Biogeensete lenduvühendite emissiooni regulatsioon stressitingimustes: geeniekspressioonist lenduvühendite emissioonivastusteni erinevate abiootiliste stresside korral

A Thesis for applying for the degree of Doctor of Philosophy in AgriculturePlants release a wide array of biogenic volatile organic compounds both constitutively and upon stress. BVOCs play key roles biologically and ecologically. In addition, the emission responses of stress volatiles act as one of the sensitive indicators for the degree of stress influenced by plants. Furthermore, there are three main photosynthetic traits, net assimilation rate, stomatal conductance to water vapour, and changes in maximum quantum yield of photosystem II, are also modified upon stress in parallel with volatile emissions. High temperature is one of the key abiotic stress factors negatively influencing the plant growth and productivity. Typically, high temperature strongly affect plant physiological and biochemical activities particularly photosynthesis, and biosynthesis and emission of volatile organic compounds. A fluctuating high temperature and in turn, the reduction of plant growth and productivity is a serious issue in tropical and mediterranean crop ecosystems. Ozone is a phytotoxic gas and one of the key abiotic stress factors. Tropospheric ozone is primarily formed through the reaction of nitrogen oxides NO and NO2 (NOX) and BVOCs in the presence of sunlight. Elevated ozone exerts oxidative stress in plants and thus, it alters biochemical adjustments and metabolic shifts. The current tropospheric ozone level is around 40 ppb in most parts of the world and this level of ozone is capable of causing oxidative stress in many plants, particularly in crop plants. Wounding is also a common mechanical stress factor, mainly caused by wind, falling debris and heavy precipitation in nature. Therefore, combined ozone and wounding treatments are predicted to result in synergistic effects on key plant physiological activities such as photosynthetic characteristics and volatile emission responses. There is a scarcity of information about the global scale of photosynthesis and volatile emission responses altered by oxidative stresses induced by heat, ozone, and wounding, and their relationship with concomitant changes in physical and chemical properties of atmosphere. In addition, there is limited information on the molecular basis of terpenoid biosynthesis upon different abiotic stresses. Therefore, understanding the gap between influence of stress and concomitant changes in plant eco-physiological activity is a vital. Aims of the study The specific aims of the Thesis were to: 1. study the impact of heat stress on time-dependent changes in photosynthetic characteristics and concomitant changes in mono- and sesquiteprene emission responses; analyze the effects of heat stress on the regulation of β-phellandrene and (E)-β-caryophyllene synthase genes in Solanum lycopersicum ‘Pontica’; and assess the correspondence between time-dependent modifications in mono-and sesquiterpene gene expression and terpenoid emissions through recovery (Paper I). 2. study the effect of combined ozone and wounding treatments on the changes in photosynthetic characteristics and stress volatile emission rates through recovery; and estimate the relationship between quantitative emission responses and stress severity, including ozone dose and stomatal ozone uptake rate in Eucalyptus globulus ‘Labil’ (Paper II). 3. assess the relationships between ozone concentration and modifications in foliage photosynthetic characteristics and emissions of LOX volatiles and mono- and sesquiterpenes; estimate the leaf ozone uptake between stomatal ozone uptake and non-stomatal ozone deposition; and relate the stomatal ozone flux and non-stomatal ozone deposition with the degree of elicitation of different volatiles in Nicotiana tabacum ‘Wisconsin’ (Paper III). Main results In this Thesis, we developed three model systems in our lab to understand the impact of abiotic stresses on primary and secondary metabolic processes of crop and economically important woody plants. • In S. lycopersicum leaves, mild heat stress did not have any significant impact on the changes in photosynthetic traits, but severe heat stress irreversibly declined photosynthetic characteristics. The modifications in foliage mono- and sesquiterpene emissions were associated with β-phellandrene and (E)-β-caryophyllene synthase gene expression at mild heat stress. Based on time-delay analysis of mono- and sesquiterpene synthase genes, the best correspondence between terpene synthase gene expression and emission was shifted by ca. 2h for mild heat stress, but the time-shift relationship between gene expression and emission was not clear-cut for severe heat stress, implying that severe heat stress significantly impair terpene metabolism (Paper I). • In E. globulus leaves, the physiological and biochemical responses to a combined effect of ozone and wounding through recovery were different from the responses in plants treated with ozone and wounding separately. Particularly, the reduction of photosynthetic characteristics such as A, gs and Fv/Fm, and increased stressed volatile emission responses were greater for combined ozone and wounding treatments followed by separate ozone and wounding treatments. Taken together, these key outcomes demonstrated that combined acute ozone and wounding treatments led to synergistic effects on leaf photosynthetic traits and stress volatile emissions. LOX emissions were scaled with the degree of stress applications, but there were no quantitative relationships observed between the severity of stress and emission responses for other stress volatiles. The absence of stress dose vs. emission relationship was likely due to removal of ozone in leaf intercellular air spaces and ROS in mesophyll cells by volatile isoprenoids through recovery (Paper II). • In N. tabacum ‘Wisconsin’ leaves, acute ozone exposure result in a dramatic decline of foliar gas-exchange characteristics, but the reductions in all these characteristics were almost fully reversible even at the highest ozone concentrations underscoring the high ozone resistance. The emission rate of LOX volatiles was dose-dependent, but dose-dependency was weaker for mono- and sesquiterpene emissions. In addition, non-stomatal ozone deposition contributed to the greater share of ozone flux into the leaf and this was associated with the release of terpenes, collectively implying that a greater share of terpenoid emission upon ozone exposures mostly reflected the modification of terpene release from trichomes on leaf surface (Paper III).Taimed eritavad mitmeid erinevaid biogeenseid orgaanilisi lenduvühendeid nii konstitutiivselt kui vastusena stressitingimustele, mistõttu on lenduvühenditel oluline bioloogiline ja ökoloogiline tähtsus. Lisaks sellele võimaldab stressivastusena taimes sünteesitavate biogeensete orgaaniliste lenduvühendite kogus määrata taime mõjutava stressi ulatust. Lenduvühendite emissiooni kõrval mõjutab stress ka teisi olulisi parameetreid: fotosünteesi, õhulõhede juhtivust ja fotosüsteem II maksimaalset kvantsaagist. Üks peamistest taime kasvu ja produktiivsust vähendavatest abiootilistest stressifaktoritest on kõrge temperatuur, mis mõjutab tugevalt taimede füsioloogilisi ja biokeemilisi protsesse, eriti fotosünteesi ja biogeensete orgaaniliste lenduvühendite sünteesi ning emissiooni. Kuumalained on tõsiseks probleemiks troopilistes ja vahemerelistes põllumajanduspiirkondades tuues kaasa suuri saagikadusid. Samuti on väga oluline abiootiline stressifaktor fütotoksiline gaas – osoon. Troposfääri osoon moodustub peamiselt lämmastiku oksiidide (NO ja NO2) ja biogeensete orgaaniliste lenduvühendite omavahelise reaktsiooni käigus päikesevalguse käes. Kõrge troposfääri osooni tase tekitab taimedes oksüdatiivset stressi, mistõttu toimuvad muutused biokeemilistes reaktsioonides ning metaboolsetes radades. Praegune troposfääri osooni tase maailmas on umbes 40 osakest miljardist, juba selline kontsentratsioon võib põhjustada oksüdatiivset stressi paljudel taimedel, eelkõige põllukultuuridel. Lisaks eelnevatele on tavaliseks stressifaktoriks ka taimeosade mehhaaniline vigastus, mille peamisteks looduslikeks põhjustajateks on tuul, langev varis ja tugevad sademed. Arvatakse, et kombineerituna on osoonil ja mehhaanilistel vigastustel vastastikune koosmõju taime füsioloogiale, fotosünteesile ja lenduvühendite emissioonile. Globaalsel skaalal on vähe informatsiooni kõrge temperatuuri, osooni ja mehhaaniliste vigastuste tagajärjel tekkinud oksüdatiivse stressi mõjudest fotosünteesile ning lenduvühendite emissioonile. Lisaks on vähe teada mil määral mõjutavad erinevad abiootilised stressifaktroid terpenoidide biosünteesi molekulaarsel tasandil. Seetõttu on äärmiselt oluline uurida täpsemalt stressi mõju taimede ökofüsioloogiale. Töö eesmärgid Antud töö täpsemad eesmärgid olid: 1. uurida ajast sõltuvat kuumastressi mõju nii fotosünteesile kui mono - ja seskviterpeenide emissioonile; analüüsida kuumastressi mõju β-fellandreeni ja (E)-β-karofülleni süntetaasi geenide regulatsioonile tomatil (Solanum lycopersicum ’Pontica’); (I artikkel). 2. uurida osooni ja mehhaaniliste vigastuste koosmõju fotosünteesile ja lenduvühendite emissioonile stressist taastumise perioodi vältel ning hinnata stressi tugevuse ja emissiooni vastuse kvantitatiivset suhet, võttes arvesse osooni doosi ja õhulõhede kaudu lehte sisenemise kiirust, liigil Eucalyptus globulus Labil (II artikkel) 3. hinnata seoseid osooni kontsentratsiooni ja lehestiku fotosünteesi, LOX-ühendite ning mono - ja seskivterpeenide emissiooni vahel; hinnata osooni jõudmist lehtedesse õhulõhede kaudu ja õhulõhedest sõltumatult; ning siduda seda erinevate volatiilsete ühendite sünteesi indutseerimisega tubakal (Nicotiana tabacum ‘Wisconsin’) (III artikkel). Peamised tulemused Antud töö käigus oleme välja töötanud kolm mudelsüsteemi mõistmaks abiootilise stressi mõju nii primaarse kui sekundaarse ainevahetuse protsessidele põllukultuuridel ja majanduslikult olulistel puittaimedel. • Kerge kuumastress tomati (S. lycopersicum) fotosünteesile olulist mõju ei avaldanud, küll aga kahjustas tugev kuumastress pöördumatult fotosünteesiaparaati. Kerge kuumastressi puhul aset leidnud muutused lehestiku mono - ja seskviterpeenide emissioonis olid seotud β-fellandreeni ja (E)-β-karofülleeni süntaasi geeni avaldumisega. Mono - ja seskviterpeenide süntetaasi geenide analüüsil eri ajahetkedel stressitöötluse järgselt ilmnes, et terpeeni süntaasi geeniekspressiooni ja emisiooni nihe kerge kuumastressi järgselt oli vaid 2 tundi, kuid tugeva kuumastressi puhul selget nihet geeni ekspressiooni ja lenduvühendi emissiooni vahel ei olnud, millest saab järeldada, et tugev kuumastress kahjustab terpeeni ainevahetust (I artikkel). • Füsioloogilised ja biokeemilised muutused ning taastumisprotsess eukalüptil (E. globulus) erinesid olenevalt sellest, kas lehti vigastati mehhaaniliselt ning töödeldi osooniga samaaegselt või rakendati stressitöötusi eraldi. Kombineeritud stressitingimuste korral vähenesid fotosünteesi karakteristikud näiteks A, gs ja Fv/Fm ning suurenes stressist tingitud lenduvühendite emissioon suuremal määral kui osooni ja mehhaanilise stressi puhul eraldi. Kokkuvõttes näitasid need tulemused, et kombineerituna on kõrgel osooni tasemel ja mehhaanilisel vigastusel sünergiline efekt fotosünteesile ning stressi poolt indutseeritud lenduvühendite emissioonile. LOX-ühendite emissioon oli vastavuses stressi tugevusega, kuid teiste stressitingimustes tekkivate lenduvühendite emissiooni ja stressi tugevuse vahel kvantitatiivset seost ei leitud. Stressi ulatuse ja lenduvühendite emissiooni vahelise kvantitatiivse seose puudumist saab seletada volatiilsete isopenoidide toimega, mis elimineerivad osooni rakuvaheruumidest ning reaktiivsed hapnikuühendeid (ROS) mesofülli rakkudest (II artikkel). • Osoon vähendas oluliselt tubaka (N. tabacum ‘Wisconsin’) lehtede gaasivahetust, kuid isegi kõige kõrgemate kontsentratsioonide korral oli see efekt pöörduv, mis viitab selle liigi tugevale osoonitaluvusele. Tubakal sõltus LOX-ühendite emissioon tugevalt osooni annusest, kuid mono - ja seskviterpeenide puhul oli kontsentratsiooni ja emissiooni vaheline kvantitatiivne suhe nõrgem. Lisaks sellele oli olulisem roll õhulõhedest sõltumatul osooni sisenemisel lehtedesse ning see kutsus esile terpeenide emissiooni, mis kokkuvõttes viitab sellele, et suurem osa osooni vastusena eritatavatest terpeenidest pärineb lehe pinnal asuvatest trihhoomidest (III artikkel).Publication of this thesis is supported by the Estonian University of Life Sciences. This research was funded by the Estonian Ministry of Science and Education (institutional grant IUT-8-3), the European Commission through the European Regional Development Fund (Centre of Excellence EcolChange, TK 131), and the European Research Council (advanced grant 322603, SIP-VOL+)

Temporal regulation of terpene synthase gene expression in Eucalyptus globulus leaves upon ozone and wounding stresses: relationships with stomatal ozone uptake and emission responses

© 2018 Elsevier B.V. Ozone and wounding are key abiotic factors but, their interactive effects on temporal changes in terpene synthase gene expression and emission responses are poorly understood. Here, we applied combined acute ozone and wounding stresses to the constitutive isoprenoid-emitter Eucalyptus globulus and studied how isoprene, 1,8-cineole, and isoledene synthase genes were regulated, and how the gene expression was associated with temporal changes in photosynthetic characteristics, product emission rates, and stomatal ozone uptake through recovery phase. Photosynthetic characteristics and emission rate of isoprene, 1,8-cineole, and isoledene were synergistically altered, while three TPS gene expressions were antagonistically altered by combined stress applications. A time-delay analysis indicated that the best correspondences between gene expression and product emission rates were observed for 0 h time-shift for wounding and 0–2 h time-shifts for separate ozone, and combined ozone and wounding treatments. The best correspondence between ozone uptake and gene expression was observed for 0–4 h time-shifts for separate ozone and combined ozone and wounding treatments. Overall, this study demonstrated that expression profiles of isoprene, the monoterpene 1,8-cineole, and the sesquiterpene isoledene synthase genes differentially influenced their corresponding product emissions for separate and combined ozone and wounding treatments through recovery

Ozone and Wounding Stresses Differently Alter the Temporal Variation in Formylated Phloroglucinols in Eucalyptus globulus Leaves

Formylated phloroglucinol compounds (FPCs) are a class of plant specialized metabolite present in the Myrtaceae family, especially in the genus Eucalyptus. FPCs are widely investigated due to their herbivore deterrence properties and various bioactivities of pharmaceutical relevance. Despite the increasing number of studies elucidating new FPCs structures and bioactivity, little is known about the role of those compounds in planta, and the effects of environmental stresses on FPC concentration. Ozone (O3) and wounding are key stress factors regularly confronted by plants. In this study, we investigated how O3, wounding, and their combination affected individual and total FPC foliar concentration of the economically important species Eucalyptus globulus. Six individual FPCs, including five macrocarpals and one sideroxylonal, showed different response patterns to the single and combined stresses. Total macrocarpals only increased under single O3 treatment, whereas total sideroxylonals only increased in response to wounding treatment, suggesting different physiological roles played by the two groups of FPCs predominantly existing in E. globulus foliage. Total FPCs increased significantly under individual wounding and O3 treatments but not under the combined treatment. A principal component analysis indicated that all different treatments had unique FPC fingerprints. Total phenolic contents increased in all O3 and wounding treatments, and a marginally positive correlation was found between total FPCs and total phenolic contents. We suggest that, depending on the concentration and composition, FPCs play multiple physiological roles in planta, including serving as antioxidants to scavenge the reactive oxygen species brought about by O3 and wounding stresses