Proteomic profiling of Arabidopsis G-protein ß subunit AGB1 mutant under salt stress.

Salt stress is a limiting environmental factor that inhibits plant growth in most ecological environments. The functioning of G-proteins and activated downstream signaling during salt stress is well established and different G-protein subunits and a few downstream effectors have been identified. Arabidopsis G-protein ß-subunit (AGB1) regulates the movement of Na+ from roots to shoots along with a significant role in controlling Na+ fluxes in roots, however, the molecular mechanism of AGB1 mediated salt stress regulation is not well understood. Here, we report the comparative proteome profiles of Arabidopsis AGB1 null mutant agb1-2 to investigate how the absence of AGB1 modulates the protein repertoire in response to salt stress. High-resolution two-dimensional gel electrophoresis (2-DE) showed 27 protein spots that were differentially modulated between the control and NaCl treated agb1-2 seedlings of which seven were identified by mass spectrometry. Functional annotation and interactome analysis indicated that the salt-responsive proteins were majorly associated with cellulose synthesis, structural maintenance of chromosomes, DNA replication/repair, organellar RNA editing and indole glucosinolate biosynthesis. Further exploration of the functioning of these proteins could serve as a potential stepping stone for dissection of molecular mechanism of AGB1 functions during salt stress and in long run could be extrapolated to crop plants for salinity stress management.

Comparative genomics, microsynteny, ancestral state reconstruction and selection pressure analysis across distinctive genomes and sub-genomes of Brassicaceae for analysis of evolutionary history of VQ gene family.

Any unfavorable condition that affects the metabolism, growth, or development of plants is considered plant stress. The molecular response of plants towards abiotic stresses involves signaling to cellular components, repressing transcription factors, and subsequently induced metabolic changes. Most valine-glutamine (VQ) motif-containing genes in plants encode regulatory proteins that interact with transcription factors and modulate their activity as transcription regulators. Several VQ proteins regulate plant development and stress responses. In spite of the functional importance of VQs, there is relatively little information about their evolutionary history in Brassicaceae or beyond. Brassicaceae is characterized by paleoploidy, mesopolyploidy, and neopolyploidy, offering a resource for studying evolution and diversification. In current study we performed phylogeny of the VQ gene family along with comparative genomics, microsynteny and evolutionary rates analysis across seven species of Brassicaceae. Our findings revealed the following; (1) a large segmental duplication in the shared common ancestor of the family Brassicaceae, resulted in paralogies of VQ1-VQ10, VQ15-VQ24, VQ16-VQ23, VQ17-VQ25, VQ18-VQ26, VQ22-VQ27; (2) chromosomal mapping revealed diverse distributions of the gene family; (3) duplicated segments undergo varying degrees of retention and loss; and (4) Out of the 12 paralogous members, most of the genes are under purifying selection. However, VQ23 in Brassicaceae stands out as it is under positive selection, indicating the need for further investigation. Overall, our results clearly establish that the ancestral VQ1/VQ10, VQ15/VQ24, VQ16/VQ23, VQ17/VQ25, VQ18/VQ26, VQ22/VQ27 genes duplicated in shared common ancestor of Brassicaceae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01347-z.

Light signaling as cellular integrator of multiple environmental cues in plants.

Plants being sessile need to rapidly adapt to the constantly changing environment through modifications in their internal clock, metabolism, and gene expression. They have evolved an intricate system to perceive and transfer the signals from the primary environmental factors namely light, temperature and water to regulate their growth development and survival. Over past few decades rigorous research using molecular genetics approaches, especially in model plant Arabidopsis, has resulted in substantial progress in discovering various photoreceptor systems and light signaling components. In parallel several molecular pathways operating in response to other environmental cues have also been elucidated. Interestingly, the studies have shown that expression profiles of genes involved in photomorphogenesis can undergo modulation in response to other cues from the environment. Recently, the photoreceptor, PHYB, has been shown to function as a thermosensor. Downstream components of light signaling pathway like COP1 and PIF have also emerged as integrating hubs for various kinds of signals. All these findings indicate that light signaling components may act as central integrator of various environmental cues to regulate plant growth and development processes. In this review, we present a perspective on cross talk of signaling mechanisms induced in response to myriad array of signals and their integration with the light signaling components. By putting light signals on the central stage, we propose the possibilities of enhancing plant resilience to the changing environment by fine-tuning the genetic manipulation of its signaling components in the future.

Molecular Characterization of NDL1-AGB1 Mediated Salt Stress Signaling: Further Exploration of the Role of NDL1 Interacting Partners.

Salt stress is considered to be the most severe abiotic stress. High soil salinity leads to osmotic and ionic toxicity, resulting in reduced plant growth and crop production. The role of G-proteins during salt stresses is well established. AGB1, a G-protein subunit, not only plays an important role during regulation of Na+ fluxes in roots, but is also involved in the translocation of Na+ from roots to shoots. N-Myc Downregulated like 1 (NDL1) is an interacting partner of G protein ßγ subunits and C-4 domain of RGS1 in Arabidopsis. Our recent in-planta expression analysis of NDL1 reported changes in patterns during salt stress. Based on these expression profiles, we have carried out functional characterization of the AGB1-NDL1 module during salinity stress. Using various available mutant and overexpression lines of NDL1 and AGB1, we found that NDL1 acts as a negative regulator during salt stress response at the seedling stage, an opposite response to that of AGB1. On the other hand, during the germination phase of the plant, this role is reversed, indicating developmental and tissue specific regulation. To elucidate the mechanism of the AGB1-NDL1 module, we investigated the possible role of the three NDL1 stress specific interactors, namely ANNAT1, SLT1, and IDH-V, using yeast as a model. The present study revealed that NDL1 acts as a modulator of salt stress response, wherein it can have both positive as well as negative functions during salinity stress. Our findings suggest that the NDL1 mediated stress response depends on its developmental stage-specific expression patterns as well as the differential presence and interaction of the stress-specific interactors.

Arabidopsis NDL-AGB1 modules Play Role in Abiotic Stress and Hormonal Responses Along with Their Specific Functions.

Arabidopsis N-MYC Downregulated Like Proteins (NDLs) are interacting partners of G-Protein core components. Animal homologs of the gene family N-myc downstream regulated gene (NDRG) has been found to be induced during hypoxia, DNA damage, in presence of reducing agent, increased intracellular calcium level and in response to metal ions like nickel and cobalt, which indicates the involvement of the gene family during stress responses. Arabidopsis NDL gene family contains three homologs NDL1, NDL2 and NDL3 which share up to 75% identity at protein level. Previous studies on NDL proteins involved detailed characterization of the role of NDL1; roles of other two members were also established in root and shoot development using miRNA knockdown approach. Role of entire family in development has been established but specific functions of NDL2 and NDL3 if any are still unknown. Our in-silico analysis of NDLs promoters reveled that all three members share some common and some specific transcription factors (TFs) binding sites, hinting towards their common as well as specific functions. Based on promoter elements characteristics, present study was designed to carry out comparative analysis of the Arabidopsis NDL family during different stages of plant development, under various abiotic stresses and plant hormonal responses, in order to find out their specific and combined roles in plant growth and development. Developmental analysis using GUS fusion revealed specific localization/expression during different stages of development for all three family members. Stress analysis after treatment with various hormonal and abiotic stresses showed stress and tissue-specific differential expression patterns for all three NDL members. All three NDL members were collectively showed role in dehydration stress along with specific responses to various treatments. Their specific expression patterns were affected by presence of interacting partner the Arabidopsis heterotrimeric G-protein ß subunit 1 (AGB1). The present study will improve our understanding of the possible molecular mechanisms of action of the independent NDL-AGB1 modules during stress and hormonal responses. These findings also suggest potential use of this knowledge for crop improvement.

Comparative expression profiling of AtRAD5B and AtNDL1: Hints towards a role in G protein mediated signaling.

Arabidopsis AtRAD5B encodes for a putative helicase of the class SWItch/Sucrose Non-Fermentable (SWI/SNF) ATPases. We identified AtRAD5B as an interactor of N-MYC DOWNREGULATED-LIKE1 (AtNDL1) in a yeast two-hybrid screen. AtNDL1 is a G protein signaling component which regulates auxin transport and gradients together with GTP binding protein beta 1 (AGB1). Auxin gradients are known to recruit SWI/SNF remodeling complexes to the chromatin and regulate expression of genes involved in flower and leaf formation. In current study, a comparative spatial and temporal co-expression/localization analysis of AtNDL1, AGB1 with AtRAD5B was carried out in order to explore the possibility of their coexistence in a common signaling network. Translational fusion (GUS) of AtNDL1 and AtRAD5B in seedlings and reproductive organs revealed that both shared similar expression patterns with the highest expression observed in male reproductive organs. Moreover, they shared similar domains of localization in roots, suggesting their potential functioning together in reproductive and root development processes. This study predicts the existence of a signaling network involving AtNDL1, AGB1 with AtRAD5B.

Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis.

Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gß subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development.

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants.

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development.

N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

BACKGROUND: N-MYC down-regulated-like (NDL) proteins interact with the Gß subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. METHODOLOGY/PRINCIPAL FINDINGS: Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. CONCLUSION/SIGNIFICANCE: NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

The heterotrimeric G-protein complex is minimally composed of Gα, Gß, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

NDR proteins: lessons learned from Arabidopsis and animal cells prompt a testable hypothesis.

N-myc Down Regulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDL proteins show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins.

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis.

Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane-localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gbeta subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.