Academic literature on the topic 'ABA-substrate'

Kohleria eriantha has rhizomes which are underground stems with buds enclosed by modified leaves, that store starch. The buds of this rhizome can develop in two morphological patterns: an aerial shoot (similar to the mother plant) or a rhizome, depending on the water level present in the substrate. Development of the shoot was inhibited by low water availability (1 mL) in the substrate. It was verified that ethylene and ABA were involved in controlling the development of the rhizome pattern under low water availability. Treatments with ethrel, PEG and ABA induced shoot development whereas treatments with AgNO3 or AVG inhibited shoot development. Increased ethylene and ABA were observed under conditions that inhibited shoot development. Moreover, it is suggested that ABA may induce the production of ethylene in the sections of the rhizome under low water availability.

Previous work has shown that exogenous abscisic acid (ABA) applications can reduce transpiration, delay wilting, and thereby extend the shelf life of unwatered plants. Paradoxically, we have seen that drenches with concentrated ABA solutions may actually induce wilting. These wilting symptoms occur despite the presence of ample water in the substrate, suggesting that ABA may interfere with the ability of roots to take up water. Our objective was to develop a better understanding of this wilting effect using tomato (Solanum lycopersicum) as a model. In the first study, ABA drenches (125–2000 mg·L−1) reduced transpiration and water use compared with the control plants, yet the relative water content (RWC) of the leaves of ABA-treated plants was lower than that of control plants at 24 h after the ABA drench. Control plants had a leaf RWC of 97%, whereas plants treated ABA had a RWC of 57% to 62%. ABA concentrations of 500 mg·L−1 or higher caused the plants to wilt within 24 h despite the presence of ample water in the substrate. Leaf ABA concentrations 24 h after the ABA application ranged from 2.6 (control) to 62.6 nmol·g−1 fresh weight (FW) in the 2000-mg·L−1 ABA treatment, indicating effective transport of ABA from the roots to the leaves. The reduced leaf RWC suggests that ABA drenches are limiting water transport through the roots to the leaves. The effects of ABA on the hydraulic conductance of the roots and stems of tomatoes were quantified to determine if ABA drenches limit water transport through the roots. The cumulative volume of water conducted by the root systems during a 4-day period ranged from 36.7 mL in the control treatments to 8.1 mL in roots systems drenched with 1000 mg·L−1 ABA, a reduction of 78%. When the conductance study was repeated using decapitated roots and excised stems, root water flux was again reduced by ABA, but water flux through internodal stem sections did not show an ABA effect. Results suggest that ABA-induced wilting is caused by a reduction in root conductance and we hypothesize that ABA affects aquaporins in the roots, limiting water uptake.

Foliar sprays or root dips of synthetic abscisic acid (s-ABA) have shown to reduce the transpiration rate and subsequently prolong postharvest longevity in a select group of herbaceous ornamental crops. The objective of our study was to determine the impact of s-ABA on postproduction performance of seed impatiens in greenhouse or low light conditions. Market ready Impatiens wallerana `Xtreme Scarlet' plants were sprayed or the root substrate was drenched with s-ABA at 250 or 500 mg·L–1 then boxed for 48 h to represent shipping conditions. Flower number was measured 3 days after application, and again after plants were hydrated following the day when the last treatment wilted 0, 2, 4, 8, 16, or 24 days after application. Visual quality ratings were made 0, 2, 3, 4, 8, 11, 16, or 19 days after application and again after plants were re-irrigated. Drenching the substrate with s-ABA at 500 mg·L–1 maintained foliage and flower turgidity up to 8 days in the greenhouse environment and 16 days in the low light environment. Substrate drenches at 500 mg·L–1 dramatically decreased flower number after removal from the shipping box under greenhouse conditions, and in the low light environment drenching the substrate at 250 mg·L–1 produced similar visual quality results to 500 mg·L–1 16 days after treatment. Plants drenched at 250 mg·L–1 also had the same number of flowers 3 and 20 days after treatment, when compared to 500 mg·L–1. Therefore, impatiens growers should drench the root substrate with s-ABA at 250 mg·L–1 to reduce labor costs associated with hand-watering and prolong postproduction performance in low light conditions, such as indoor retail conditions.

The plant hormone, abscisic acid (ABA), is not only important for promoting abiotic stress responses but also plays a versatile and crucial role in plant immunity. The pathogen infection-induced dynamic accumulation of ABA mediates the degradation of non-expresser of PR genes 1 (NPR1) through the CUL3NPR3NPR4 proteasome pathway. However, the functional significance of NPR1 degradation by other E3 ligases in response to ABA remains unclear. Here, we report that NPR1 is induced transcriptionally by ABA and that npr1-1 mutation results in ABA insensitivity during seed germination and seedling growth. Mutants lacking NPR1 downregulate the expression of ABA-responsive transcription factors ABA INSENSITIVE4 (ABI4) and ABA INSENSITIVE5 (ABI5), and that of their downstream targets EM6, RAB18, RD26, and RD29B. The npr1-1 mutation also affects the transcriptional activity of WRKY18, which activates WRKY60 in the presence of ABA. Furthermore, NPR1 directly interacts with and is degraded by HOS15, a substrate receptor for the DDB1-CUL4 ubiquitin E3 ligase complex. Collectively, our findings demonstrate that NPR1 acts as a positive regulator of ABA-responsive genes, whereas HOS15 promotes NPR1 degradation in a proteasome-dependent manner.

The natural composition of nutrients present in food is a key factor determining the immune function and stress responses in the honeybee ( Apis mellifera ). We previously demonstrated that a supplement of abscisic acid (ABA), a natural component of nectar, pollen, and honey, increases honeybee colony survival overwinter. Here we further explored the role of ABA in in vitro -reared larvae exposed to low temperatures. Four-day-old larvae (L4) exposed to 25°C for 3 days showed lower survival rates and delayed development compared to individuals growing at a standard temperature (34°C). Cold-stressed larvae maintained higher levels of ABA for longer than do larvae reared at 34°C, suggesting a biological significance for ABA. Larvae fed with an ABA-supplemented diet completely prevent the low survival rate due to cold stress and accelerate adult emergence. ABA modulates the expression of genes involved in metabolic adjustments and stress responses: Hexamerin 70b, Insulin Receptor Substrate, Vitellogenin , and Heat Shock Proteins 70. AmLANCL2, the honeybee ABA receptor, is also regulated by cold stress and ABA. These results support a role for ABA increasing the tolerance of honeybee larvae to low temperatures through priming effects.

Abscisic acid (ABA) is a plant hormone involved in regulating stomatal responses to environmental stress. By inducing stomatal closure, applications of exogenous ABA can reduce plant water use and delay the onset of drought stress when plants are not watered. However, ABA can also cause unwanted side effects, including chlorosis. Pansy (Viola ×wittrockiana) has been shown to be particularly susceptible to ABA-induced chlorosis. The objective of this study was to determine if fertilization rate affects the severity of ABA-induced chlorosis in this species. ‘Delta Premium Pure Yellow’ pansy seedlings were fertilized with controlled-release fertilizer incorporated at rates from 0 to 8 g·L−1 of substrate. When plants had reached a salable size, half the plants were sprayed with a solution containing 1 g·L−1 ABA, whereas the other plants were sprayed with water. Leaf chlorophyll content was monitored for 2 weeks following ABA application. Leaf chlorophyll content increased greatly as fertilizer rate increased from 0 to 2 g·L−1, with little increase in leaf chlorophyll at even higher fertilizer rates. ABA induced chlorosis, irrespective of the fertilizer rate. Plant dry weight was lowest when no controlled-release fertilizer was incorporated, but similar in all fertilized treatments. ABA treatment reduced shoot dry weight by ≈24%, regardless of fertilization rate. This may be due to ABA-induced stomatal closure, which limits carbon dioxide (CO2) diffusion into the leaves. We conclude that ABA sprays induce chlorosis, regardless of which fertilizer rate is used. However, because leaf chlorophyll concentration increases with increasing fertilizer rates, higher fertilizer rates can mask ABA-induced chlorosis.

A cell-free preparation from avocado fruit incorporates [14C]mevalonate into ABA. A number of specific inhibitors have been used to probe the system and tungstate ions at 100 µM reduce the 14C in ABA by 80% The inhibitory effect was overcome by the alkaloid cinchonine (2000 µM) which binds tungstate strongly and selectively. More 14C from mevalonate was present in xanthoxal (4600 dpm), less in ABA (340 dpm) when the cell-free system was inhibited by tungstate (100 µM) than in controls (1810 dpm in xanthoxal, 1200 dpm in ABA), which shows that xanthoxal is the substrate for the aldehyde oxidase. Xanthoxic acid, therefore, is the next intermediate and AB-aldehyde is not a normal precursor. The potential for using the tungstate/cinchonine reaction to probe other biosynthetic pathways which require a molybdate ion is discussed.

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.

Abstract A lack of adequate watering reduces the shelf life of many ornamental plants during retail. Our goals were to determine whether sprays or drenches with abscisic acid (ABA) can reduce transpiration and extend the shelf life of hydrangea (Hydrangea macrophylla). During the first 5 days after treatment, ABA drenches of 125 to 1000 ppm reduced stomatal conductance (gs) by 50 to 80% as compared to water. ABA-induced stomatal closure reduced plant water uptake from the substrate; control plants took up half of the plant-available water during the first 7 days after treatment, while it took 14 days for plants drenched with 1000 ppm to take up half of the available water. Control plants wilted after 12 days and time to wilting of drenched plants increased with increasing ABA concentrations, up to 23 days in the 1000 ppm treatment. Spray treatments had little effect on gs and no detectable effect on water uptake or time to wilting. Some yellowing of older leaves was seen with ABA drenches of 500 or 1000 ppm. Despite this side effect, ABA drenches have potential to extend the shelf life of hydrangeas in retail environments.

We analysed the role and relationship between hydrogen peroxide (H2O2) reduction and the inhibition of abscisic acid (ABA)-induced stomatal closure by ethylene. Like ascorbic acid (ASA), the most important reducing substrate for H2O2 removal, catalase, one of the H2O2 scavenging enzymes and diphenylene iodonium, an inhibitor of the H2O2-generating enzyme NADPH oxidase, both ethylene-releasing compound 2-chloroethylene phosphonic acid (ethephon, ETH) and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, were found to inhibit stomatal closure by ABA and to reduce H2O2 levels by ABA in guard cells, indicating that ethylene-caused inhibition of ABA-induced stomatal closure involves reduction of H2O2 levels in guard cells. Additionally, similar to ASA and catalase, ACC/ETH not only suppressed H2O2-induced stomatal closure and H2O2 levels in guard cells treated with exogenous H2O2 in light, but also reopened the stomata which had been closed by ABA and reduced H2O2 levels that had been generated by ABA. The abovementioned effects of ACC and ETH were dissimilar to that of diphenylene iodonium, an inhibitor of the H2O2-generating enzyme NADPH oxidase, which not only had incapability to reduce H2O2 levels by exogenous H2O2 but also could not abolish H2O2 that had been generated by ABA. So we suggest that ethylene probably induces H2O2 removal and reduces H2O2 levels in Vicia faba guard cells, and finally inhibits stomatal closure induced by ABA.

[ES] La ubiquitinación inducida por hormonas desempeña un papel crucial en la vida media de los reguladores negativos clave de la propia señalización hormonal. En la señalización por ABA, los reguladores negativos clave son las PP2Cs del clado A, como PP2CA o ABI1, y su degradación es un mecanismo complementario a la inhibición de su actividad mediada por PYR/PYL/RCAR. El ABA promueve la degradación de ABI1 a través de las E3 ligasas PUB12/13, y PP2CA a través de las E3 ligasas RGLG1/5. Sin embargo, se predice que otras E3 ligasas no identificadas también regularán la vida media de las PP2Cs del clado A. En pasos posteriores de la señalización por ABA, el ABA también induce la regulación positiva de los niveles de transcrito y proteína de las PP2Cs como un mecanismo de retroalimentación negativa. Por lo tanto, restablecer la señalización de ABA también requiere la degradación de las PP2Cs para evitar su acumulación excesiva inducida por el propio ABA. En este trabajo identificamos las proteínas BTB/POZ AND MATH DOMAIN (BPM), adaptadores del sustrato de las E3 ligasas multiméricas CULLIN3-RING E3 (CRL3), como proteínas que interactúan con las PP2Cs. BPM3 y BPM5 interactúan en el núcleo con PP2CA, así como con ABI1, ABI2 y HAB1. Además, BPM3 y BPM5 aceleran la degradación de las PP2Cs de una manera dependiente del ABA y su sobreexpresión conduce a una mayor sensibilidad al ABA. Además, las plantas mutantes bpm3 bpm5 mostraron una mayor acumulación de PP2CA, ABI1 y HAB1, lo que conduce a una sensibilidad global disminuida al ABA. Finalmente, utilizando ensayos bioquímicos y genéticos, demostramos que las BPM aumentaban la ubiquitinación de PP2CA. Dado que la formación de los complejos ternarios receptor-ABA-fosfatasa se ve notablemente afectada por la abundancia de sus componentes proteicos y la concentración de ABA, revelamos que las BPM y las E3 ligasas multiméricas CRL3 son moduladores importantes de los niveles del correceptor PP2C para regular la señalización temprana de ABA, así como durante los consiguientes pasos de restablecimiento. Al contrario que con PUB12/13, no se sabe cómo el ABA aumenta la degradación de PP2CA a través de RGLG1/5. En el caso de RGLG1, esta proteína se encuentra predominantemente en la membrana plasmática, mientras que PP2CA se encuentra predominantemente en el núcleo. Nosotros demostramos que el ABA modifica la localización subcelular de RGLG1, promoviendo la interacción nuclear con PP2CA. En primer lugar, encontramos que RGLG1 está miristoilado in vivo, lo que facilita su unión a la membrana plasmática, sin embargo, el ABA inhibe esta miristoilación. El ABA también regula negativamente a N-myristoyltransferase 1, la enzima activa y central en la miristoilación de proteínas, esto puede ayudar a promover la translocación de RGLG1 al núcleo. Allí, RGLG1 puede interactuar con ciertos receptores monoméricos del ABA, como PYL8. El reclutamiento nuclear de la E3 ligasa también fue promovido por el aumento de los niveles de proteína PP2CA y por la formación de complejos RGLG1-PYL8-PP2CA en presencia de ABA. Además, nosotros encontramos que RGLG1Gly2Ala, mutada en el sitio de miristoilación N-terminal, muestra localización nuclear constitutiva y provoca una respuesta más sensible al ABA y al estrés salino y osmótico. En resumen, proporcionamos evidencia de que una ligasa E3 puede reubicarse dinámicamente en respuesta al ABA, el estrés salino y osmótico, y el aumento de los niveles de su sustrato, lo que revela un mecanismo para explicar cómo el ABA mejora la interacción RGLG1-PP2CA y, por lo tanto, la degradación de PP2CA.
[CAT] L' ubiquitinació induïda per hormones té un paper crucial en la vida mitjana dels reguladors negatius clau de la pròpia senyalització hormonal. En la senyalització per ABA, els reguladors negatius clau són les PP2Cs del clado A, com PP2CA o ABI1, i la seva degradació és un mecanisme complementari a la inhibició de la seva activitat mediada per PYR/PYL/RCAR. El ABA promou la degradació de ABI1 a través de les E3 lligases PUB12/13, i PP2CA per mitja de les E3 lligases RGLG1/5. No obstant això, es prediu que altres E3 lligases no identificades també regularan la vida mitjana de les PP2Cs del clado A. En passos posteriors de la senyalització per ABA, l'ABA també indueix la regulació positiva de nivells de transcrit i proteïna de les PP2Cs com un mecanisme de retroalimentació negativa. Per tant, restablir la senyalització d'ABA també requereix la degradació de les PP2Cs per evitar la seva acumulació excessiva induïda pel propi ABA. En aquest treball identifiquem les proteïnes BTB/POZ AND MATH DOMAIN (BPM), adaptadors del substrat de les E3 lligases multimèriques CULLIN3-RING E3 (CRL3), com proteïnes que interactuen amb les PP2Cs. BPM3 i BPM5 interactuen en el nucli amb PP2CA, així com amb ABI1, ABI2 i HAB1. A més, BPM3 i BPM5 acceleren la degradació de les PP2Cs d'una manera dependent del ABA i la seva sobreexpressió porta a una major sensibilitat al ABA. A més, les plantes mutants bpm3 bpm5 van mostrar una major acumulació de PP2CA, ABI1 i HAB1, el que porta a una sensibilitat global disminuïda a ABA. Finalment, utilitzant assajos bioquímics i genètics, aconseguint que les BPM augmentaven l' ubiquitinació de PP2CA. Atès que la formació dels complexos ternaris receptor-ABA-fosfatasa es veu notablement afectada per l'abundància dels seus components proteics i la concentració d'ABA, revelem que les BPM i les E3 lligases multimèriques CRL3 són moduladors importants dels nivells del coreceptor PP2C per regular la senyalització primerenca de ABA, així com durant els consegüents passos de restabliment. Al contrari que amb PUB12/13, no se sap com el ABA augmenta la degradació de PP2CA a través d'RGLG1/5. En el cas de RGLG1, aquesta proteïna es troba predominantment en la membrana plasmàtica, mentre que PP2CA es troba predominantment en el nucli. Nosaltres vam demostrar que l'ABA modifica la localització subcelular de RGLG1, promovent la interacció nuclear amb PP2CA. En primer lloc, trobem que RGLG1 està miristoilado in vivo, el que facilita la seva unió a la membrana plasmàtica, però, el ABA inhibeix aquesta miristoilación. El ABA també regula negativament N-myristoyltransferase 1, l' enzim actiu i central en la miristoilación de proteïnes, això pot ajudar a promoure la translocació de RGLG1 al nucli. Allà, RGLG1 pot interactuar amb certs receptors monomèrics de l'ABA, com PYL8. El reclutament nuclear de l'E3 lligasa també va ser promogut per l'augment dels nivells de proteïna PP2CA i per la formació de complexos RGLG1-PYL8-PP2CA en presència d'ABA. A més, nosaltres trobem que RGLG1Gly2Ala, mutada en el lloc de miristoilació N-terminal, mostra localització nuclear constitutiva i provoca una resposta més sensible al ABA i l'estrès salí i osmòtic. En resum, proporcionem evidència que una ligasa E3 pot reubicar dinàmicament en resposta al ABA, l'estrès salí i osmòtic, i l'augment dels nivells de la seva substrat, el que revela un mecanisme per explicar com el ABA millora la interacció RGLG1-PP2CA i, per tant, la degradació de PP2CA.
[EN] Hormone-induced ubiquitination plays a crucial role to determine the half-life of key negative regulators of hormone signaling. In case of ABA signaling, the key negative regulators are the clade-A PP2Cs, such as PP2CA or ABI1, and their degradation is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of their activity. ABA promotes the degradation of ABI1 through the PUB12/13 E3 ligases, and PP2CA through the RGLG1/5 E3 ligases. However, other unidentified E3 ligases are predicted to regulate clade A PP2Cs half-life as well. At later steps of ABA signaling, ABA also induces upregulation 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. In this work we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric CULLIN3-RING E3 ligases (CRL3s), as PP2C-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2 and HAB1. Additionally, BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity. Moreover, bpm3 bpm5 mutant plants showed increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Finally, using biochemical and genetic assays we demonstrated that BPMs enhance the ubiquitination of PP2CA. 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 co-receptor levels to regulate early ABA signaling as well as the subsequent resetting steps. In contrast to PUB12/13, it was not known how ABA enhances the degradation of PP2CA by RGLG1/5. RGLG1 is predominantly found in the plasma membrane whereas PP2CA is predominant in the nucleus. We demonstrate that ABA modifies the subcellular localization of RGLG1, promoting nuclear interaction with PP2CA. Firstly, we found that RGLG1 is myristoylated in vivo, which facilitates its attachment to the plasma membrane, nevertheless, ABA inhibits its myristoylation. ABA also downregulates N-myristoyltransferase 1, the central active enzyme of protein myristoylation, which may help to promote RGLG1 translocation to the nucleus. There, RGLG1 can interact with certain monomeric ABA receptors, as PYL8. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-PYL8-PP2CA complexes in the presence of ABA. Additionally, we found that RGLG1Gly2Ala protein, mutated at the N-terminal myristoylation site, shows constitutive nuclear localization and causes an enhanced response to ABA and salt and osmotic stresses. In summary, we provided evidence that an E3 ligase can dynamically relocalize in response to ABA, salt and osmotic stress, and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation.
Julián Valenzuela, J. (2020). Regulation of ABA signaling through degradation of clade A PP2Cs by the RGLG1 and CRL3 BPM E3 ligases [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137777
TESIS

[ES] El crecimiento de las plantas se ve afectado por el estrés abiótico, sequía, salinidad o altas temperaturas. La transducción de señales de estrés abiótico es fundamental para generar una respuesta fisiológica adecuada, que implica la participación de diferentes hormonas vegetales, siendo el ácido abscísico (ABA) el regulador hormonal crítico en la regulación de la respuesta de la planta a situaciones de estrés por déficit hídrico. La vía de señalización de ABA y los componentes principales están bien caracterizados molecular y bioquímicamente. Los receptores de ABA "Pyrabactin Resistance 1"(PYR)/"PYR1-LIKE" (PYL)/ "Regulatory Component of ABA Receptor" (RCAR) juegan un papel importante en la regulación cuantitativa de la señalización ABA tanto en semillas como en tejidos vegetativos. Aunque la función bioquímica de los receptores PYR/PYL/RCARs de ABA, está bien caracterizada, se conoce poco sobre otros aspectos con relevancia biológica, como sus modificaciones postraduccionales o la regulación de su vida media. Uno de los avances recientes en este campo ha sido el descubrimiento de una nueva familia de E3 ligasas llamadas RSL1/RFAs ("RING-finger-ABA-related") que consta de al menos 10 miembros, reguladores clave de la estabilidad de los receptores PYR/PYL/RCAR de ABA en tejidos de raíces y hojas, regulando su degradación en diferentes ubicaciones celulares. Un estudio detallado de esta familia génica reveló que RSL1/RFA se caracterizan estructuralmente por la presencia de tres dominios RING putativos en tándem, denominados "RING1-IN BETWEEN RING-RING2" (RBR), y en consecuencia pertenecen a la familia de E3 ligasas de tipo RBR. Cinco miembros de la familia RSL1/RFA, RSL1 y RFA6-RFA9, contienen un dominio TM en el extremo C-terminal, lo que sugiere que RFA6-RFA9 también se localizan en la membrana plasmática. Sin embargo, otros miembros de las E3 ligasas como RFA1-RFA5 carecen del dominio TM C-terminal y su caracterización funcional, así como su ubicación celular, aún no se conocen. Nosotros mostramos que la E3 ligasa RFA1 se localiza en núcleo y citosol, mientras que RFA4 muestra una localización específica en el núcleo promoviendo la degradación nuclear de los receptores ABA. Por lo tanto, los miembros de la familia RSL1/RFA interactúan con los receptores ABA en la membrana plasmática, el citosol y el núcleo, dirigiéndolos a su degradación a través de la vía endosomal/vacuolar (en el caso de RSL1) o el proteosoma 26S (para RFA1 y RFA4). Proporcionamos información sobre la función fisiológica de estas E3 ligasas de tipo RBR. Realizando tanto mutagénesis como ensayos bioquímicos para identificar la cisteína 361 (Cys361) en RFA4 como la Cys del sitio activo, que es una característica distintiva de las E3 ligasas de tipo RBR. Demostramos mediante análisis de inmunotransferencia del mutante con pérdida de función de rfa1rfa4 que los niveles endógenos de los receptores de ABA PYR1 y PYL4 aumentan en comparación con las plantas de tipo silvestre. Hemos identificado una enzima E2, "Ubiquitin Conjugating Enzyme 26" (UBC26), como la enzima nuclear canónica E2 que interactúa con la E3 ligasa RFA4 y forma complejos UBC26-RFA4-Receptor, formando agregados nucleares. Generamos alelos ubc26 con pérdida de función que mostraban una mayor sensibilidad a ABA y acumulación de receptores ABA en comparación con el tipo silvestre. En definitiva, hemos revelado un sofisticado sistema de ubiquitinación de receptores ABA en diferentes ubicaciones subcelulares llevado a cabo a través de la familia de E3 ligasas RSL1/RFA de tipo RBR. Por otro lado, hemos iniciado pruebas bioquímicas para identificar la S-acilación en el dominio TM de RSL1. Generando RSL1C334S, RSL1 C5S y RSL1C6S mediante mutagénesis y RSL1ΔTM que presenta una delección del dominio TM. Los estudios iniciales han demostrado que los residuos de Cys cercanos al dominio TM están S-acilados. Finalmente, generamos nu
[CA] El creixement de les plantes es pot veure afectat per l'estrès abiòtic, sequera, salinitat o altes temperatures. La transducció de senyals d'estrès abiòtic és fonamental per a generar una resposta fisiològica adequada, que implica la participació de diferents hormones vegetals, sent l'àcid abscísic (ABA) el regulador hormonal crític en la regulació de la resposta de la planta a situacions d'estrès per dèficit hídric. La ruta de senyalització d'ABA i els components principals de la ruta estan ben caracteritzats molecularment i bioquímica. Els receptors "Pyrabactin Resistance 1"(PYR)/"PYR1-LIKE"(PYL)/"Regulatory Component of ABA Receptor" (RCAR) exerceixen un paper important en la regulació quantitativa en resposta a l'estrès tant en llavors com en planta. Encara que la funció bioquímica dels receptors PYR/PYL/RCARs d'ABA, està ben caracteritzada en els últims anys, es coneix poc sobre altres aspectes amb rellevància biològica, com les seues modificacions postraduccionals o la regulació de la seua vida mitjana. Un dels avanços recents en aquest camp ha sigut el descobriment d'una nova família d'E3 ligases anomenades RSL1/RFAs ("RING-finger-ABA-related") que consta d'almenys 10 membres, que són reguladors clau de l'estabilitat dels receptors PYR/PYL/RCAR d'ABA en teixits d'arrels i fulles, regulant la seua degradació en diferents ubicacions cel·lulars. Un estudi més detallat d'aquesta família gènica va revelar que RSL1/RFAs es caracteritzen estructuralment per la presència de tres dominis RING putatius en tàndem, denominats "RING1-IN BETWEEN RING-RING2" (RBR), i en conseqüència pertanyen a la família d'E3 ligases de tipus RBR. Cinc membres de la família RSL1/RFA, RSL1 i RFA6-RFA9, contenen un domini TM en l'extrem C-terminal, la qual cosa suggereix que RFA6-RFA9 també es localitzen en la membrana plasmàtica. No obstant això, altres membres d'aquesta família d'E3 ligases com RFA1-RFA5 manquen del domini TM C-terminal i la seua caracterització funcional, així com la seua ubicació cel·lular, encara no ha sigut investigada. Vam mostrar que l'E3 ligasa RFA1 es localitza tant en el nucli com en el citosol, mentre que RFA4 mostra una localització específica en el nucli promovent la degradació nuclear dels receptors ABA. Per tant, els membres de la família RSL1/RFA interactuen amb els receptors ABA en la membrana plasmàtica, el citosol i el nucli, dirigint-los a la seua degradació a través de la vía endosomal/vacuolar (en el cas de RSL1) o el proteosoma 26S (per a RFA1 i RFA4). Proporcionem informació sobre la funció fisiològica d'aquestes E3 ligases de tipus RBR. Realitzant tant mutagènesis com a assajos bioquímics per a identificar la cisteïna 361 (Cys361) en RFA4 com la Cys del lloc actiu, que és una característica distintiva de les E3 ligases de tipus RBR. Hem demostrat mitjançant una anàlisi d'immuno-transferència del mutant amb pèrdua de funció de rfa1rfa4 que els nivells endògens dels receptors d'ABA PYR1 i PYL4 augmenten en comparació amb les plantes de tipus silvestre. D'altra banda, hem identificat un enzim E2, "Ubiquitin Conjugating Enzyme 26" (UBC26), com l'enzim nuclear canònic E2 que interactua amb l'E3 ligasa RFA4 i forma complexos UBC26-RFA4-Receptor, formant agregats nuclears. També generem al·lels ubc26 amb pèrdua de funció que mostraven una major sensibilitat a ABA i acumulació de receptors ABA en comparació amb el tipus silvestre. En definitiva, hem revelat un sofisticat sistema d'ubiquitinació de receptors ABA en diferents ubicacions subcel·lulars dut a terme a través de la família d'E3 ligases RSL1/RFA de tipus RBR. Hem iniciat proves bioquímiques per a identificar la S-acilació en el domini TM de RSL1. Hem generat RSL1C334S, RSL1 C5S i RSL1C6S mitjançant mutagènesis, així com RSL1ΔTM que presenta una delecció del domini TM. Els estudis inicials han demostrat que els residus de Cys pròxims al domini TM estan S-acilados. Final
[EN] Plant growth is affected by abiotic stress, drought, salinity or high temperature. Signal transduction of abiotic stress is crucial to generate an appropriated physiological response, which involves the participation of different plant hormones, being abscisic acid (ABA) the critical hormonal regulator in regulating the plant's response to situations of stress due to water deficit. The ABA signaling pathway and the major components of the pathway are well characterized molecularly and biochemically. Pyrabactin Resistance 1 (PYR)/PYR1-LIKE (PYL)/Regulatory Component of ABA Receptor (RCAR) ABA receptors play an important role in quantitative regulation of ABA signaling both in seeds and vegetative tissues. Although the biochemical function of the PYR/PYL/RCAR ABA receptors has been well established in recent years, little is known about other aspects with biological relevance, such as their post-translational modifications or the regulation of their half-life. One of the recent advances in this field has been the discovery of a new family of E3 ligases called RSL1/RFAs (RING-finger-ABA-related) that consists of at least 10 members, which are key regulators of the stability of PYR/PYL/RCARs in root and leaf tissues, and regulate the degradation of ABA receptors at different cellular locations. Further inspection of the gene family revealed that RSL1/RFAs are structurally characterized by the presence of three putative RING domains in tandem, named as RING1-IN BETWEEN RING (IBR)-RING2, and accordingly they belong to the RBR-type E3 ligase family. Five members of the RSL1/RFA family, that is, RSL1 and RFA6-RFA9, contain a TM domain at the C-terminal end of the proteins, which suggests that RFA6-RFA9 are also localized in plasma membrane. However, other members of this family of E3 ligases such as RFA1-RFA5 lack the C-terminal TM domain and their functional characterization, as well as their cellular location, has not been investigated yet. In this study we show that the E3 ligase RFA1 is localized both in the nucleus and in the cytosol, while RFA4 shows a specific localization in the nucleus promoting the nuclear degradation of ABA receptors. Therefore, we members of the RSL1/RFA family interact with ABA receptors at the plasma membrane, cytosol and nucleus, targeting them for degradation via the endosomal/vacuolar pathway (in the case of RSL1) or the 26S- proteasome (for RFA1 and RFA4). We provide information on the physiological function of these RBR-type E3 ligases, which are hardly explored in plants. Additionally, we performed mutagenesis and biochemical assays to identify Cys361 in RFA4 as the active site cysteine, which is a distinctive feature of RBR-type E3 ligases. We have shown by immunoblot analysis of the rfa1rfa4 loss-of-function mutant that endogenous levels of ABA receptors PYR1 and PYL4 are increased compared to wild-type plants. On the other hand, we have identified an E2 enzyme, Ubiquitin Conjugating Enzyme 26 (UBC26), as the canonical nuclear enzyme E2 that interacts with the E3 ligase RFA4 and forms UBC26-RFA4-Receptor complexes, forming nuclear aggregates. We also generated loss-of function ubc26 alleles that exhibited higher sensitivity to ABA and accumulation of ABA receptors compared to wild type. We have revealed a sophisticated ubiquitination system of ABA receptors in different subcellular locations carried out through the RBR-type RSL1/RFA family of E3 ligases. We have proceeded with the biochemical and genetic study of the different members of the family. We have started biochemical tests to identify the S-acylation in the TM domain of RSL1. To this end, we have generated RSL1C334S, RSL1 C5S and RSL1C6S by mutagenesis as well as RSL1ΔTM, a deletion of the TM domain. Initial studies have shown that Cys residues close to the TM domain are S-acylated. Finally, we have also generated new combined mutants: rsl1rfa1, rsl1rfa5, rfa1rfa5 and rsl1rfa1rfa5.
Fernández López, MA. (2021). Regulación de la señalización del ABA mediante mecanismos que controlan vida media y actividad de los receptores PYR/PYL [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172364
TESIS

Cryptic elements of proteins are not visible in unbound structures but become apparent in ligand/substrate-bound structures. These binary states of cryptic elements in protein structures can impact important aspects of proteins such as substrate recognition, catalytic mechanism and are also utilized for drug-design against difficult protein targets. The thesis explores distinct facets of cryptic elements in different proteins through a combination of experimental and computational approaches. More specifically, while addressing a problem specific to a given protein, the work presented in the thesis shows how cryptic elements of that protein assist in finding a solution. Targeting cryptic sites for drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, it is difficult to identify cryptic sites. Use of small glycols as probe molecules to identify cryptic sites in proteins is demonstrated through crystallography experiments, molecular dynamics simulations, protein dataset construction and analysis. The study suggests the use of small glycols in both experimental as well as computational methods to identify cryptic sites in proteins, thus facilitating drug-design for undruggable and/or difficult protein targets and expanding the druggable proteome. Abrin is an extremely cytotoxic ribosome inactivating protein. Abrin A-chain (ABA) inactivates eukaryotic ribosomes leading to cell death. The crystal structures of apo-ABA and ABA complexed with substrate analogs were determined in order to study ABA’s substrate binding and catalytic mechanism. The analyses of ABA-substrate analog crystal structures show the presence of “two binding pockets" feature not apparent in unbound-ABA structure, making it a cryptic feature of ABA and sheds light on aspects of substrate recognition by ABA. Catalytic water was clearly located in ABA-Adenine crystal structure but was not observed in unbound-ABA crystal structure, making it another cryptic feature of ABA structure and was used to explain the catalytic mechanism of ABA. A monoclonal antibody (mAb) D6F10 is known to neutralize the toxicity of abrin. The structures of ABA-Adenine complex and ribosome were used to construct ABA-Ribosome complex. A 3D homology model of variable region (Fv) of mAb D6F10 was generated and was docked with apo-ABA structure to obtain computational model of ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. Furthermore, crystal structure of fragment crystallizable (Fc) region of mAb D6F10 at 1.95 Å resolution was determined. Crystal structure based glycan analysis of mAb D6F10 Fc confirmed the presence of speculated terminal galactose on glycans of mAb D6F10 Fc fragment thereby pointing to the proposed model of abrin neutralization at 1:100 molar ratio of abrin:D6F10. Rv0731c, a protein from human pathogen Mycobacterium tuberculosis, is annotated as putative S-adenosyl-L-methionine(SAM)-dependent methyltransferase. A high resolution crystal structure of Rv0731c at 1.63 Å was determined and bioinformatics was used to predict its biological function. The structural analysis of Rv0731c revealed a conserved carboxy-terminal SAM binding domain and a methyl-accepting substrate binding domain similar to that of the leucine carboxyl methyltransferases from human and yeast. In apo-Rv0731c, the substrate binding site is occluded by a flexible loop-helix flap segment and substrate binding may displace the flap revealing the site thereby making it a cryptic feature of Rv0731c and sheds light on the features of substrate recognition by Rv0731c