Abstsiishappe, CO2 ning kutiikula roll taime transpiratsiooni regulatsioonis

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Veatu gaasivahetuse regulatsioon on äärmiselt oluline taimele elus püsimiseks ning aluseks põllumajandustaimede kõrgele saagikusele. Gaasivahetuse all peetakse silmas süsihappegaasi sisenemist taime lehte ning vee väljumist lehest atmosfääri. Süsihappegaasi on taimel vaja fotosünteesi käimas hoidmiseks, kuid veekadu peab taim võimalusel piirama, et vältida kuivale jäämist ja põua tõttu suremist. Enamik taime veekaost toimub läbi taime pinnal olevate väikeste pooride, õhulõhede, kuna epidermiserakke katab vett hülgav lipiidne kutiikula kiht. Selleks, et saaksime tõsta saagikust või parandada taimede ellujäämust ekstreemsetes oludes, on oluline mõista molekulaarseid mehhanisme, millega kontrollitakse taime gaasivahetust nii õhulõhede kui kutiikula kaudu. Käesoleva doktoritöö raames kinnitati, et taimehormooni abstsiishappe signaaliraja komponendid omavad olulist rolli madala õhuniiskuse, pimeduse ja õhusaasteaine osooni toimel õhulõhede sulgumise reaktsioonides. Kuid kõrge süsihappegaasi mõjul toimuvas õhulõhede sulgumises osalevad vaid mõned abstsiishappe signaaliraja komponendid ning tõenäoliselt esineb ka paralleelne signaalirada. Lisaks pakuti välja mudel, mille kohaselt signaal süsihappegaasi kontsentratsiooni tõusust edastatakse harilikus müürloogas (Arabidopsis thaliana) läbi MAP kinaasvalkude MPK12 ja MPK4. Aktiveeritud MPK12 ja MPK4 on võimelised inhibeerima CO2-spetsiifilist kinaasi HT1 ning seeläbi võimaldatakse anioonkanali SLAC1 aktivatsioon sulgrakkudes ja selle tulemusena ka õhulõhede sulgumine. Leidmaks uusi molekulaarseid komponente, mis reguleerivad taime gaasivahetust, on mutantide analüüs panustanud tähelepanuväärselt palju teadusmaailma varasalve. Seega, töötati välja uus taimemutantide skriiningmeetod, mis võtab arvesse taimelehtede temperatuuri, lõikamisjärgset närtsimist ning kaalukaotust. Üks skriiningust isoleeritud mutantidest, cool breath 5 oli defektse BODYGUARD geeni ekspressiooniga. BODYGUARD valk on oluline faktor C18 küllastumata kutiikula rasvhapete normaalse hulga saavutamisel eelkõige noortes lehtedes ja õites. Tulemuste põhjal pakuti välja, et BODYGUARD võiks kaasa aidata endoplasmaatilises retiikulumis toimuvale kutiikula monomeeride biosünteesile. Kokkuvõtteks, abstsiishape, MPK-d ja kutiikula on kõik olulised faktorid reguleerimaks taime transpiratsiooni ja gaasivahetust.Flawless coordination and regulation of gas exchange is highly important for the survival of land plants and for maintaining high agricultural crop yield. Plants need to ensure the entrance of carbon dioxide to feed photosynthesis, but at the same time water loss must be minimized to avoid desiccation and eventually death. Water loss through the epidermal cells is inhibited by the water-repellent layer called cuticle, which is covering all the aboveground parts of plants. Thus, most of the water exits plants through the small pores in the epidermis called stomata. In order to be able to increase crop yield or viability of plants in extreme conditions, it is important to understand the molecular mechanisms controlling the gas exchange between the plant and the atmosphere both through the stomata and the cuticle. The current thesis confirmed that the signaling pathway of plant hormone abscisic acid controls the closure of stomata in response to reduced air humidity, darkness, and air pollutant O3. Elevated CO2-induced stomatal closure involves only partially the abscisic acid pathway and the presence of a parallel signaling pathway is possible. Furthermore, a model for stomatal CO2 signaling is suggested, where the signal of elevated CO2 is conducted via novel players, MAP kinases MPK12 and MPK4, in Arabidopsis thaliana. Activated MPK12 and MPK4 are able to inhibit the CO2-specific kinase HT1, which results in activation of the anion channel SLAC1 in the guard cells and eventually in stomatal closure. In order to find new molecular components determining the rate of gas exchange between the plant and the atmosphere, reverse genetics approaches have contributed remarkably to it. Hence, a new screening method for screening mutants for impaired transpiration was introduced, which was based on temperature-dependent water spot condensation, visual wilting and weight loss of excised leaves. One of the mutants from this screen, named cool breath 5 was defective in the expression of BODYGUARD gene. BODYGUARD protein plays a crucial role in determining the occurrence of C18 unsaturated fatty acids in the cuticle, especially in young developing leaf tissues and flowers. It was hypothesized that BODYGUARD could facilitate a biosynthetic step of these cuticular monomers in the endoplasmic reticulum. All-in-all, abscisic acid, MPKs and cuticle all have their distinct role in regulating plant transpiration and coordinating gas exchange of plants

Differential role of MAX2 and strigolactones in pathogen, ozone, and stomatal responses

Strigolactones are a group of phytohormones that control developmental processes including shoot branching and various plant-environment interactions in plants. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria. Here we show that both strigolactone biosynthesis (max3 and max4) and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. Further analysis of gene expression revealed no major role for strigolactone in regulation of defense gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4, and d14 mutants. We analyzed stomatal responses to stimuli that cause stomatal closure. While the response to abscisic acid (ABA) was not impaired in any of the mutants, the response to darkness and high CO2 was impaired in max2 and d14-1 mutants, and to CO2 also in strigolactone synthesis (max3, max4) mutants. To position the role of MAX2 in the guard cell signaling network, max2 was crossed with mutants defective in ABA biosynthesis or signaling. This revealed that MAX2 acts in a signaling pathway that functions in parallel to the guard cell ABA signaling pathway. We propose that the impaired defense responses of max2 are related to higher stomatal conductance that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signaling (related to CO2 signaling), this protein could be one of the components that allow guard cells to distinguish between different environmental conditions.Peer reviewe

The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana

Peer reviewe

Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling

Author Summary Human activities have increased the concentrations of CO2 and harmful air pollutants such as ozone in the troposphere. These changes can have detrimental consequences for agricultural productivity. Guard cells, which form stomatal pores on leaves, regulate plant gas exchange. To maintain photosynthesis, stomata open to allow CO2 uptake, but at the same time, open stomata lead to loss of water and allow the entrance of ozone. Elevated atmospheric CO2 levels reduce stomatal apertures, which can improve plant water balance but also increases leaf temperature. Using genetic approaches—in which we exploit natural variation and mutant analysis of thale cress (Arabidopsis thaliana)—we find that MITOGEN-ACTIVATED PROTEIN KINASE 12 (MPK12) and its inhibitory interaction with another kinase, HIGH LEAF TEMPERATURE 1 (HT1) (involved in guard cell CO2 signaling), play a key role in this regulatory process. We have therefore identified a mechanism in which guard cell CO2 signaling regulates how efficiently plants use water and cope with the air pollutant ozone.Peer reviewe

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

ALMT-independent guard cell R-type anion currents

Plant transpiration is controlled by stomata, with S- and R-type anion channels playing key roles in guard cell action. Arabidopsis mutants lacking the ALMT12/QUAC1 R-type anion channel function in guard cells show only a partial reduction in R-type channel currents. The molecular nature of these remaining R-type anion currents is still unclear. To further elucidate this, patch clamp, transcript and gas-exchange measurements were performed with wild-type (WT) and different almt mutant plants. The R-type current fraction in the almt12 mutant exhibited the same voltage dependence, susceptibility to ATP block and lacked a chloride permeability as the WT. Therefore, we asked whether the R-type anion currents in the ALMT12/QUAC1-free mutant are caused by additional ALMT isoforms. In WT guard cells, ALMT12, ALMT13 and ALMT14 transcripts were detected, whereas only ALMT13 was found expressed in the almt12 mutant. Substantial R-type anion currents still remained active in the almt12/13 and almt12/14 double mutants as well as the almt12/13/14 triple mutant. In good agreement, CO2-triggered stomatal closure required the activity of ALMT12 but not ALMT13 or ALMT14. The results suggest that, with the exception of ALMT12, channel species other than ALMTs carry the guard cell R-type anion currents.Peer reviewe

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

A conserved glycine is important for MPK4 and MPK12 function.

<p>(A) Inhibition of HT1 kinase activity in vitro by MPK4 and MPK4 G55R. Upper panel: autoradiography of the SDS PAGE gel; lower panel: Coomassie-stained SDS PAGE. Reaction mixture was incubated for 30 min. (B) Whole protein (left) and close-up (right) view of the superposition of models for MPK12 wild-type (secondary structure and surface in white) and MPK12 G53R (secondary structure in green). There is a close structural similarity between the structures except where the arginine at position 53 protrudes from the mutant protein surface and changes the loop region for the mutant. (C) Whole protein (left) and close-up (right) view of the superposition of models for MPK4 wild-type (secondary structure and surface in white) and MPK4 G55R (secondary structure in yellow). Similar to MPK12 G53R, the arginine at position 55 in MPK4 protrudes from the mutant protein surface and changes the loop region.</p

Responsiveness of the NIL Col-S2 and <i>mpk12</i> mutants to stomatal opening and closing stimuli.

<p>(A) Stomatal opening induced by 100 ppm CO₂ in whole plants (58 min after induction; <i>n</i> = 12–13). (B) Light-induced stomatal opening inhibited by 2.5 μM ABA in whole plants (24 min after induction; <i>n</i> = 16–18). (C) Stomatal closure induced by 800 ppm CO₂ in whole plants (10 min after induction; <i>n</i> = 12–13). (D) Stomatal closure induced by spraying whole plants with 5 μM ABA solution (24 min after induction; <i>n</i> = 12–14). (E) MPK12 is required for the bicarbonate (HCO₃^-)-induced slow type anion channel activation in guard cell protoplasts. Upper panels show typical whole guard cell protoplast recordings with 11.5 mM free HCO₃^- added to the pipette solution, and lower panels show average steady-state current-voltage relationships for wild-type (Col-0), NIL Col-S2, and <i>mpk12-4</i> after treatment with mock or 11.5 mM HCO₃^- (<i>n</i> = 4–8 per line and treatment). Small letters (A, C) and asterisks (B, D) indicate statistically significant differences according to one-way ANOVA and two-way ANOVA with Tukey HSD for unequal sample size post hoc tests (<i>p</i> < 0.05), respectively. Error bars mark ± SEM. The raw data for panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

MPK12 interacts with HT1.

<p>(A) Split-ubiquitin yeast two-hybrid assay on the SD-LeuTrp plate (left and middle panels) indicates the presence of both bait and prey plasmids; X-gal overlay assay (middle) and growth assay on the SD-LeuTrpHisAde plate (right) show HT1 interaction with MPK12 that is similar to the positive control (pAI-Alg5). Only weak or no interaction was detected with MPK12 G53R and MPK11, similar to the negative control (pDL2-Alg5). (B) Quantitative β-galactosidase assay from pools of ten colonies each. Activities are shown as the percentage of the positive control (± SEM; <i>n</i> = 3). (C) High-magnification (63x objective) BiFC images from a single infiltrated <i>N</i>. <i>benthamiana</i> leaf with identical confocal microscopy acquisition settings. Scale bar = 50 μm. (D) Ratiometric BiFC shows weaker interaction of MPK12 G53R than MPK12 with HT1, while MPK11 exhibits a weak interaction with HT1. The plasma membrane–localized SLAC1-CFP was used as an internal control. Eighteen images (from three leaves) of each construct set were analyzed. (E) Western blot together with Coomassie staining of proteins extracted from BiFC samples used for confocal imaging and controls with single construct shows expression of all fusion proteins. (F) Steady-state stomatal conductance of Col-S2 <i>ht1-2</i>, <i>mpk12-4 ht1-2</i>, and Col-S2 <i>abi1-1</i> (<i>ABA insensitive 1–1</i>) double mutants (mean ± SEM, <i>n</i> = 11–13). Experiments were repeated at least three times. Letters in B, D, and F denote statistically significant differences with one-way ANOVA and Tukey HSD post hoc test for equal B, D, or unequal F sample size. The raw data for panels B, D, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p