RESUMEN
Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Genes de Plantas , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Receptores de Glutamato , Transducción de Señal , Animales , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Conductividad Eléctrica , Fenómenos Electrofisiológicos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Herbivoria/fisiología , Isoleucina/análogos & derivados , Isoleucina/metabolismo , Modelos Animales , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacología , Enfermedades de las Plantas/etiología , Enfermedades de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/efectos de los fármacos , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sinapsis/metabolismo , Transmisión Sináptica , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
The grape berry provides a model for investigating the physiology of non-climacteric fruits. Increased K(+) accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K(+) channel from the Shaker family, VvK1.2, and two CBL-interacting protein kinase (CIPK)/calcineurin B-like calcium sensor (CBL) pairs, VvCIPK04-VvCBL01 and VvCIPK03-VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K(+) channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K(+) currents when co-expressed with the pairs VvCIPK03-VvCBL02 or VvCIPK04-VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK-CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK-CBL partners, VvK1.2 acts as a voltage-gated inwardly rectifying K(+) channel that is activated at voltages more negative than -100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K(+) transport in the berry and to contribute to the extensive re-organization of the translocation pathways and transport mechanisms that occurs at veraison.
Asunto(s)
Frutas/citología , Proteínas de Plantas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Vitis/metabolismo , Animales , Proteínas de Arabidopsis/genética , Proteínas de Unión al Calcio/genética , Clonación Molecular , Sequías , Femenino , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Transporte Iónico , Datos de Secuencia Molecular , Oocitos/fisiología , Filogenia , Proteínas de Plantas/genética , Canales de Potasio de Rectificación Interna/genética , Proteínas Serina-Treonina Quinasas/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo , Vitis/genética , Vitis/crecimiento & desarrolloRESUMEN
Potassium (K(+)) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K(+) channels remain poorly understood. Here, we show that the calcium (Ca(2+)) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K(+) channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca(2+)-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca(2+) sensor modulates K(+) channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Calcio/metabolismo , Canales de Potasio/metabolismo , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Fenotipo , Fosforilación , Canales de Potasio/genética , Proteínas Quinasas/genética , Transporte de Proteínas , Transducción de SeñalRESUMEN
Grapevine (Vitis vinifera), the genome sequence of which has recently been reported, is considered as a model species to study fleshy fruit development and acid fruit physiology. Grape berry acidity is quantitatively and qualitatively affected upon increased K(+) accumulation, resulting in deleterious effects on fruit (and wine) quality. Aiming at identifying molecular determinants of K(+) transport in grapevine, we have identified a K(+) channel, named VvK1.1, from the Shaker family. In silico analyses indicated that VvK1.1 is the grapevine counterpart of the Arabidopsis AKT1 channel, known to dominate the plasma membrane inward conductance to K(+) in root periphery cells, and to play a major role in K(+) uptake from the soil solution. VvK1.1 shares common functional properties with AKT1, such as inward rectification (resulting from voltage sensitivity) or regulation by calcineurin B-like (CBL)-interacting protein kinase (CIPK) and Ca(2+)-sensing CBL partners (shown upon heterologous expression in Xenopus oocytes). It also displays distinctive features such as activation at much more negative membrane voltages or expression strongly sensitive to drought stress and ABA (upregulation in aerial parts, downregulation in roots). In roots, VvK1.1 is mainly expressed in cortical cells, like AKT1. In aerial parts, VvK1.1 transcripts were detected in most organs, with expression levels being the highest in the berries. VvK1.1 expression in the berry is localized in the phloem vasculature and pip teguments, and displays strong upregulation upon drought stress, by about 10-fold.VvK1.1 could thus play a major role in K(+) loading into berry tissues, especially upon drought stress.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Sequías , Proteínas de Plantas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Canales de Potasio de la Superfamilia Shaker/fisiología , Vitis/genética , Ácido Abscísico/farmacología , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Biología Computacional , Frutas/efectos de los fármacos , Frutas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Genoma de Planta/genética , Hibridación in Situ , Filogenia , Componentes Aéreos de las Plantas/efectos de los fármacos , Componentes Aéreos de las Plantas/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Reacción en Cadena de la Polimerasa , Canales de Potasio/clasificación , Canales de Potasio/genética , Canales de Potasio/fisiología , Proteínas Serina-Treonina Quinasas/genética , Canales de Potasio de la Superfamilia Shaker/clasificación , Canales de Potasio de la Superfamilia Shaker/genética , Cloruro de Sodio/farmacología , Vitis/efectos de los fármacosRESUMEN
Guard cells adjust their volume by changing their ion content due to intense fluxes that, for K(+), are believed to flow through inward or outward Shaker channels. Because Shaker channels can be homo- or heterotetramers and Arabidopsis guard cells express at least five genes encoding inward Shaker subunits, including the two major ones, KAT1 and KAT2, the molecular identity of inward Shaker channels operating therein is not yet completely elucidated. Here, we first addressed the properties of KAT1-KAT2 heteromers by expressing KAT1-KAT2 tandems in Xenopus oocytes. Then, computer analyses of the data suggested that coexpression of free KAT1 and KAT2 subunits resulted mainly in heteromeric channels made of two subunits of each type due to some preferential association of KAT1-KAT2 heterodimers at the first step of channel assembly. This was further supported by the analysis of KAT2 effect on KAT1 targeting in tobacco cells. Finally, patch-clamp recordings of native inward channels in wild-type and mutant genotypes strongly suggested that this preferential heteromerization occurs in planta and that Arabidopsis guard cell inward Shaker channels are mainly heteromers of KAT1 and KAT2 subunits.