RESUMO
Oxygen limitation (hypoxia), arising as a key stress factor due to flooding, negatively affects plant development. Consequently, maintaining root growth under such stress is crucial for plant survival, yet we know little about the root system's adaptions to low-oxygen conditions and its regulation by phytohormones. In this study, we examine the impact of hypoxia and, herein, the regulatory role of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors on root growth in Arabidopsis. We found lateral root (LR) elongation to be actively maintained by hypoxia via ERFVII factors, as erfVII seedlings possess hypersensitivity towards hypoxia regarding their LR growth. Pharmacological inhibition of abscisic acid (ABA) biosynthesis revealed ERFVII-driven counteraction of hypoxia-induced inhibition of LR formation in an ABA-dependent manner. However, postemergence LR growth under hypoxia mediated by ERFVIIs was independent of ABA. In roots, ERFVIIs mediate, among others, the induction of ABA-degrading ABA 8'-hydroxylases CYP707A1 expression. RAP2.12 could activate the pCYC707A1:LUC reporter gene, indicating, combined with single mutant analyses, that this transcription factor regulates ABA levels through corresponding transcript upregulation. Collectively, hypoxia-induced adaptation of the Arabidopsis root system is shaped by developmental reprogramming, whereby ERFVII-dependent promotion of LR emergence, but not elongation, is partly executed through regulation of ABA degradation.
Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Homeostase , Raízes de Plantas , Fatores de Transcrição , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Ácido Abscísico/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Oxigênio/metabolismoRESUMO
Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Serina Endopeptidases , Fatores de Transcrição , Humanos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Fibrinogênio/metabolismo , Regulação da Expressão Gênica de Plantas , Hipóxia/metabolismo , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Fotossíntese , Oxirredutases/metabolismo , Hipóxia/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
A key trait conferring flood tolerance is the ability to grow adventitious roots as a response to submergence. The genetic traits of deepwater rice determining the development and characteristics of aquatic adventitious roots (AAR) had not been evaluated. We used near-isogenic lines introgressed to test the hypothesis that the impressive shoot elongation ability of deepwater rice linked to quantitative trait loci 1 and 12 also promote the development of AAR. The deepwater rice genotype NIL-12 possessed expanded regions at the stem nodes where numerous AAR developed as a response to submergence. Two types (AR1 and AR2) of roots with distinct timing of emergence and large differences in morphological and anatomical traits formed within 3 (AR1) to 7 (AR2) d of submergence. The mechanical impedance provided by the leaf sheath caused AR2 to emerge later promoting thicker roots, higher elongation capacity and higher desiccation tolerance. Upregulation of key genes suggests a joint contribution in activating the meristem in AAR enhancing the development of these in response to submergence. The morphological and anatomical traits suggested that AR2 is better adapted to long-term flooding than AR1. We therefore propose that AR2 in deepwater rice functions as an evolutionary defence strategy to tackle periodic submergence.
Assuntos
Oryza , Locos de Características Quantitativas , Locos de Características Quantitativas/genética , Oryza/fisiologia , Inundações , Fenótipo , GenótipoRESUMO
Flooding is constantly threatening the growth and yield of crops worldwide. When flooding kicks in, the soil becomes water-saturated and, therefore, the roots are the first organs to be exposed to excess water. Soon after flooding, the soil turns anoxic and the roots can no longer obtain molecular oxygen for respiration from the rhizosphere, rendering the roots dysfunctional. Rice, however, is a semi-aquatic plant and therefore relatively tolerant to flooding due to adaptive traits developed during evolution. In the present review, we have identified three key root traits, viz. cortical aerenchyma formation, a barrier to radial oxygen loss and adventitious root growth. The understanding of the physiological function, the molecular mechanisms, and the genetic regulation of these three traits has grown substantially and therefore forms the backbone of this review. Our synthesis of the recent literature shows each of the three key root traits contributes to flood tolerance in rice. One trait, however, is generally insufficient to enhance plant tolerance to flooding. Consequently, we suggest comprehensive use of all three adaptive traits in a pyramiding approach in order to improve tolerance to flooding in our major crops, in general, and in rice, in particular.
RESUMO
Fluctuations in oxygen (O2) availability occur as a result of flooding, which is periodically encountered by terrestrial plants. Plant respiration and mitochondrial energy generation rely on O2 availability. Therefore, decreased O2 concentrations severely affect mitochondrial function. Low O2 concentrations (hypoxia) induce cellular stress due to decreased ATP production, depletion of energy reserves and accumulation of metabolic intermediates. In addition, the transition from low to high O2 in combination with light changes-as experienced during re-oxygenation-leads to the excess formation of reactive oxygen species (ROS). In this review, we will update our current knowledge about the mechanisms enabling plants to adapt to low-O2 environments, and how to survive re-oxygenation. New insights into the role of mitochondrial retrograde signaling, chromatin modification, as well as moonlighting proteins and mitochondrial alternative electron transport pathways (and their contribution to low O2 tolerance and survival of re-oxygenation), are presented.
RESUMO
Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSK receptors (PSKRs) are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST; encoding tyrosylprotein sulfotransferase). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 169 PSK-regulated genes, several had functions in root growth and development, in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs, and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23), and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1, revealing unexpected synergistic effects of ligand and PSKR deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth, suggesting that PSKR1 may be active in the absence of sulfated ligands.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Secas , Serina Proteases/metabolismo , Estresse Fisiológico , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Serina Proteases/genética , Transdução de SinaisRESUMO
Flooding causes oxygen deprivation in soils. Plants adapt to low soil oxygen availability by changes in root morphology, anatomy, and architecture to maintain root system functioning. Essential traits include aerenchyma formation, a barrier to radial oxygen loss, and outgrowth of adventitious roots into the soil or the floodwater. We highlight recent findings of mechanisms of constitutive aerenchyma formation and of changes in root architecture. Moreover, we use modelling of internal aeration to demonstrate the beneficial effect of increasing cortex-to-stele ratio on sustaining root growth in waterlogged soils. We know the genes for some of the beneficial traits, and the next step is to manipulate these genes in breeding in order to enhance the flood tolerance of our crops.
Assuntos
Oxigênio , Solo , Inundações , Melhoramento Vegetal , Raízes de PlantasRESUMO
Flooding is an environmental stress that leads to a shortage of O2 that can be detrimental for plants. When flooded, deepwater rice grow floating adventitious roots to replace the dysfunctional soil-borne root system, but the features that ensure O2 supply and hence growth of aquatic roots have not been explored. We investigate the sources of O2 in aquatic adventitious roots and relate aerenchyma and barriers for gas diffusion to local O2 gradients, as measured by microsensor technology, to link O2 distribution in distinct root zones to their anatomical features. The mature root part receives O2 exclusively from the stem. It has aerenchyma that, together with suberin and lignin depositions at the water-root and cortex-stele interfaces, provides a path for longitudinal O2 movement toward the tip. The root tip has no diffusion barriers and receives O2 from the stem and floodwater, resulting in improved aeration of the root tip over mature tissues. Local formation of aerenchyma and diffusion barriers in the mature root channel O2 towards the tip which also obtains O2 from the floodwater. These features explain aeration of floating roots and their ability to grow under water.
Assuntos
Oryza , Inundações , Oxigênio , Raízes de Plantas , SoloRESUMO
Sulfated peptides are plant hormones that are active at nanomolar concentrations. The sulfation at one or more tyrosine residues is catalysed by tyrosylprotein sulfotransferase (TPST), which is encoded by a single-copy gene. The sulfate group is provided by the co-substrate 3´-phosphoadenosine 5´-phosphosulfate (PAPS), which links synthesis of sulfated signaling peptides to sulfur metabolism. The precursor proteins share a conserved DY-motif that is implicated in specifying tyrosine sulfation. Several sulfated peptides undergo additional modification such as hydroxylation of proline and glycosylation of hydroxyproline. The modifications render the secreted signaling molecules active and stable. Several sulfated signaling peptides have been shown to be perceived by leucine-rich repeat receptor-like kinases (LRR-RLKs) but have signaling pathways that, for the most part, are yet to be elucidated. Sulfated peptide hormones regulate growth and a wide variety of developmental processes, and intricately modulate immunity to pathogens. While basic research on sulfated peptides has made steady progress, their potential in agricultural and pharmaceutical applications has yet to be explored.
Assuntos
Hormônios Peptídicos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas/metabolismo , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Sulfatos/metabolismoRESUMO
Flooding is a severe limitation for crop production worldwide. Unlike other crop plants, rice (Oryza sativa L.) is well adapted to partial submergence rendering it a suitable crop plant to understand flooding tolerance. Formation of adventitious roots (ARs), that support or replace the main root system, is a characteristic response to flooding. In rice, AR emergence is induced by ethylene and in the dark where roots grow upward. We used the synthetic auxins 2,4-D and α-NAA, and the auxin transport inhibitor naphthylphtalamic acid (NPA) to study emergence, growth rate and growth angle of ARs. While α-NAA had no effect, NPA and 2,4-D reduced the root elongation rate and the angle with a stronger effect on root angle in the dark than in the light. Furthermore, NPA delayed emergence of AR primordia suggesting that efflux carrier-mediated auxin transport is required for all aspects of directed AR growth. Expression analysis using OsPIN:GUS reporter lines revealed that OsPIN1b and OsPIN1c promoters were active in the stele and root cap in accord with their predicted role in acropetal auxin transport. OsPIN2 was expressed at the root tip and was reduced in the presence of NPA. Auxin activity, detected with DR5:VENUS, increased in primordia following growth induction. By contrast, auxin activity was high in epidermal cells above primordia and declined following growth induction suggesting that auxin levels are antagonistically regulated in AR primordia and in epidermal cells above AR primordia suggesting that auxin signaling contributes to the coordinated processes of epidermal cell death and AR emergence.
RESUMO
Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estresse Fisiológico , Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , NADPH Oxidases/genéticaRESUMO
Rice (Oryza sativa) is a semiaquatic plant that is well adapted to partial flooding. Rice stems develop adventitious root (AR) primordia at each node that slowly mature but emerge only when the plant gets flooded, leading to the formation of a whole new secondary root system upon flooding. AR growth is induced by ethylene that accumulates in submerged plant tissues due to its lowered diffusion rate in water. Here, we report that the architecture of the secondary root system in flooded rice plants is controlled not only by altered gas diffusion but also by gravity and light. While ethylene promotes the emergence and growth of ARs, gravity and light determine their gravitropic setpoint angle (i.e. the deviation of growth direction relative to vertical). ARs grow upward at about 120° in the dark and downward at 54° in the light. The upward growth direction is conserved in indica and japonica rice varieties, suggestive of a conserved trait in rice. Experiments with a klinostat and with inverted stem orientation revealed that gravity promotes upward growth by about 10°. Red, far-red, and blue light lead to negative phototropism in a dose-dependent manner, with blue light being most effective, indicating that phytochrome and blue light signaling control AR system architecture. The cpt1 (coleoptile phototropism1) mutant, which lacks one of the phototropin-interacting CPT proteins, shows reduced sensitivity to blue light. Hence, the gravitropic setpoint angle of rice ARs is controlled by genetic and environmental factors that likely balance the need for oxygen supply (upward growth) with avoidance of root desiccation (downward growth).
Assuntos
Escuridão , Gravitação , Luz , Oryza/fisiologia , Raízes de Plantas/fisiologia , Etilenos/metabolismo , Inundações , Mutação , Oryza/anatomia & histologia , Oryza/crescimento & desenvolvimento , Fototropismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Transdução de SinaisRESUMO
When plants encounter soil water logging or flooding, roots are the first organs to be confronted with reduced gas diffusion resulting in limited oxygen supply. Since roots do not generate photosynthetic oxygen, they are rapidly faced with oxygen shortage rendering roots particularly prone to damage. While metabolic adaptations to low oxygen conditions, which ensure basic energy supply, have been well characterized, adaptation of root growth and development have received less attention. In this study, we show that hypoxic conditions cause the primary root to grow sidewise in a low oxygen environment, possibly to escape soil patches with reduced oxygen availability. This growth behavior is reversible in that gravitropic growth resumes when seedlings are returned to normoxic conditions. Hypoxic root bending is inhibited by the group VII ethylene response factor (ERFVII) RAP2.12, as rap2.12-1 seedlings show exaggerated primary root bending. Furthermore, overexpression of the ERFVII member HRE2 inhibits root bending, suggesting that primary root growth direction at hypoxic conditions is antagonistically regulated by hypoxia and hypoxia-activated ERFVIIs. Root bending is preceded by the establishment of an auxin gradient across the root tip as quantified with DII-VENUS and is synergistically enhanced by hypoxia and the auxin transport inhibitor naphthylphthalamic acid. The protein abundance of the auxin efflux carrier PIN2 is reduced at hypoxic conditions, a response that is suppressed by RAP2.12 overexpression, suggesting antagonistic control of auxin flux by hypoxia and ERFVII. Taken together, we show that hypoxia triggers an escape response of the primary root that is controlled by ERFVII activity and mediated by auxin signaling in the root tip.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gravitropismo , Oxigênio/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Ligação a DNA , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Fotossíntese , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plântula/genética , Plântula/fisiologia , Fatores de Transcrição/genéticaRESUMO
Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.
Assuntos
Bioensaio , Calmodulina/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Sítios de Ligação , Calmodulina/genética , Corantes Fluorescentes/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Microscopia de Fluorescência , Proteínas de Plantas/genética , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/genética , Transformação GenéticaRESUMO
Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca2+.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Bioensaio , Cálcio/metabolismo , Calmodulina/metabolismo , Escherichia coli/genética , Receptores de Superfície Celular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Calmodulina/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transformação GenéticaRESUMO
The phytosulfokine peptide receptor PSKR1 is modified by phosphorylation of its cytoplasmic kinase domain. We analyzed defined phosphorylation sites by site-directed mutagenesis with regard to kinase activity in vitro and receptor activity in planta. S696 and S698 in the juxtamembrane (JM) domain are phosphorylated in planta. The phosphomimetic S696D/S698D replacements resulted in reduced transphosphorylation activity of PSKR1 kinase in vitro but did not reduce autophosphorylation activity. Growth-promoting activity of the PSKR1(S696D/S698D) receptor isoform was impaired in the shoot but not in the root. The JM domain thus seems to be important for phosphorylation of a target protein required for shoot growth promotion. The phosphomimetic replacement T998D at the C-terminus (CT) abolished kinase activity in vitro but not receptor function in planta, indicating that additional levels of regulation exist in planta. A possible mode of receptor regulation is the interaction with regulatory proteins such as the calcium sensor calmodulin (CaM). We show that the previously reported binding of CaM2 to PSKR1 is calcium-dependent, occurs predominately to the hypophosphorylated soluble PSKR1 kinase, and does not significantly change PSKR1 kinase activity. In conclusion, our results show that peptide signaling of growth by PSKR1 is regulated by differential phosphorylation of the juxtamembrane and C-terminal domains of the intracellular receptor part and suggest that interaction of PSKR1 with CaM serves a function other than the regulation of kinase activity.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Receptores de Superfície Celular/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Calmodulina/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Receptores de Superfície Celular/metabolismoRESUMO
PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.