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In vivo monitoring of electrical activity between plant tissues and organs can be used to study information transport mechanisms and physiological responses to external stimuli. These signals are mainly characterized as action potential (AP) and variation potential (VP), related to long- and short-distance signaling, respectively. Spontaneous action potentials (SAPs) may also occur in the absence of known stimuli at frequencies adjusted to environmental conditions. By contrast, the mechanism of spontaneous depolarizations (SDs) remain unknown. In this work, spontaneous generation and evocation of electrical signals via electrical stimulation were monitored for 72 h using Ag/AgCl electrodes inserted into the stem and petioles of two mutant tomatoes, cv. Micro-Tom notabilis (MTnot) and MTsp12::NCED (MTNCED), and wild type (MTwt). MTnot generated more SAPs than did MTwt and MTNCED, propagated through the stem and petioles, with a transmission speed of 0.50 cm min− 1. SDs propagation occurred mainly in the early morning, most often in MTwt. MTNCED gave a lower number of SDs that were associated with membrane depolarization level. The electrical stimuli promoted the generation of APs in all studied genotypes. Mutants MTnot and MTNCED were less sensitive to electrical stimulation, requiring greater stimuli to reach the excitation threshold.

Highlights • Following the step by step described in this protocol is it is possible to measure long-distance electrical signaling induced by several stimuli, including wounding, current application, irrigation, and others. • Extracellular electrical signal measurements were associated to gas exchange measurements using an infra-red gas analyzer (IRGA, model Li-6400, Li-Cor) and turgor pressure measurements using a patch clamp pressure probe (ZIM-probe, YARA ZIM-plant Technology). • The technique is interesting to study electrical signaling in association with other physiological parameters in plants. • We present results that validate the protocol and discuss the troubleshooting that may compromise measurements and data collection., We present a detailed protocol for measuring extracellular electrical signals in plants using the electrode insertion technique. Using this approach, it is possible to measure long-distance electrical signaling induced by several stimuli, including wounding, current application, irrigation, burning, and others. Additionally, we describe how to associate gas exchange measurements using an infra-red gas analyzer (IRGA, Model Li-6400, Li-Cor) and turgor pressure measurements using a patch clamp pressure probe (ZIM-probe, YARA ZIM-plant Technology) to measure extracellular electrical signals. • The method requires a complete electrical circuit that includes a measuring device (amplifier and voltmeter) and electrodes that provide a contact between the biological material and the equipment. • The infra-red gas analyzer (IRGA), needs to be grounded because it is an important source of noise for electrophysiological measurements. • The ZIM-probe did not cause any interference in electrical signal measure. • Our approach is useful for plant physiologists wishing to implement the technique of measuring electrical signals in plants, in association with other parameters of plant physiology. In addition, our text was written for agricultural and biological scientists who are not electronics specialists., Graphical abstract Image, graphical abstract

Drought is considered the major abiotic stress limiting crop productivity. This study seeks to identify proteins involved in the drought response in sugarcane stems submitted to drought stress. The integration of nuclei enrichment sample preparation with the shotgun proteomic approach results in great coverage of the sugarcane stem proteome with 5381 protein groups identified. A total of 1204 differentially accumulated proteins are detected in response to drought, among which 586 and 618 are increased and reduced in abundance, respectively. A total of 115 exclusive proteins are detected, being 41 exclusives of drought-stressed plants and 74 exclusives of control plants. In the control plants, most of these proteins are related to cell wall metabolism, indicating that drought affects negatively the cell wall metabolism. Also, 37 transcription factors (TFs) are identified, which are low abundant nuclear proteins and are differentially accumulated in response to drought stress. These TFs are associated to protein domains such as leucine-rich (bZIP), C2H2, NAC, C3H, LIM, Myb-related, heat shock factor (HSF) and auxin response factor (ARF). Increased abundance of chromatin remodeling and RNA processing proteins are also observed. It is suggested that these variations result from an imbalance of protein synthesis and degradation processes induced by drought.

During their life cycles, plants are often subjected to periods of fluctuation between water deficit and re-irrigation events. Efficient communication and rapid signaling between the shoot and root systems are needed for adaptive stress responses. Abscisic acid (ABA) and the hydraulic signal are the main long-distance signals responsive to water deficit. Nevertheless, ABA is susceptible to interactions with electrical signals and may not be sufficient as a sign of stress in plants. Therefore, we aimed to investigate the relationship between hydraulic, chemical and electrical signals after a re-irrigation stimulus to induce changes in gas exchange of ABA mutant microtomato plants. Three genotypes of tomato, cultivar Micro-Tom (MT) (MTwt), the isogenic mutant MTnotabilis (MTnot) and transgenic MTsp12::NCED (MTNCED), were exposed to 7, 4 and 11 days under simulated drought conditions, respectively. Then, the plants were re-irrigated and we simultaneously evaluated gas exchange, electric potential and the propagation of action potentials. We found that water deficit affected the water potential of the plants and induced increased depolarization of the plasma membrane electric potential in Mtwt and MTnot. In response to re-irrigation, all genotypes propagated action potentials from root to shoot. In MTwt, the electrical signal induced increases in photosynthesis, stomatal conductance and transpiration rate. By contrast, in ABA-mutants, the gas exchange variables were reduced. These results were related to the effect of ABA and possibly JA on stomatal control and pH changes inactivating photosynthesis.

Uma propriedade fundamental dos seres vivos é a condução de sinais elétricos através de seus tecidos. Mas esse fato pouco é lembrado quando se trata de organismos vegetais. Outro item fundamental é a geração de sinais que possam transmitir informações entre os tecidos e órgãos para um ajuste fino do metabolismo. Nas plantas esses sinais podem ser de natureza química, hidráulica e elétrica. Nesse último caso são conhecidos potenciais de ação (PA), de variação (PV), de ferimentos (WP) e sistêmicos (SP), cada um com sua particularidade quanto à amplitude, velocidade e rotas de propagação, bem como seu papel no metabolismo. Os sinais elétricos podem afetar a respiração, fotossíntese, absorção de água, ativação de genes e fechamento de folhas de plantas insetívoras. PAs podem se propagar com velocidade relativamente constante e sem decréscimo. Eles seguem a lei do tudo-ou-nada, ou seja, todo estímulo que desencadeia um PA deve atingir um limiar de excitação para desencadear o sinal. Assim que o limiar é atingido o sinal se autoperpetua ao passo que estímulos supra limiares desencadeiam PAs de amplitude constante. A via de propagação do PA é o vaso do floema. O objetivo do presente trabalho foi avaliar a sinalização elétrica em resposta à irrigação em plantas de girassol sob déficit hídrico e caracterizar esse sinal quanto à amplitude, duração, velocidade e direção de propagação. Para tanto 37 plantas foram avaliadas por meio de eletrodos extracelulares. Elas foram monitoras eletrofisiologicamente durante um período em que eram irrigadas e em um período sob déficit hídrico. Desse montante, onze responderam à irrigação pela geração de potenciais de ação (PA), ou seja, 30% delas. Oito delas geraram PAs em direção ao ápice (propagação acrópeta) enquanto duas geraram na direção basípeta. Uma delas gerou nos dois sentidos. O PA foi gerado também pós-irrigação mesmo com a planta não tendo passado por déficit hídrico, porém só aconteceu em uma das onze plantas. Os sinais se propagaram no caule, pecíolo e nervura central das folhas. O potencial de ação é gerado após irrigação em plantas de girassol com maior frequência quando elas passam por período de déficit hídrico e se propagam por toda a planta. Isso evidencia o papel do PA na sinalização de longa distância nos vegetais. A fundamental property in the leaving beings is the conduction of electrical signals through their tissues. However, this fact is not always remembered when it comes to plant organisms. Another key process is the generation of signals that can transmit information among tissues and organs to a fine-tuning of the metabolism. In plants, these signals can be chemical, hydraulic and electrical. Concerning the last one, action potentials (AP), variation potentials (VP), wound potentials (WP) and system potentials (SP) are known; each one with its particularity regarding amplitude, velocity and propagation routes as well as its role in metabolism. The electrical signals may affect respiration, photosynthesis, water uptake, activation of genes and leaf closure in insectivorous plants. APs can spread with relatively constant speed and no decrement. They follow the all-or-nothing law, in another words, every stimulus that triggers an AP must reach a threshold to trigger the signal. Once the threshold is reached, the signal is self-perpetuating while stimuli above threshold trigger APs with constant amplitude. The propagation path of the AP is the phloem vessel. The aim of this work was to evaluate the electrical signaling in response to irrigation in sunflower plants under water deficit and characterize the AP regarding its amplitude, duration, velocity and propagation direction. Thirty seven plants were analyzed using extracellular electrodes. They were electrophysiologically monitored during a period when irrigated and in a period under drought. Eleven plants generated AP after irrigation, i.e. 30%. Eight of them generated AP that propagated acropetally while two generated in basipetal direction. One generated in both directions. The action potential was also generated in a plant that was not under drought stress, however it just happened in one of the eleven plants. The signals propagated in the stem, petiole and midrib of the leaves. The action potential is generated after irrigation in sunflower plants more frequently when they go through a period of water deficit and propagate throughout the plant. This highlights the role of AP in long-distance signaling in plants.

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.

Made available in DSpace on 2015-08-21T17:53:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2015-08-24T14:41:35Z : No. of bitstreams: 1 ISSN1981-3163-2015-31-01-259-267.pdf: 216497 bytes, checksum: f2756e796047288e39f9a3388ab765cc (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Kielmeyera coriacea Mart. (Clusiaceae), popularly known as “pau-santo”, is a typical Brazilian cerrado tree known due to its varied secondary metabolites. This study aimed to determine the allelopathic potential of the hydroalcoholic extract of leaves of K. coriacea through bioassays of seed germination, seedling growth and mitotic index of Lactuca sativa L. (lettuce). In addition it was done the tetrazolium assay and a phytochemical screening. The extract concentrations caused alterations in germination parameters, in root growth and in the mitotic index. The phytochemical screening showed the presence of triterpenes, coumarins, steroids, flavonoids and condensed tannins, compounds known to confer allelopathic characteristics upon other species. These data indicate that K. coriacea presents an allelopathic potential because its leaf extracts interfere with germination and growth without any interference of pH and osmotic potential in the results. Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Ciências e Letras de Assis, Assis, Avenida Dom Antonio, 2100, Vila Universitária, CEP 19806-900, SP, Brasil Programa de Pós-Graduação em Fisiologia e Bioquímica de Plantas, Escola Superior deAgricultura “Luiz de Queiroz” - ESALQ, Universidade de São Paulo - USP, Piracicaba, SP, Brasil Fundação Educacional do Município de Assis - FEMA, Assis, SP, Brasil Programa de Pós-Graduação em Ciências Biológicas (Botânica), Universidade Estadual Paulista - UNESP, Botucatu, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Ciências e Letras de Assis, Assis, Avenida Dom Antonio, 2100, Vila Universitária, CEP 19806-900, SP, Brasil Programa de Pós-Graduação em Ciências Biológicas (Botânica), Universidade Estadual Paulista - UNESP, Botucatu, SP, Brasi

O estudo alelopático investiga os efeitos positivos e negativos que metabólitos secundários de plantas, microrganismos ou fungos exercem sobre o desenvolvimento de indivíduos vizinhos. Pyrostegia venusta (Ker-Gaw) Miers (Bignoniaceae) é uma espécie invasora, utilizada na medicina popular e com metabólitos secundários bem caracterizados. Nesse trabalho foram investigados os efeitos de extrato hidroalcoólico de flores de P. venusta sobre a germinação e desenvolvimento do sistema radicular de sementes de Brassica chinensis L. var. parachinensis (Bailey) Sinskaja (couve-da-malásia). O extrato foi preparado adicionando o material vegetal seco e moído (flor) à solução de etanol e água destilada a 70% na proporção de 1:10 (p/v) e após 24 horas foi filtrado em baixa pressão sob vácuo. Para a realização dos bioensaios, o extrato foi diluídos em 1 mL de água destilada nas concentrações de 5, 10 ou 20 mg mL-1. As diferentes concentrações do extrato mostraram alteração nos parâmetros germinativos e no comprimento radicular da planta-teste couve-da-malásia. Os resultados indicam a existência de potencial alelopático em P. venusta, uma vez que as diferentes concentrações do extrato causaram alterações metabólicas e morfológicas nas sementes e plântulas de couve-da-malásia e, portanto, a P. venusta pode contribuir de forma significativa no estudo das interações químicas entre plantas e na possível produção de um bioherbicida.

Atualmente a soja é uma das principais culturas de interesse econômico cultivadas no Brasil, sendo que o país configura como o segundo maior produtor da oleaginosa, ficando atrás apenas da produção obtida pelos Estados Unidos da América. Para se proteger essa alta produtividade e manter o Brasil em um cenário econômico competitivo, os produtores brasileiros utilizam diversos defensivos agrícolas. Destes, os fungicidas são principalmente utilizados de maneira preventiva, ou seja, sem a presença do patógeno alvo, isso ocorre principalemnte devido ao modo de ação dos princípios ativos disponíveis comercialmente na atualidade. Essa utilização preferencial de fungicidas de maneira preventiva, fez com que crescesce também o interesse pelo estudo dos efeitos, sejam eles positivos ou negativos, na fisiologia e bioquímica das plantas cultivadas. O presente experimento foi instalado em casa de vegetação no município de Holambra-SP, tendo por objetivo avaliar os efeitos fisiológicos e bioquímicos provocados pela aplicação dos fungicidas benzovindiflupir+azoxistrobina (STL+AZT) e protioconazol+trifloxistrobina (PTZ+TFS), na cultura da soja. O delineamento experimental foi o de blocos casualizados contando com seis repetições cada. Os tratamentos com fungicidas utilizados foram: [T1] controle (testemunha); [T2] plantas que receberam aplicação de STL+AZT (30 + 60 g de ingrediente ativo por hectare), com adição do adjuvante Nimbus a 600 ml por hectare, conforme recomendação do fabricante; [T3] plantas que receberam aplicação de PTZ+TFS (70 + 60 g de ingradiente ativo por hectare), com adição do adjuvante Aureo a 400 ml por hectare, confrome recomendação do fabricante. Foram realizadas duas aplicações, sendo a primeira 42 dias após a emergência das plantas, quando estas se encontravam na fase de início de florescimento (R1) e a segunda com 15 dias de intervalo da primeira. Os tratamentos foram avaliados atraves de trocas gasosas, fluorescência da clorofila a e enzimas do sistema antioxidante. Não foram observadas alterações significativas na eficiência quântica potencial (Fv/Fm) e eficiência quântica máxima (Fv\'/Fm\') do fotossistema II (PSII) para nenhum dos fungicidas testados. Entretanto observou-se que a utilização de PTZ+TFS reduziu de maneira significativa no rendimento quântico efetivo do PSII (ΦPSII), na taxa aparente de transporte de elétrons (ETR) e na proporção de centro de reações do PSII \"abertos\" (qP), sendo que reduções na condutância estomática (gs), transpiração (E), taxa de assimilação de CO2 (A) e na velocidade máxima de carboxilação da rubisco (Vcmáx) também foram observadas quando se aplicou esse fungicida. Além das reduções observadas, constatou-se também que esse fungicida provoca uma elevação da temperatura foliar (Tf). Além dessas alterações fotoquímicas, foi também observado um aumento da atividade das enzimas dismutase da superoxido (SOD) e glutationa redutase (GR). Em contrapartida a utilização de STL+AZT aumentou ΦPSII, ETR e qP, assim como elevação em gs, E, Vcmáx e consequentemente em A também foram observados. Currently soybean is one of the main crops of economic interest cultivated in Brazil, and the country is the second largest producer of this oilseed, behind only of the production obtained by the United States of America. In order to protect this high productivity and keep Brazil in a competitive economic scenario, brazilian growers use several agricultural pesticides. Among these pesticides, fungicides are mainly used in a preventative way, that is, without the presence of the target pathogen, this is mainly due to the mode of action of the currently commercially available active ingredients. This preferential use of preventative fungicides has also increased the interest in studying the effects, whether positive or negative, on the physiology and biochemistry of cultivated plants. The present experiment was carried out in a greenhouse in the municipality of Holambra-SP, Brazil, to evaluate the physiological and biochemical effects of the fungicides benzovindiflupir + azoxystrobin (STL+AZT) and prothioconazole + trifloxystrobin (PTZ +TFS) on soybeans. The experimental design was a randomized block design with six replicates each. The treatments with fungicides used were: [T1] control (control); [T2] plants receiving STL + AZT (30 + 60 g of active ingredient per hectare), with addition of the Nimbus adjuvant at 600 ml per hectare, according to the manufacturer\'s recommendation; [T3] plants receiving PTZ + TFS (70 + 60 g active in per hectare), with addition of the Aureo adjuvant at 400 ml per hectare, according to the manufacturer\'s recommendation. Two applications were performed, the first was 42 days after the emergence of the plants when they were in the begning of flowering (R1) and the second after 15 days of the first one. The treatments were evaluated through gas exchange, chlorophyll a fluorescence and antioxidant system enzymes. No significant changes were observed in the potential quantum efficiency (Fv/Fm) and the maximum quantum efficiency (Fv\'/Fm\') of photosystem II (PSII) for none of the fungicides tested. However, it was observed that the use of PTZ+TFS significantly reduced the effective quantum yield of PSII (ΦPSII), the apparent electron transport rate (ETR) , and reductions in stomatal conductance (gs), transpiration (E), CO2 assimilation rate (A) and the maximum rate of carboxylation of rubisco (Vcmáx) were also observed when applying this fungicide. In addition to the observed reductions, it was also verified that this fungicide causes a rise in leaf temperature (Tf). In addition to these photochemical alterations, an increased activity of the enzymes superoxide dismutase (SOD) and glutathione reductase (GR) was also observed. In contrast, the use of STL + AZT increased ΦPSII, ETR and qP, as well as elevation in gs, E, Vcmax and consequently in A were also observed.