF3-1 was produced via gene duplication events in Oryza species, and that it is expressed throughout the day. A comparison between the oself3-1 mutant and transgenic rice lines in which OsELF3-1 and OsELF3-2 are simultaneously silenced uncovered a role for OsELF3-1 in addition to the canonical ELF3 function as an evolutionarily conserved role for a nighttime repressor that regulates the rice circadian clock. Our study confirmed that an ELF3 paralog, OsELF3-1, had a unique role involving the suppression of phytochrome signaling.
Arabidopsis RACK1 (Receptors for Activated C Kinase 1) are versatile scaffold proteins that have been shown to be involved in the regulation of plant response to plant hormones including auxin, ABA, gibberellin and brassinosteroid, but not ethylene. By characterizing the double and triple mutants of RACK1 genes, we found that rack1 mutants showed reduced sensitivity to ethylene. By characterizing double and high order mutants generated between ein2, a loss-of-function mutant of the key ethylene signaling regulator gene EIN2 (Ethylene INsensitive 2), and rack1 mutants, we found that loss-of-function of EIN2 partially recovered some phenotypes observed in the rack1 mutants, such as low-fertility and reduced root length and rosette size. On the other hand, the ein2 rack1 mutants produced more rosette leaves, and flowered late when compared with ein2 and the corresponding rack1 mutants. We also found that the curled leaves and twisted petioles phenotypes observed in the ein2 mutants were enhanced in the ein2 rack1 mutants. However, assays in yeast indicated that EIN2 may not physically interact with RACK1. On the other hand, RT-PCR results showed that the expression level of EIN2 was reduced in the rack1 mutants. Taken together, our results suggest that RACKl may integrate ethylene signaling to regulate plant growth and development in Arabidopsis.
Plants have evolved many receptor‐like cytoplasmic kinases (RLCKs) to modulate their growth, development, and innate immunity. Broad‐Spectrum Resistance 1 (BSR1) encodes a rice RLCK whose overexpression confers resistance to multiple diseases, including fungal rice blast and bacterial leaf blight; however, the mechanisms underlying the resistance remain largely unknown. In the present study, we report that BSR1 is a functional protein kinase that autophosphorylates and transphosphorylates an artificial substrate, in vitro. Although BSR1 is classified as a serine/threonine kinase, it was shown to autophosphorylate on tyrosine as well as on serine/threonine residues when expressed in bacteria, demonstrating that it is a dual‐specificity kinase. Protein kinase activity was found to be indispensable for resistance to rice blast and leaf blight in BSR1‐overexpressing plants. Importantly, tyrosine phosphorylation of BSR1 was critical for proper localization of BSR1 in rice cells and played a crucial role in BSR1‐mediated resistance to multiple diseases, as evidenced by compromised disease resistance in transgenic plants overexpressing a mutant BSR1 in which Tyr‐63 was substituted with Ala. Overall, our data indicate that BSR1 is a nonreceptor dual‐specificity kinase and that both tyrosine and serine/threonine kinase activities are critical for the normal functioning of BSR1 in the resistance to multiple pathogens. Our results support the notion that tyrosine phosphorylation plays a major regulatory role in the transduction of defense signals from cell‐surface receptor complexes to downstream signaling components in plants.
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Maize (Zea mays ssp. mays) was domesticated in southwestern Mexico ∼9,000 years ago from its wild ancestor, teosinte (Zea mays ssp. parviglumis) [1]. From its center of origin, maize experienced a rapid range expansion and spread over 90° of latitude in the Americas [2-4], which required a novel flowering-time adaptation. ZEA CENTRORADIALIS 8 (ZCN8) is the maize florigen gene and has a central role in mediating flowering [5, 6]. Here, we show that ZCN8 underlies a major quantitative trait locus (QTL) (qDTA8) for flowering time that was consistently detected in multiple maize-teosinte experimental populations. Through association analysis in a large diverse panel of maize inbred lines, we identified a SNP (SNP-1245) in the ZCN8 promoter that showed the strongest association with flowering time. SNP-1245 co-segregated with qDTA8 in maize-teosinte mapping populations. We demonstrate that SNP-1245 is associated with differential binding by the flowering activator ZmMADS1. SNP-1245 was a target of selection during early domestication, which drove the pre-existing early flowering allele to near fixation in maize. Interestingly, we detected an independent association block upstream of SNP-1245, wherein the early flowering allele that most likely originated from Zea mays ssp. mexicana introgressed into the early flowering haplotype of SNP-1245 and contributed to maize adaptation to northern high latitudes. Our study demonstrates how independent cis-regulatory variants at a gene can be selected at different evolutionary times for local adaptation, highlighting how complex cis-regulatory control mechanisms evolve. Finally, we propose a polygenic map for the pre-Columbian spread of maize throughout the Americas.
Abscisic acid (ABA) and ethylene play key roles in growth and development of plants. Several attempts have been made to investigate the ABA and ethylene-induced signaling in plants, however, the involvement of phosphorylation and dephosphorylation in fine-tuning of the induced response has not been investigated much. Here, a phosphoproteomic analysis was carried out to identify the phosphoproteins in response to ABA, ethylene (ET) and combined ABA + ET treatments in soybean leaves. Phosphoproteome analysis led to the identification of 802 phosphopeptides, representing 422 unique protein groups. A comparative analysis led to the identification of 40 phosphosites that significantly changed in response to given hormone treatments. Functional annotation of the identified phosphoproteins showed that these were majorly involved in nucleic acid binding, signaling, transport and stress response. Localization prediction showed that 67% of the identified phosphoproteins were nuclear, indicating their potential involvement in gene regulation. Taken together, these results provide an overview of the ABA, ET and combined ABA + ET signaling in soybean leaves at phosphoproteome level.
Non-availability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NP) and phosphoproteins (NPPs) execute crucial cellular functions and form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. This study aimed at identifying dehydration-responsive nuclear phosphoproteins in a food legume, chickpea. Four-week-old seedlings were subjected to gradual dehydration and nuclear proteins were extracted from unstressed control as well as 72 and 144 h of stressed tissues. Proteomic analyses led to the identification of 4832 proteins and 478 phosphosites, corresponding to 299 unique phosphoproteins involved in a wide array of cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases and transcription factors, besides 660 uncharacterised proteins. Members of spliceosome complex and splicing related proteins were dominant among the differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of the NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also regulatory proteins of key processes viz., protein degradation, regulation of flowering time and circadian clock were observed to undergo dehydration-induced dephosphorylation.