SLAC1 anioonkanali ja tema regulaatorite roll õhulõhede avanemisel ja sulgumisel müürloogas (Arabidopsis thaliana)

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Taimelehtede pinnal on mikroskoopilised poorid, mida kutsutakse õhulõhedeks. Nende kaudu toimub CO2 omastamine ja transpiratsioon. Igat õhulõhet ümbritseb paar sulgrakke, mille osmootsest rõhust ja veesisaldusest sõltub poori suurus. Õhulõhede avanemise ja sulgumise regulatsiooni detailse mehhanismi selgumine aitab kaasa uue põlvkonna põuatolerantsete sortide aretamisele. Me uurisime õhulõhede avanemise ja sulgumise signaalradu. Selleks panime müürlooga taime hermeetilisse kambrisse ja mõõtsime taimest väljuva veeauru hulka. Pimeduse, kõrge CO2 taseme, vähese õhuniiskuse, taimehormoon abstsiishappe (ABA) ja osooni toimel õhulõhed sulguvad ning taimest eralduva veeauru hulk väheneb. Seevastu valgus, madal CO2 tase ja kõrge õhuniiskus stimuleerivad õhulõhesid avanema, mis meie mõõteaparatuuris kajastub suurema taimest väljuva veeauru hulgana. Uurisime ABA toimel käivituvat signaalrada ja selle rolli õhulõhede sulgumises keskkonnafaktorite toimel. Näitame, et valgud, mida on varem kirjeldatud pelgalt ABA signaalraja osana, osalevad ka muudes signaalradades. Näiteks kui mutantses taimes puuduvad ABA retseptorvalgud , ei sulge see taim enam oma õhulõhesid ei osooni, pimeduse, kõrge CO2 taseme ega vähese õhuniiskuse toimel. Lisaks uurisime õhulõhede avanemist anioonide väljavoolukanali SLAC1 mutantides, mis pole võimelised õhulõhesid kiirelt sulgema. Nendes mutantides oli kummalisel kombel ka õhulõhede avanemine aeglasem, kuigi SLAC1 kanali puudumine takistab ainult ioonide väljavoolu. San Diego (USA) koostööpartnerite abil selgitasime välja, et õhulõhede aeglase avanemise põhjuseks on vähenenud kaaliumi sissevool, mis on tingitud hoopis muutustest sulgrakkude kaltsiumi tundlikkuses ning kontsentratsioonis SLAC1 mutandis. Seega on õhulõhede avanemise ja sulgumise signaalrajad omavahel läbi põimunud ning mutatsioon võivad taimes mõjutada isegi protsesse, mis ei ole puuduva valguga otseselt seotud.Stomata are microscopic pores on the surface of leaves, mediating CO2 uptake and transpirational water loss. Stomata close when ions are released from the two guard cells surrounding each pore. Understanding the mechanisms of stomatal regulation would help to design next generation drought tolerant cultivars in crop breeding. We studied the signaling pathways leading to stomatal opening or closure. Thale cress plants were placed into hermetically sealed flow-through chambers and the amount of water evaporating from the plant was measured. Darkness, elevated CO2, low air humidity, plant hormone abscisic acid (ABA), and ozone induce stomatal closure, resulting in decreased evaporation from the plant. Conversely, light, reduced CO2, and high humidity induce stomatal opening, which is manifested by larger amount of water transpired from plant. We studied the ABA signaling pathway and showed that the components of ABA signaling pathway participate in stomatal closure induced by different abiotic factors. This can be illustrated with mutant plants lacking ABA receptors – these plants also lack stomatal closure in response to ozone, darkness, low air humidity and have reduced response to elevated CO2. Stomatal opening and closure are opposite processes as regards the direction of ion movement, but are partly co-regulated. SLAC1 anion channel mutants with impaired stomatal closure were also slow in stomatal opening, even though the mutated anion channel only mediates ion efflux. We showed with our San Diego (USA) collaborators that the slow stomatal opening is caused by reduced K+ uptake due to increased Ca2+ concentration and sensitivity in guard cells of plants without functional SLAC1. The intertwined nature of stomatal opening and closure pathways creates a challenge for both plant genetic research and genetic engineering as even single mutations can lead to changes which are not directly related with the missing protein

BODYGUARD is required for the biosynthesis of cutin in Arabidopsis

International audienceThe cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an a/bhydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated x-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration

Abscisic acid-independent stomatal CO2 signal transduction pathway and convergence of CO2 and ABA signaling downstream of OST1 kinase

Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. CO2 elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO2/ABA interaction remain unclear. Two models have been considered: (i) CO2 elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (ii) CO2 signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutants nced3/nced5 and aba2-1 remain responsive to CO2 elevation. Rapid CO2-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays suggest that CO2 elevation does not trigger [ABA] increases in guard cells, in contrast to control ABA exposures. Moreover, CO2 activates guard cell S-type anion channels in nced3/nced5 and ABA receptor hextuple mutants. Unexpectedly, in-gel protein kinase assays show that unlike ABA, elevated CO2 does not activate OST1/SnRK2 kinases in guard cells. The present study points to a model in which rapid CO2 signal transduction leading to stomatal closure occurs via an ABA-independent pathway downstream of OST1/SnRK2.6. Basal ABA signaling and OST1/SnRK2 activity are required to facilitate the stomatal response to elevated CO2 These findings provide insights into the interaction between CO2/ABA signal transduction in light of the continuing rise in atmospheric [CO2]

PYR/RCAR receptors contribute to Ozone-, Reduced Air Humidity-, Darkness- and CO2-Induced Stomatal Regulation

[EN] Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O-3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca2+-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O-3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O-3 and are involved in responses to elevated CO2.This work was supported by the Estonian Ministry of Science and Education (grant no. IUT2-21), by the European Regional (Center of Excellence in Environmental Adaptation) and Social (Mobilitas Top Researchers grant no. MTT9) Fund, by the National Science Foundation (grant no. MCB0918220 to J.I.S.) and the National Institutes of Health (grant no. R01GM060396 to J.I.S.), by Shanxi Scholarship Council of China (grant no. 2011-012 to S.X.), by Shanxi Technology Foundation and Natural Science Foundation of Shanxi (grant no. 2012011006-4 to S.X.), and by Ministerio de Economia y Competitividad (grant no. BIO2011-23446 to P.L.R.).Merilo, E.; Laanemets, K.; Hu, H.; Shaowu, X.; Jakobson, L.; Tulva, I.; González Guzmán, M.... (2013). PYR/RCAR receptors contribute to Ozone-, Reduced Air Humidity-, Darkness- and CO2-Induced Stomatal Regulation. Plant Physiology. 162(3):1652-1668. https://doi.org/10.1104/pp.113.220608S16521668162