Kip- related proteins ( KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin- dependent kinase ( CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula ( Mt) by a yeast two- hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two- hybrid test, the KRPMt protein interacted with CDK ( Medsa; CDKA; 1) and D- type cyclins. However, in the pull- down assays, B- type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa; CDKA; 1/ A; 2 complex was strongly inhibited, whereas the mitotic Medsa; CDKB2; 1 complex was the most sensitive to inhibition. Function of Medsa; CDKB1; 1 complex was not inhibited by the KRPMt protein. The mitotic Medsa; CYCB2 and Medsa; CYCA2; 1 complexes responded weakly to this inhibitor protein. Kinase complexes from G2/ M cells showed increased sensitivity towards the inhibitor compared with those isolated from G1/ S- phase cells. In vitro phosphorylation of Medicago retinoblastoma- related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin- like domain protein kinase ( MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin- dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross- talk between Ca2+ signalling and regulation of cell- cycle progression in plants.

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro. Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1 heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human positive transcription elongation factor P-TEFb.

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an essential regulator of the cell cycle from metaphase until S phase in yeast and metazoans. APC mediates degradation of numerous cell cycle-related proteins, including mitotic cyclins and its activation and substrate-specificity are determined by two adaptor proteins, Cdc20 and Cdh1. Plants have multiple APC activators and the Cdh1-type proteins, in addition, are represented by two subclasses, known as Ccs52A and Ccs52B. The Arabidopsis genome contains five cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. In Schizosaccharomyces pombe, expression of the three Atccs52 genes elicited distinct phenotypes supporting nonredundant function of the AtCcs52 proteins. Consistent with these activities, the AtCcs52 proteins were able to bind both to the yeast and the Arabidopsis APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts were present from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1 and ccs52A2 were from late M until early G2, suggesting consecutive action of these APC activators in the plant cell cycle. The AtCcs52 proteins interacted with different subsets of mitotic cyclins, in accordance with their expression profiles, either in free- or CDK-bound forms. Expression of most APC subunits was constitutive, whereas cdc27a and cdc27b, corresponding to two forms of apc3, and ubc19 and ubc20 encoding E2-C type ubiquitin-conjugating enzymes displayed differences in their cell cycle regulation. These data indicate the existence of numerous APC(Cdc20/Ccs52/Cdc27) forms in Arabidopsis, which in conjunction with different E2 enzymes might have distinct or complementary functions at distinct stages of the cell cycle.

The cambium of woody plants cycles between active and dormant states. Dormancy can be subdivided into eco- and endodormant stages. Ecodormant trees resume growth upon exposure to growth-promotive signals, while the establishment of endodormant state results in loss of the ability to respond to these signals. In this paper, we analysed the regulation of cyclin-dependent kinases (CDKs) to understand the differential response of cell division machinery to growth-promotive signals during the distinct stages of dormancy in hybrid aspen. We show that 4 weeks of short-day (SD) treatment causes termination of the cambial cell division and establishment of the ecodormant state. This coincides with a steady decline in the histone H1 kinase activity of the PSTAIRE-type poplar CDKA (PttCDKA) and the PPTTLRE-type PttCDKB kinase complexes. However, neither the transcript nor the polypeptide levels of PttCDKA and PttCDKB are reduced during ecodormancy. In contrast, 6 weeks of SD treatment establishes endodormancy, which is marked by the reduction and disappearance of the PttCDKA and PttCDKB protein levels and the PttCDKB transcript levels. The transition to endodormancy is preceded by an elevated E2F (adenosine E2 promoter binding factor) phosphorylation activity of the PttCDKA kinase that reduces the DNA-binding activity of E2F in vitro. The transition to endodormancy is followed by a reduction of retinoblastoma (Rb) phosphorylation activity of PttCDKA protein complexes. Both phosphorylation events could contribute to block the G1 to S phase transition upon the establishment of endodormancy. Our results indicate that eco- and endodormant stages of cambial dormancy involve a stage-specific regulation of the cell cycle effectors at multiple levels.

The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium -transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo . These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro. AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-beta II isoenzyme involved in human insulin signaling.

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.

Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription factors in yeast and mammalian cells. Here we describe partial purification, identification, and characterization of a protein kinase that phosphorylates the Arabidopsis heat-shock factor AtHSF1 at multiple serine residues. The HSF1 kinase forms a stable complex with AtHSF1, which can be detected by kinase pull-down assays using a histidine-tagged AtHSF1 substrate. The HSF1 kinase interacts with the cell-cycle control protein Suc1p and is immunoprecipitated by an antibody specific for the Arabidopsis cyclin-dependent CDC2a kinase. Phosphorylation by CDC2a in vitro inhibits DNA binding of AtHSF1 to the cognate heat-shock elements, suggesting a possible regulatory interaction between heat-shock response and cell-cycle control in plants.

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.

A β-glucoside encoded by a cloned Zea mays complementary DNA ( Zm-p60.1 ) cleaved the biologically inactive hormone conjugates cytokinin-O-glucosides and kinetin-N3-glucoside, releasing active cytokinin. Tobacco protoplasts that transiently expressed Zm-p60.1 could use the inactive cytokinin glucosides to initiate cell division. The ability of protoplasts to sustain growth in response to cytokinin glucosides persisted indefinitely after the likely disappearance of the expression vector. In the roots of maize seedlings, Zm-p60.1 was localized to the meristematic cells and may function in vivo to supply the developing maize embryo with active cytokinin.

Virtually all aspects of plant growth and development are influenced by structurally relatively simple substances termed phytohormones. It has been argued that the wide range of responses elicited by these substances requires a mode of action that is radically different from those of animal hormones. Current evidence indicates that enzymes that can synthesize and modify phytohormones and their antagonists, or hydrolyze phytohormone conjugates to release active hormones, play a role in initiating important regulatory pathways. They are also likely to provide invaluable tools with which to study the mechanisms underlying growth and differentiation in plants. Here we describe recent biochemical progress in the characterization of the molecular targets of phytohormones.

Hormones as auxins and cytokinins trigger the division cycle in differentiated plant cells and are required for the maintenance of proliferation in cultured cells in vitro. Northern analysis showed that the expression pattern of alfalfa cdc2 genes is significantly different in cells of primary explants exposed to hormone treatment and of rapidly cycling suspension culture. Transcription of at least one of the cdc2 genes is activated by hormones in cultured leaf mesophyll protoplasts or in root tissues treated with auxins and cytokinins. In a suspension culture of alfalfa cells, cdc2 transcripts are at a constitutively high level, irrespective of actual cell cycle phase. In addition to the transcriptional control of cdc2 genes, in hormone‐induced cells cdc2‐related kinase complexes were identified as potential phase‐specific components of post‐transcriptional regulation of the cell cycle. The known specific interaction between eukaryotic p34cdc2 protein complexes and the yeast p13suc1 protein was exploited for purification of cell cycle regulatory protein kinases from alfalfa. Alfalfa suspension cells were synchronized either for G1 phase by double‐phosphate‐starvation or for S phase by hydroxyurea treatment. The p13suc1‐Sepharose affinity matrix bound two cdc2 protein‐related complexes, one with increased histone H1 kinase activity in S, the other in G2/M phase. The complex from S phase cells showed higher kinase activity than the G2/M phase complex. Immunoblotting of p13suc1‐Sepharose‐bound protein complex showed the presence of a 33–34 kDa doublet that is recognized by anti‐PSTAIR antibodies and the co‐purification of two proteins (apparent molecular mass 65 and 42 kDa) cross‐reacting with human cyclin A antibodies. Immunoprecipitation with these cyclin A antibodies allowed the detection of a third cdc2‐related kinase complex that appeared during G1/S and early S phases of the cell cycle and phosphorylated histone H1. The results suggest that a multi‐component regulatory system is in control of the cell cycle in plants, which includes a hormone‐activated transcriptional control of cdc2 genes and phase‐specific cdc2‐related kinase complexes.

Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription.

A biologically active and photolabile auxin analog, 5‐azido‐[7‐3H]indole‐3‐acetic acid ([3H]N3IAA), was used to search for auxin‐binding proteins in cytosolic extracts from maize coleoptiles (Zea mays L.) and identified a protein with a molecular mass of 60 kDa (p60). Binding of [3H]N3IAA is highly specific as demonstrated by competition analysis with functionally relevant auxin analogs. p60 is found in coleoptiles and roots of etiolated maize seedlings and was detected in cytosolic as well as in microsomal fractions. The protein binds to 1‐naphthylacetic acid (1‐NAA) sepharose and is eluted with auxins. A purification scheme resulting in homogenous p60 protein was devised and it was shown that p60 has β‐d‐glucoside glucohydrolase activity (E.C.3.2.1.21). The hydrolytic activity of p60 for the synthetic substrate p‐nitro‐phenyl‐β‐d‐glucopyranoside is diminished by 1‐NAA. p60 shows high substrate specificity since it hydrolyzes indoxyl‐O‐glucoside, but not β‐(1,4)‐cellobiose, IAA‐inositol or IAA‐amino acid conjugates. The present data suggest that p60 might be involved in the hydrolysis of auxin conjugates.

Conference Article| February 01 1992 Auxin-binding proteins of Zea mays identified by photoaffinity labelling Ian Moore; Ian Moore 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Joachim Feldwisch; Joachim Feldwisch 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Narciso Campos; Narciso Campos 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Rolf Zettl; Rolf Zettl 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Bretislav Brzobohaty; Bretislav Brzobohaty 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Laszlo Bakó; Laszlo Bakó 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Jeff Schell; Jeff Schell 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Klaus Palme Klaus Palme 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (1): 70–73. https://doi.org/10.1042/bst0200070 Article history Received: September 19 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Ian Moore, Joachim Feldwisch, Narciso Campos, Rolf Zettl, Bretislav Brzobohaty, Laszlo Bakó, Jeff Schell, Klaus Palme; Auxin-binding proteins of Zea mays identified by photoaffinity labelling. Biochem Soc Trans 1 February 1992; 20 (1): 70–73. doi: https://doi.org/10.1042/bst0200070 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: IAA, indole-3-acetic acid, TIBA, 2,3,5-triiodobenzoic acid, NPA, 1-N-naphthylphthalamic acid, azido-IAA, 5-azido-7-[3H]indole-3-acetic acid, PCR, polymerase chain reaction, NAA, naphthylacetic acid, azido-NPA, 5′-azido-[3,6-3H]NPA This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.