In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcriptio... more In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family, most of which interact with 29 auxin/indole acetic acid (Aux/IAA) repressors, themselves forming, in the presence of auxin, coreceptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX PROTEINS (TIR1/AFB). Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. Considerable efforts have been made to discern the temporal and spatial specificity of auxin action. However, owing to a lack of obvious phenotype in single loss-of-function mutants in Aux/IAA genes, most genetic studies have relied on gain-of-function mutants, which are highly pleiotropic. In this article, we describe a molecular framework for the role of several members of the auxin sensing machinery. Using loss-of-function mutants, we demonstrate that TIR1 and AFB2 are positive regulators, w...
The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence ... more The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the p...
Summary The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary ... more Summary The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary elements. Yet overexpression of XND1 blocks differentiation of tracheary elements. The molecular mechanism of XND1 action was investigated. Phylogenetic and motif analyses indicated that XND1 and its homologs are present only in angiosperms and possess a highly conserved C‐terminal region containing linear motifs (CKII‐acidic, LXCXE, E2FTD‐like and LXCXE‐mimic) predicted to interact with the cell cycle and differentiation regulator RETINOBLASTOMA‐RELATED (RBR). Protein–protein interaction and functional analyses of XND1 deletion mutants were used to test the importance of RBR‐interaction motifs. Deletion of either the LXCXE or the LXCXE‐mimic motif reduced both the XND1–RBR interaction and XND1 efficacy as a repressor of differentiation, with loss of the LXCXE motif having the strongest negative impacts. The function of the XND1 C‐terminal domain could be partially replaced by RBR fused ...
Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors... more Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the BLADE-ON-PETIOLE (BOP) genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate...
We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicl... more 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, sug...
The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell grow... more 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 circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex... more The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation o...
Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are is... more Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PICKLE interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress theLATERAL ORGAN BOUNDARIES-DOMAIN 16(LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL-RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL-RBR1 is counteracted by auxin indicating that in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.
Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription fac... more 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.
In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcriptio... more In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family, most of which interact with 29 auxin/indole acetic acid (Aux/IAA) repressors, themselves forming, in the presence of auxin, coreceptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX PROTEINS (TIR1/AFB). Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. Considerable efforts have been made to discern the temporal and spatial specificity of auxin action. However, owing to a lack of obvious phenotype in single loss-of-function mutants in Aux/IAA genes, most genetic studies have relied on gain-of-function mutants, which are highly pleiotropic. In this article, we describe a molecular framework for the role of several members of the auxin sensing machinery. Using loss-of-function mutants, we demonstrate that TIR1 and AFB2 are positive regulators, w...
The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence ... more The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the p...
Summary The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary ... more Summary The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary elements. Yet overexpression of XND1 blocks differentiation of tracheary elements. The molecular mechanism of XND1 action was investigated. Phylogenetic and motif analyses indicated that XND1 and its homologs are present only in angiosperms and possess a highly conserved C‐terminal region containing linear motifs (CKII‐acidic, LXCXE, E2FTD‐like and LXCXE‐mimic) predicted to interact with the cell cycle and differentiation regulator RETINOBLASTOMA‐RELATED (RBR). Protein–protein interaction and functional analyses of XND1 deletion mutants were used to test the importance of RBR‐interaction motifs. Deletion of either the LXCXE or the LXCXE‐mimic motif reduced both the XND1–RBR interaction and XND1 efficacy as a repressor of differentiation, with loss of the LXCXE motif having the strongest negative impacts. The function of the XND1 C‐terminal domain could be partially replaced by RBR fused ...
Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors... more Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the BLADE-ON-PETIOLE (BOP) genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate...
We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicl... more 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, sug...
The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell grow... more 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 circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex... more The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation o...
Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are is... more Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PICKLE interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress theLATERAL ORGAN BOUNDARIES-DOMAIN 16(LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL-RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL-RBR1 is counteracted by auxin indicating that in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.
Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription fac... more 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.
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