Les mouvements stomatiques en réponse aux stimuli environnementaux sont cruciaux pour le contrôle du statut hydrique de la plante et sa protection contre les pathogènes. L'acide abscissique (ABA) et les Motifs Moléculaires Associés aux Pathogènes (PAMPs), comme le peptide flg22, déclenchent, via le peroxyde d'hydrogène (H2O2), des processus de signalisation qui conduisent à la fermeture stomatique. Bien que les mouvements stomatiques impliquent d'importants flux membranaires d'eau et changements de volume cellulaire, le rôle des canaux hydriques membranaires (aquaporines, AQP) est resté hypothétique. Des expérimentations sur épidermes isolés montrent que les plantes pip2;1 d'Arabidopsis thaliana invalidées pour le gène codant pour PIP2;1, une AQP de la membrane plasmique, présentent un défaut de fermeture stomatique en réponse à l'ABA et au flg22, mais des réponses normales à l'obscurité, la lumière ou le dioxyde de carbone. Les protoplastes de cellules de garde de plantes sauvages présentent, contrairement aux protoplastes de plantes pip2;1, une augmentation de perméabilité hydrique (Pf) de deux fois en réponse à l'ABA. Une sonde génétique sensible à l'H2O2 (HyPer) a permis de révéler une accumulation d'H2O2 dans les cellules de garde de plantes sauvages en réponse à l'ABA et au flg22, accumulation qui est abolie chez les plantes pip2;1. SnRK2;6 (OST1), une protéine kinase étroitement liée aux récepteurs de l'ABA, est capable de phosphoryler in vitro un peptide cytoplasmique de PIP2;1, au niveau du résidu Ser121. SnRK2;6 augmente aussi l'activité de transport d'eau de PIP2;1 après co-expression en ovocytes de xénope. L'expression dans des plantes pip2;1 de formes mutées de PIP2;1 mimant un état phosphorylé (Ser121Asp) ou non phosphorylé (Ser121Ala) a permis de montrer que l'augmentation de Pf en réponse à l'ABA, l'accumulation intracellulaire d'H2O2 et la fermeture stomatique induites par l'ABA ou le flg22, requièrent la phosphorylation de PIP2;1 en Ser121. Ce travail fournit les premières évidences génétiques et physiologiques directes d'un rôle des AQPs dans les mouvements stomatiques. Nous proposons que PIP2;1 joue à la fois un rôle hydraulique et signalétique en facilitant le transport d'eau et H2O2 au travers de la membrane plasmique des cellules de garde. Sa phosphorylation en Ser121, par SnRK2.6 notamment, serait nécessaire à son activation dans ce contexte. / Stomatal movements in response to changing environmental conditions are crucial for controlling the plant water status and protecting plants against pathogens. Abscisic acid (ABA) or Pathogen-Associated Molecular Patterns (PAMPs) such as the flg22 peptide, induce, via hydrogen peroxide (H2O2), signaling networks that lead to stomatal closure. Although stomatal movements involve marked water fluxes and changes in cell volume, the role of membrane water channels (aquaporins, AQP) has remained hypothetical. Functional assays in epidermal peels showed that Arabidopsis thaliana pip2;1 plants invalidated for plasma membrane AQP PIP2;1 have a defect in stomatal closure in response to ABA and flg22, but normal responses to dark, light or carbon dioxide. Guard cell protoplasts from wild-type plants showed a two-fold increase in osmotic water permeability (Pf) in response to ABA which was fully abrogated in pip2;1 plants. Plants expressing a H2O2 sensitive probe (HyPer) revealed an accumulation of H2O2 in guard cells of wild-type plants in response to ABA and flg22, which was abolished in pip2;1 plants. OST1 (SnRK2.6), a protein kinase closely linked to ABA receptors, was able to phosphorylate, in vitro and at Ser121, a cytosolic peptide of PIP2;1. SnRK2.6 also enhanced PIP2;1 water transport activity after co-expression in Xenopus oocytes. Expression in pip2;1 plants of a phosphomimetic (Ser121Asp) or a phosphodeficient form (Ser121Ala) of PIP2;1 allowed to demonstrate that the increase in guard cell protoplast Pf, the intracellular accumulation of H2O2 and stomatal closure induced by ABA and flg22 require the phosphorylation of PIP2;1 at Ser121. This work provides the first direct genetic and physiological evidence for aquaporin function in guard cells. We propose that PIP2;1 plays both a hydraulic and signaling role in guard cells, by facilitating the transport of water and H2O2 across the guard cell plasma membrane. PIP2;1 phosphorylation at Ser121, by SnRK2.6 in particular, would be required in this context.

Stomata are structures in plant epidermis which regulate contact between inner and outer environment of the plant by mediating their stomatal aperture. Many inner and outer signals contribute to the ontogenesis of the stomatal pattern. Guard cells undergo significant change of volume and surface during stomatal movement. This change of surface must be compensated by intracellular trafficking of membrane material because biological membrane has limited elasticity. Most of this trafficking takes place between plasma membrane and endosomal compartments. Complex exocyst is protein complex that ensures proper targeting of secretory vesicles to their destination on the plasma membrane. Function of this complex is essential for many cellular processes that require precise targeting of secretion. Mutation in gene Exo70B1 causes different development of the stomatal pattern. Plants with mutated Exo70B1 differ in stomatal size depending on the cultivation conditions more than wild type plant. Protein EXO70B1 is also directly involved in stomatal dynamics because mutants exo70B1 have retarded stomatal opening in response to light. This direct connection can be observed on the fluorescently labeled protein EXOB1 which significantly changes its localization during stomatal movements. None of these observed phenotypes is...

For optimal growth, plants have evolved strategies to integrate environmental signals to coordinate complex developmental and defensive processes to cope with the changing surroundings. Under challenges, plants prioritize their defense over growth. This trade-off involves complex interactions between multiple hormonal pathways and developmental networks. We discovered that JACKDAW (JKD), the core component of the SHORTROOT (SHR)-SCARECROW (SCR)- JKD plant developmental regulatory network is linking defense responses to the developmental programming. Unlike the well-studied function of JKD in root development, its function in leaves is yet to be understood. We found that JKD is expressed on the abaxial side of the leaf ground tissue. It has conserved functions in promoting SHR nuclear retention and restricting cyclinD6 expression in the leaf. Additionally, JKD has a function in leaf internal architecture establishment, including suppression of the bundle sheath cell division and shaping of the leaf ground tissue. We also found that SHR is a universal asymmetric cell division (ACD) activator, as ectopic SHR expression in the leaf ground tissue promotes stomata development via promoting the ACD to produce more stomata precursor cells. We showed that the knockout mutant of JKD has larger rosettes and better photosynthesis capacity, while the basal defense level and resistance to Botrytis cinerea, a necrotrophic pathogen, are enhanced. Our transcriptome and transcription studies revealed that JKD suppresses the expression of the plant defense hormone Jasmonic acid (JA) response genes and is itself downregulated by JA. This suggests that JKD is involved in the JA signaling, which mediates defense responses for wounding and herbivore attacks. Together, our study indicates that the loss of JKD uncoupled the plant growth-defense trade-off. JKD is a new link between plant development and defense. To verify whether this function of JKD is conserved in crops, JKD orthologues in tomatoes are identified, CRISPR-Cas9 and TILLING mutants are created and analyzed. The results showed that the functions of JKD in root development and resistance to botrytis are conserved. The broad presence of JKD orthologs makes them a great target for molecular breeding to generate crops that do not have to sacrifice their normal growth to defense response.

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published_or_final_version / Social Work / Master / Master of Social Work

Chez les végétaux supérieurs, la régulation des mouvements stomatiques permet de contrôler les échanges de CO2 et la montée de la sève brute tout en limitant les pertes excessives d'eau par transpiration. Ce contrôle est assuré par des variations rapides de la turgescence des deux cellules de garde formant le stomate dues à l'activité de nombreux canaux et transporteurs ioniques. Nous avons identifié un nouveau membre de la famille des CLC chez A. thaliana, AtCLC-c exprimé dans la cellule de garde. L'étude de l'expression d'AtCLC-c et du phénotype de mutants invalidés ont permis de démontrer son rôle dans l'ouverture stomatique à la lumière et la fermeture en réponse à l'ABA. Les mutants clcc accumulent moins d'ions Cl- dans leurs cellules de garde par rapport aux plantes sauvages et sont hypersensibles à un stress salin. Enfin, nous avons confirmé par des études d'électrophysiologie la sélectivité d'AtCLC-c aux ions Cl-. L'ensemble de ce travail montre l'importance du transporteur vacuolaire d'ions Cl- AtCLC-c dans les mouvements stomatiques et la tolérance au stress salin. / In plants, the high turgor is assured by ion transport and involves the creation and maintenance of a large vacuolar volume. In recent years, various chloride channels and transporters have been identified to be involved in specific functions such as plant nutrition, stomatal movements or metal tolerance. We have characterized a new member of the CLC family in A. thaliana, AtCLC-c, highly expressed in guard cell and up-regulated by ABA and salt treatment in the whole plant. Knock-out mutants in AtCLC-c are impaired in light-induced stomatal opening and ABA-induced stomatal closing correlated to a large decrease in guard cell Cl- content. Furthermore, clcc mutants are hypersensitive to salt stress compared to wild-type. Finally, using electrophysiological studies, we demonstrated that AtCLC-c is selective to Cl-. Altogether, this work shows that AtCLC-c is a tonoplastic Cl- transporter involved in stomatal movements and salt tolerance.

A ocorrência de potenciais de ação e neurotransmissores, componentes principais do sistema nervoso animal, em plantas, bactérias e fungos mostra a universalidade dos princípios de sinalização e transmissão de informações na forma de sinais químicos e elétricos em todos os organismos. Esses tópicos de estudo, juntamente com inteligência em plantas e transporte vesicular de auxina, constituem as linhas de pesquisa principais da recém-criada Neurobiologia Vegetal. Entre os neurotransmissores encontrados em plantas, a acetilcolina atua, entre outras situações, no crescimento e desenvolvimento controlado pelo fitocromo e na permeabilidade iônica de membranas. Nesse contexto, foi sugerido que a acetilcolina pode desempenhar um papel importante na regulação do movimento estomático, tendo efeito estimulatório na abertura dos estômatos além de poder atuar na sinalização entre raiz e parte aérea. Dessa forma, foi proposto identificar a presença deste neurotransmissor em plantas de milho hidratadas e submetidas a estresse hídrico, com o objetivo de correlacionar a presença de acetilcolina com as respostas estomáticas de tais plantas. Além disso, foi objetivo do trabalho avaliar parâmetros fisiológicos como potencial hídrico, condutância estomática, transpiração e fotossíntese líquida e suas possíveis relações com a acetilcolina em três folhas das plantas hidratadas e estressadas. Para tanto, foi montado um experimento em blocos casualizados em esquema fatorial (2x3). Os fatores foram: água, nos níveis de hidratação e estresse, e idade das folhas nos níveis folha 4 (mais velha), folha 5 (idade intermediária) e folha 7 (mais jovem). As plantas foram divididas em 20 blocos, contendo uma planta hidratada e uma sob estresse cada e as análises fisiológicas feitas nas três folhas. As plantas foram colocadas em câmara de crescimento tipo BOD com controle de iluminação e temperatura. Após análises fisiológicas, as folhas foram utilizadas para extração e determinação de acetilcolina. Os extratos purificados e secos foram submetidos à pirólise e cromatografia gasosa e as substâncias identificadas por espectrometria de massas. Não foi detectada acetilcolina nas plantas, apesar de estudos anteriores demonstrarem sua ocorrência em folhas e sementes de milho. Hipóteses foram levantadas para explicar tal fato. Quanto as variáveis fisiológicas, o déficit hídrico reduziu em aproximadamente 59% a transpiração, em 65% a condutância estomática e em 59% a fotossíntese das plantas. Condutância estomática e transpiração, condutância e fotossíntese, e transpiração e fotossíntese apresentaram intensa correlação. Já o potencial hídrico teve baixa correlação com essas variáveis. Quanto ao fator idade, folhas 7 apresentaram maiores valores de fotossíntese, condutância e transpiração que as folhas 4 e 5. / The occurrence of action potential and neurotransmitters, the major components of animal nervous system, in plants, bacteria and fungi, shows the universality of signaling principles and information transmission in the way of chemical and electrical signals in all organisms. These study topics, along with plant intelligence and vesicular-based auxin transport, constitute the major research lines of the newly created Plant Neurobiology. Among the neurotransmitters found in plants, the acetylcholine plays a role in phytochromecontrolled growth and development and in membrane ion permeability. In this context, it was suggested that acetylcholine can play an important role in the regulation of stomatal movements, having stimulatory effect in the stomatal opening. In addition it can play a role in root-to-shoot signaling process. Therefore, it was proposed to identify the presence of this neurotransmitter in maize plants hydrated and under water stress, with the aim of correlating the presence of acetylcholine with the stomatal responses of such plants. Moreover, another aim of the study was to evaluate physiological parameters like water potential, stomatal conductance, transpiration rate and net photosynthetic rate and their possible relationship with the acetylcholine in three leaves of hydrated and stressed plants. Therefore, an experiment was set up in randomized block design in 2x3 factorial. The factors were: water, in the levels of hydration and stress, and leaves age in the levels leaf 4 (older), leaf 5 (intermediary age) and leaf 7 (younger). The plants were divided in 20 blocks, and each one has had one hydrated plant and one stressed plant and the physiological analysis was made in three leaves. The plants were placed in B.O.D. growth chamber under controlled conditions of light and temperature. After the physiological analysis, the leaves were used to extraction and determination of acetylcholine. The dried and purified extracts were subjected to pyrolysis and gas chromatography and the substances identified by mass spectrometry. The acetylcholine was not detected in plants, although earlier studies have had demonstrated its occurrence in maize leaves and seeds. Hypotheses were elaborated to explain such fact. Regarding the physiological variables, water stress reduced the plants transpiration rate in 59%, stomatal conductance in 65% and net photosynthesis in 59%. Stomatal conductance, transpiration and photosynthesis were strongly related. On the other hand, the water potential showed weak correlation with that variable. As for the age factor, leaves 7 had higher photosynthetic rates, conductance and transpiration than the leaves 4 and 5.

Crop yields are coming under pressure to continue to grow in the face of climate change, competition, disease and pressure to reduce inputs. Photosynthetic efficiency is being targeted for improvement to increase yields. This study examined the variation in parameters of photosynthetic efficiency including canopy structure (leaf length, canopy angle, and chlorophyll content and growth rate) and gas exchange (photosynthetic rate, stomatal density and chlorophyll fluorescence) in Spring Barley (Hordeum vulgare ssp vulgare). These were first established for modern cultivars representing the most widely grown lines in the last 60 years. As cultivars are developed from a small pool of parents they may have limited genetic variation available for breeding. Landraces have been suggested as sources of variation. Using field and growth cabinet based studies the photosynthetic efficiencies of canopy structure and gas exchange were established for a range of European landraces under high and low nutrient inputs. This study demonstrated that in modern cultivars the leaf length increased with year of release from 23.2 to 29.6 cm and the chlorophyll content decreased from 46.9 to 34.8 SPAD units. Once the ear had emerged no difference was seen in canopy structure or photosynthetic rate. There was variation in landrace canopy establishment rate, leaf angle and number of leaves present within the canopy. The landraces from Northern European latitudes pushed though booting and reached full canopy establishment up to 8 days sooner than those from Southern Europe. This may be a response to a shorter growth season at Northern latitudes requiring the canopy to be established quickly. The landraces held the leaves within their canopy in a more horizontal position than the Southern European lines with leaf angle ranging from 18-45 degrees at GS39 and 31-84 degrees at GS59. This regressed negatively with temperature so it may be that a vertical canopy structure is beneficial in areas with higher temperatures. The photosynthetic rate of the landraces showed no variation but when chlorophyll fluorescence examined the efficiency of photosystem II (PSII) there was a positive regression of Fv/Fm ratio with latitude. This suggested that lines from Southern Europe were experiencing a greater stress with a ratio of up to 0.822 compared to those lines from the North with ratios from 0.767. The stomatal density of the landraces showed a large difference in ranges from 22-41 stomata between the lines. When high and low nutrient inputs were compared reductions from a ratio of 0.48 to 0.47 in Harvest Index and from 55g to 52g in 1000 grain weight were seen. The chlorophyll content of the lines was also reduced from 41.7 to 39.2 SPAD units at GS39 and from 44.9 to 39.8 SPAD units at GS59 by the reduction in nutrient inputs which may be a result of less N available for the production of chlorophyll. In conclusion there is variation present in canopy structure in European landraces that may be useful for future breeding or in identifying landrace collections which could be targeted for traits of interest in photosynthetic efficiency. These landraces may provide traits which could be used to develop cultivars which are locally adapted to climate and environmental conditions.

Stomatal development and its regulation by environmental conditions (light, CO2 concentration) and physiological processes in the leaf of higher plants were investigated. The study was based on the assumptions that stomatal development should be regulated by signals coming from both external environment and leaf interior, and that the signal should be transduced from cotyledons to leaves. Transgenerational effect in stomatal development was also studied. Molecular and physiological approaches were applied to reveal the relationship between leaf environment, stomatal development, stomatal function and leaf physiology.