RESUMEN
Abscisic acid (ABA) is a crucial player in plant responses to the environment. It accumulates under stress, activating downstream signaling to implement molecular responses that restore homeostasis. Natural variance in ABA sensitivity remains barely understood, and the ABA pathway has been mainly studied at the transcriptional level, despite evidence that posttranscriptional regulation, namely, via alternative splicing, contributes to plant stress tolerance. Here, we identified the Arabidopsis accession Kn-0 as less sensitive to ABA than the reference Col-0, as shown by reduced effects of the hormone on seedling establishment, root branching, and stomatal closure, as well as by decreased induction of ABA marker genes. An in-depth comparative transcriptome analysis of the ABA response in the two variants revealed lower expression changes and fewer genes affected for the least ABA-sensitive ecotype. Notably, Kn-0 exhibited reduced levels of the ABA-signaling SnRK2 protein kinases and lower basal expression of ABA-reactivation genes, consistent with our finding that Kn-0 contains less endogenous ABA than Col-0. ABA also markedly affected alternative splicing, primarily intron retention, with Kn-0 being less responsive regarding both the number and magnitude of alternative splicing events, particularly exon skipping. We find that alternative splicing introduces a more ecotype-specific layer of ABA regulation and identify ABA-responsive splicing changes in key ABA pathway regulators that provide a functional and mechanistic link to the differential sensitivity of the two ecotypes. Our results offer new insight into the natural variation of ABA responses and corroborate a key role for alternative splicing in implementing ABA-mediated stress responses.
Asunto(s)
Ácido Abscísico , Empalme Alternativo , Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Perfilación de la Expresión Génica , Plantones/genética , Plantones/efectos de los fármacos , Plantones/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Flowers, and hence, fruits and seeds, are produced by the activity of the inflorescence meristem after the floral transition. In plants with indeterminate inflorescences, the final number of flowers produced by the inflorescence meristem is determined by the length of the flowering period, which ends with inflorescence arrest. Inflorescence arrest depends on many different factors, such as the presence of seeds, the influence of the environment, or endogenous factors such as phytohormone levels and age, which modulate inflorescence meristem activity. The FRUITFULL-APETALA2 (FUL-AP2) pathway plays a major role in regulating the end of flowering, likely integrating both endogenous cues and those related to seed formation. Among AP2 targets, HOMEOBOX PROTEIN21 (HB21) has been identified as a putative mediator of AP2 function in the control of inflorescence arrest. HB21 is a homeodomain leucine zipper transcription factor involved in establishing axillary bud dormancy. Here, we characterized the role of HB21 in the control of the inflorescence arrest at the end of flowering in Arabidopsis (Arabidopsis thaliana). HB21, together with HB40 and HB53, are upregulated in the inflorescence apex at the end of flowering, promoting floral bud arrest. We also show that abscisic acid (ABA) accumulation occurs in the inflorescence apex in an HB-dependent manner. Our work suggests a physiological role of ABA in floral bud arrest at the end of flowering, pointing to ABA as a regulator of inflorescence arrest downstream of the HB21/40/53 genes.
Asunto(s)
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Flores , Regulación de la Expresión Génica de las Plantas , Inflorescencia , Factores de Transcripción , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Inflorescencia/genética , Inflorescencia/metabolismo , Inflorescencia/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Flores/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Meristema/genética , Meristema/metabolismo , Meristema/crecimiento & desarrolloRESUMEN
The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.
Asunto(s)
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Endosomas , Estomas de Plantas , Transducción de Señal , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endosomas/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Estomas de Plantas/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Sequías , Mutación/genética , Proteolisis , Transporte de ProteínasRESUMEN
White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.
Asunto(s)
Acetolactato Sintasa , Resistencia a los Herbicidas , Herbicidas , Malatión , Mutación , Sinapis , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Acetolactato Sintasa/antagonistas & inhibidores , Herbicidas/farmacología , Resistencia a los Herbicidas/genética , Sinapis/efectos de los fármacos , Sinapis/genética , Malatión/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arilsulfonatos/farmacología , Simulación del Acoplamiento Molecular , Imidazoles/farmacologíaRESUMEN
Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Óxido Nítrico/metabolismo , Factores de Transcripción/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Germinación/efectos de los fármacos , Germinación/fisiología , Óxido Nítrico/farmacología , Oxígeno/farmacología , Estomas de Plantas/efectos de los fármacos , Proteolisis , Transducción de Señal , Factores de Transcripción/efectos de los fármacosRESUMEN
Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas bpm3 bpm5 plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.
Asunto(s)
Ácido Abscísico/farmacocinética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Cullin/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteolisis , Secuencias de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Cullin/genética , Fosfoproteínas Fosfatasas/genéticaRESUMEN
Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.
Asunto(s)
Resistencia a los Herbicidas , Papaver , Resistencia a los Herbicidas/genética , Ácidos Indolacéticos , Ligandos , Mutación , Ácido 2,4-Diclorofenoxiacético/farmacologíaRESUMEN
The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Núcleo Celular/metabolismo , Modelos Biológicos , Mutación/genética , Células Vegetales/metabolismo , Proteolisis , Fracciones Subcelulares/metabolismo , UbiquitinaciónRESUMEN
The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Phoeniceae , Ácido Abscísico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas , Phoeniceae/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés FisiológicoRESUMEN
The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL85KR mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Abscísico/agonistas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Genes de Plantas , Ligandos , Meristema/metabolismo , Mutación , Reguladores del Crecimiento de las Plantas/agonistas , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Estabilidad Proteica/efectos de los fármacos , Quinolonas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , UbiquitinaciónRESUMEN
Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.
Asunto(s)
Ácido Abscísico/metabolismo , Proteína Fosfatasa 2C/metabolismo , Estrés Fisiológico/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fosforilación , Reguladores del Crecimiento de las Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Pyrenophoric acid (P-Acid), P-Acid B, and P-Acid C are three phytotoxic sesquiterpenoids produced by the ascomycete seed pathogen Pyrenophora semeniperda, a fungus proposed as a mycoherbicide for biocontrol of cheatgrass, an extremely invasive weed. When tested in cheatgrass bioassays, these metabolites were able to delay seed germination, with P-Acid B being the most active compound. Here, we have investigated the cross-kingdom activity of P-Acid B and its mode of action, and found that it activates the abscisic acid (ABA) signaling pathway in order to inhibit seedling establishment. P-Acid B inhibits seedling establishment in wild-type Arabidopsis thaliana, while several mutants affected in the early perception as well as in downstream ABA signaling components were insensitive to the fungal compound. However, in spite of structural similarities between ABA and P-Acid B, the latter is not able to activate the PYR/PYL family of ABA receptors. Instead, we have found that P-Acid B uses the ABA biosynthesis pathway at the level of alcohol dehydrogenase ABA2 to reduce seedling establishment. We propose that the fungus P. semeniperda manipulates plant ABA biosynthesis as a strategy to reduce seed germination, increasing its ability to cause seed mortality and thereby increase its fitness through higher reproductive success.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/crecimiento & desarrollo , Ascomicetos/fisiología , Vías Biosintéticas , Germinación , Sesquiterpenos/metabolismo , Arabidopsis/microbiologíaRESUMEN
Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.
Asunto(s)
Arabidopsis/fisiología , Compuestos Epoxi/farmacología , Macrólidos/farmacología , Empalme del ARN/efectos de los fármacos , Transducción de Señal/genética , Estrés Fisiológico/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Intrones , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Estomas de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The extensive support for abscisic acid (ABA) involvement in the complex regulatory networks controlling stress responses and development in plants contrasts with the relatively recent role assigned to nitric oxide (NO). Because treatment with exogenous ABA leads to enhanced production of NO, it has been widely considered that NO participates downstream of ABA in controlling processes such as stomata movement, seed dormancy, and germination. However, data on leaf senescence and responses to stress suggest that the functional interaction between ABA and NO is more complex than previously thought, including not only cooperation but also antagonism. The functional relationship is probably determined by several factors including the time- and place-dependent pattern of accumulation of both molecules, the threshold levels, and the regulatory factors important for perception. These factors will determine the actions exerted by each regulator. Here, several examples of well-documented functional interactions between NO and ABA are analysed in light of the most recent reported data on seed dormancy and germination, stomata movements, leaf senescence, and responses to abiotic and biotic stresses.
Asunto(s)
Ácido Abscísico/metabolismo , Regulación de la Expresión Génica de las Plantas , Óxido Nítrico/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/metabolismo , Estrés Fisiológico , Germinación , Modelos Biológicos , Desarrollo de la Planta , Latencia en las Plantas , Inmunidad de la Planta , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/inmunología , Hojas de la Planta/fisiología , Fenómenos Fisiológicos de las Plantas , Estomas de Plantas/crecimiento & desarrollo , Estomas de Plantas/inmunología , Estomas de Plantas/fisiología , Plantas/inmunología , Semillas/crecimiento & desarrollo , Semillas/inmunología , Semillas/fisiología , Factores de TiempoRESUMEN
Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta.
Asunto(s)
Ácido Abscísico/química , Arabidopsis/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Receptor de Androstano Constitutivo , Análisis Mutacional de ADN , Espectroscopía de Resonancia Magnética/métodos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfoproteínas Fosfatasas/metabolismo , Conformación Proteica , Proteína Fosfatasa 2C , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Semillas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Water deficit represents a serious limitation for agriculture and both genetic and chemical approaches are being used to cope with this stress and maintain plant yield. Next-generation agrochemicals that control stomatal aperture are promising for controlling water use efficiency. For example, chemical control of abscisic acid (ABA) signaling through ABA-receptor agonists is a powerful method to activate plant adaptation to water deficit. Such agonists are molecules able to bind and activate ABA receptors and, although their development has experienced significant advances in the last decade, few translational studies have been performed in crops. Here, we describe protection by the ABA mimic-fluorine derivative 4 (AMF4) agonist of the vegetative growth in tomato plants subjected to water restriction. Photosynthesis in mock-treated plants is markedly impaired under water deficit conditions, whereas AMF4 treatment notably improves CO2 assimilation, the relative plant water content and growth. As expected for an antitranspirant molecule, AMF4 treatment diminishes stomatal conductance and transpiration in the first phase of the experiment; however, when photosynthesis declines in mock-treated plants as stress persists, higher photosynthetic and transpiration parameters are recorded in agonist-treated plants. Additionally, AMF4 increases proline levels over those achieved in mock-treated plants in response to water deficit. Thus water deficit and AMF4 cooperate to upregulate P5CS1 through both ABA-independent and ABA-dependent pathways, and therefore, higher proline levels are produced Finally, analysis of macronutrients reveals higher levels of Ca, K and Mg in AMF4- compared to mock-treated plants subjected to water deficit. Overall, these physiological analyses reveal a protective effect of AMF4 over photosynthesis under water deficit and enhanced water use efficiency after agonist treatment. In summary, AMF4 treatment is a promising approach for farmers to protect the vegetative growth of tomatoes under water deficit stress.
RESUMEN
The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.
RESUMEN
Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Ligandos , Sequías , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.
Asunto(s)
Arabidopsis/enzimología , Liasas de Carbono-Oxígeno/antagonistas & inhibidores , Liasas de Carbono-Oxígeno/metabolismo , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacología , Tirosina/metabolismo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Sitios de Unión , Biocatálisis , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/genética , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Mutación , Extractos Vegetales/metabolismo , Conformación Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
The transition from etiolated to green seedlings involves a shift from hypocotyl growth-promoting conditions to growth restraint. These changes occur through a complex light-driven process involving multiple and tightly coordinated hormonal signaling pathways. Nitric oxide (NO) has been lately characterized as a regulator of plant development interacting with hormone signaling. Here, we show that Arabidopsis (Arabidopsis thaliana) NO-deficient mutant hypocotyls are longer than those from wild-type seedlings under red light but not under blue or far-red light. Accordingly, exogenous treatment with the NO donor sodium nitroprusside and mutant plants with increased endogenous NO levels resulted in reduced hypocotyl length. In addition to increased hypocotyl elongation, NO deficiency led to increased anthocyanin levels and reduced PHYB content under red light, all processes governed by phytochrome-interacting factors (PIFs). NO-deficient plants accordingly showed an enhanced expression of PIF3, PIF1, and PIF4. Moreover, exogenous NO increased the levels of the gibberellin (GA)-regulated DELLA proteins and shortened hypocotyls, likely through the negative regulation of the GA Insensitive Dwarf1 (GID1)-Sleepy1 (SLY1) module. Consequently, NO-deficient seedlings displayed up-regulation of SLY1, defective DELLA accumulation, and altered GA sensitivity, thus resulting in defective deetiolation under red light. Accumulation of NO in wild-type seedlings undergoing red light-triggered deetiolation and elevated levels of NO in the GA-deficient ga1-3 mutant in darkness suggest a mutual NO-GA antagonism in controlling photomorphogenesis. PHYB-dependent NO production promotes photomorphogenesis by a GID1-GA-SLY1-mediated mechanism based on the coordinated repression of growth-promoting PIF genes and the increase in the content of DELLA proteins.