MENTHA X PIPERITA, OCIMUM BASILICUM E SALVIA DESERTA, (LAMIACEAE): ABORDAGENS FISIOLÓGICAS E FITOQUÍMICAS JENNIFER BÚFALO Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Doutor em Ciências Biológicas (Botânica) Área de concentração: Fisiologia Vegetal. BOTUCATU – SP 2015 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU MENTHA X PIPERITA, OCIMUM BASILICUM E SALVIA DESERTA, (LAMIACEAE): ABORDAGENS FISIOLÓGICAS E FITOQUÍMICAS JENNIFER BÚFALO ORIENTADORA: PROFA. DRA. CARMEN SÍLVIA FERNANDES BOARO Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Doutor em Ciências Biológicas (Botânica) Área de concentração: Fisiologia Vegetal. BOTUCATU – SP 2015 DDeeddiiccaattóórriiaass Dedico, Aos meus pais, Vital José Búfalo e Maria de Lourdes Provasi Búfalo, pelos exemplos de vida, dignidade, apoio incondicional e força durante todos esses anos, por compreenderem a minha ausência, quando não estive presente. A vocês a minha sincera admiração, meu respeito e eterna gratidão. Amo vocês. AAggrraaddeecciimmeennttooss AAggrraaddeecciimmeennttooss À Deus. Ao CNPq (140495/2011-8), pela bolsa de estudos concedida no Brasil, e à CAPES (4873-13-0) pela bolsa de Doutorado Sanduíche no Exterior. Ás minhas irmãs Graziela e Michelle Em especial a Michelle, por participar intensamente de todos os momentos da minha vida, e ao cunhado, Jose Augusto Poles por todo auxílio prestado. As minhas tias, Lilian Teresa Búfalo e Maria Eliza Búfalo, a minha gratidão é pequena diante da grandeza do que fizeram por mim. À Profª Dra. Carmen Sílvia Fernandes Boaro pela orientação, paciência e compreensão. Pela confiança depositada em mim, que soube dosar liberdade a qual proporcionou meu crescimento profissional e a realização deste trabalho. Ao pesquisador Dr. Charles L. Cantrell do USDA por ter me recebido tão bem em seu laboratório e, que muito contribuiu para a minha formação acadêmica. Agradecimento especial ao Dr. Stephen O. Duke pela oportunidade de trabalhar no USDA e principalmente pela oportunidade de conviver com Mrs. Amber Reichley, Mr. Solomon Green III, Mr. Jason Martin, Dr. Franck E. Dayan, Mrs. Gloria Redmond and Mr. Robert D. Johnson. Dr .Valtcho D. Zheljazkov da Oregon State University que colaborou durante o estágio sanduíche nos EUA e me deu oportunidade de participar de outros projetos. Aos Prof. Dr. João Rodrigues Domingos e a Profª Dra. Elizabeth Orika Ono pelas considerações no Exame Geral de Qualificação. Ao Prof. Dr. Luiz Fernando Rolim de Almeida e Dra. Marcia Ortiz de Mayo Marques por acompanharem meu crescimento desde a época do mestrado, pelo incentivo e por acreditarem em minha capacidade como pesquisadora, pelas conversas e amizade. À Profª Dra. Tatiane Maria Rodrigues por todas as oportunidades e por estar sempre disponível em ajudar em tudo o que for preciso. A todos os meus AMIGOS que estiveram ao meu lado, mesmo com a distância, muitos não me abandonaram e me deram força quando precisei, e aos novos AMIGOS conquistados, aqui e nos EUA. À todos funcionários, alunos e professores do Departamento de Botânica, pela convivência, ensinamentos e auxílios. À Profª Dra. Lídia R. Carvalho pela orientação na análise estatística do experimento. À equipe do Centro de Microscopia Eletrônica, IB, UNESP, pela assistência no processamento do material. Enfim, agradeço a todos que colaboraram, na elaboração deste trabalho. Obrigada! SSuummáárriioo SUMÁRIO RESUMO E ABSTRACT ........................................................................................................1 INTRODUÇÃO.........................................................................................................................4 REVISÃO BIBLIOGRÁFICA.................................................................................................5 Família Lamiaceae..................................................................................................................5 Mentha x piperita L................................................................................................................5 Ocimum basilicum L...............................................................................................................8 Salvia deserta Shang............................................................................................................10 REFERÊNCIAS BIBLIOGRÁFICAS..................................................................................12 CAPÍTULO I – MANUSCRITO I PEG-induced osmotic stress in Mentha x piperita L.: structural features and metabolic responses……………………………………………………………………………………...20 CAPÍTULO II – MANUSCRITO II Organic versus conventional fertilization effects on sweet basil (Ocimum basilicum L.) growth in a greenhouse system ………………………………………………........................54 CAPÍTULO III – MANUSCRITO III Antimicrobial and antileishmanial activities of diterpenoids isolated from the roots of Salvia deserta Shang…………………………………………………...………….…………………70 CONSIDERAÇÕES FINAIS.................................................................................................89 RReessuummoo ee AAbbssttrraacctt 1 BUFALO, J. MENTHA X PIPERITA, OCIMUM BASILICUM E SALVIA DESERTA, (LAMIACEAE). ABORDAGENS FISIOLÓGICAS E FITOQUÍMICAS. 2015, 89 p. - INSTITUTO DE BIOCIÊNCIAS, UNESP – UNIVERSIDADE ESTADUAL PAULISTA, BOTUCATU. RESUMO - As plantas medicinais e aromáticas como hortelã-pimenta (Mentha x piperita), manjericão doce (Ocimum basilicum) e salvia (Salvia deserta) possuem grande importância no contexto mundial devido à demanda das indústrias de alimentos, químicas e farmacêuticas. Hortelã-pimenta, além de ser utilizada pela medicina popular, é cultivada principalmente pela extração de óleo essencial. O estudo realizado objetivou avaliar se o estresse osmótico, aplicado em duas concentrações de polietilenoglicol (PEG) em curto período de tempo, influencia na anatomia e ultraestrutura foliar e interfere no padrão fisiológico da M. x piperita, modificando o perfil do óleo essencial. Os resultados indicaram que as respostas ao estresse osmótico, foram dose dependente, pois as plantas submetidas ao PEG 50 g L-1 mantiveram os aspectos estruturais e funções metabólicas semelhantes às plantas do tratamento controle. Plantas submetidas ao PEG 100 g L-1, apresentaram alterações anatômicas, danos ultraestruturais, como degradação e lise de organelas, as quais estão de acordo com a redução do potencial água das folhas e das trocas gasosas, aumento no conteúdo de açúcares totais e ativação no sistema de defesa antioxidante. Essas plantas apresentaram diminuição do conteúdo e qualidade do óleo essencial. O manjericão doce, planta medicinal importante utilizada principalmente na culinária, foi cultivado em casa de vegetação em sistema de fertilização orgânica e convencional, em duas doses de nitrogênio (150 e 250 kg ha-1 N). Os resultados revelaram que plantas cultivadas com fertilizante convencional na dose de 250 kg ha-1 N apresentaram maior massa fresca. Os tratamentos aplicados não afetaram o conteúdo, produção e composição do óleo essencial, sendo o linalol, o composto majoritário encontrado no estudo. Os resultados demostraram que independente do fertilizante, orgânico ou convencional, não houve modificação da composição do óleo essencial. A bioprospecção realizada com raízes de S. deserta identificou a presença de quatro diterpenos com atividades biológicas. Taxodione apresentou atividade antileishmanicida, antifúngica e antimicrobiana e o ferruginol apresentou a maior atividade (24-h IC50 1.29 mg L-1) contra bactéria presente em peixes, Streptococcus iniae. A fração do extrato bruto qual continha os compostos isolados 7- O-acetylhorminone e horminone mostraram forte atividade antibacteriana (1.28 µg mL-1 para Staphylococcus aureus e 1.12 µg mL-1 para S. Aureus resistente á meticilina (MRSA) do que os compostos testados isoladamente. 7-O-acetylhorminone e horminone exibiram ação sinérgica contra MRSA (FIC 0.2 µg mL-1) e horminone apresentou melhor atividade contra S. aureus em relação aos outros compostos isolados de raízes de S. deserta. O estudo mostra que raízes de S. deserta são fontes potenciais de diterpenos com atividades biológicas e possibilidade para o desenvolvimento de novos compostos dessa planta com atividade pesticida. De modo geral, o estudo permite concluir que o metabolismo das plantas medicinais e aromáticas responde à variação das condições abióticas, déficit hídrico e nutrição, modificando o óleo essencial sintetizado, cuja bioprospecção pode revelar atividades biológicas de grande importância econômica. Palavras-chave: hortelã-pimenta, salvia, manjericão, óleo essencial, ultraestrutura, diterpenos. 2 BUFALO, J. MENTHA X PIPERITA, OCIMUM BASILICUM AND SALVIA DESERTA, (LAMIACEAE). PHYTOCHEMICAL AND PHYSIOLOGICAL APPROACHES. 2015, 89 p. - INSTITUTO DE BIOCIÊNCIAS, UNESP – UNIVERSIDADE ESTADUAL PAULISTA, BOTUCATU. ABSTRACT - Medicinal and aromatic plants such as peppermint (Mentha x piperita), sweet basil (Ocimum basilicum) and salvia (Salvia deserta) have great importance in the global context due to the demand of the food, chemical, and pharmaceutical industries. Peppermint is used by folk medicine and is grown mainly for essential oil extraction. The study investigated whether osmotic stress induced by two polyethyleneglycol (PEG) levels, in a short time, in peppermint changes the physiological pattern, anatomy, leaf ultrastructure and essential oil content and composition. The results indicated that osmotic stress responses were dose dependent, as plants subjected to PEG 50 g L-1 maintained structural features and metabolic functions similar to those of control plants. Plants exposed to PEG 100 g L-1 showed anatomical changes and ultrastructural damage as degradation and organelles lysis, which are in agreement with the low leaf water potential, gas exchange reduction, increase of total sugars, and activity of antioxidant enzymes. These plants showed lower content and quality of essential oil. Sweet basil, an important medicinal plant used mainly in culinary arts, was grown in a greenhouse using both organic and conventional fertilization systems with two nitrogen rates each (150 and 250 kg N/ha). The results showed that the highest fresh weight was obtained from the plants grown with conventional fertilizer at a rate of 250 kg N/ha. The treatments did not affect the essential oil content, yield, and composition and linalool was the major compound found in the study. The results showed that regardless of fertilizer, organic or conventional, there was no change in the composition of the oil. The bioprospecting conducted with S. deserta roots identified the presence of four diterpenes with biological activities. Taxodione showed leishmanicidal, antifungal, and antimicrobial activity, and the ferruginol displayed the greatest activity (24-h IC50 1.29 mg/L) against the fish pathogenic bacteria Streptococcus iniae. The crude extract fraction which contained the isolated compounds 7-O-acetylhorminone and horminone showed stronger in vitro antibacterial activity (1.28 µg/mL for Staphylococcus aureus and 1.12 µg/mL for methicillin-resistant S. aureus - MRSA) than the compounds tested alone. 7-O-Acetylhorminone and horminone exhibited a synergistic effect against MRSA (FIC 0.2 µg/mL), and horminone had better activity against S. aureus with respect to other compounds isolated from S. deserta roots. The study shows that S. deserta roots are a good source of diterpenoids with biological activities and that are possibilities for the development of novel antipesticidal compounds from this plant. Overall, the study suggests that the metabolism of medicinal and aromatic plants respond to changes in abiotic conditions, water deficit, and nutrition by modifying the essential oil synthesized, where bioprospecting can reveal biological activities of great economic importance. Keywords: peppermint, salvia, basil, essential oil, ultrastructure, diterpenes. IInnttrroodduuççããoo ee RReevviissããoo BBiibblliiooggrrááffiiccaa __________________________ _ _ _Introdução e Revisão Bibliográfica 4 Introdução As plantas produzem grande diversidade de compostos do metabolismo secundário, a partir da biossíntese provenientes de compostos do metabolismo primário. Esses compostos secundários, encontrados em concentrações relativamente baixas e em determinados grupos de plantas, podem apresentar marcantes atividades biológicas (SANTOS, 2007). A biossíntese desses compostos depende de inúmeros fatores do ambiente, como condições climáticas, sazonais, geográficas e de solo (GOBBO-NETO; LOPES, 2007). No entanto, os fatores genéticos também interferem na produção de compostos do metabolismo secundário (TRAPP; CROTEAU, 2001). Os metabolitos secundários estão envolvidos na resistência a pragas e doenças, na atração de polinizadores, na interação com microorganismos, entre outros (SANTOS, 2007). Atualmente, o interesse nos metabólitos secundários tem aumentado devido à grande importância que desempenham na indústria farmacêutica, alimentícia, bebidas, perfumaria e cosméticos (SANTOS, 2007). Entretanto, ainda são necessários estudos que integrem conhecimento sobre fisiologia, anatomia e morfologia das plantas, bem como química de produtos naturais, como forma de identificar os compostos naturais, compreender os fatores que interferem no crescimento e desenvolvimento de plantas que produzem princípios ativos com interesse medicinal. Espera-se que as variáveis fisiológicas, anatômicas e químicas avaliadas neste trabalho, contribuam para melhor entendimento do metabolismo das espécies da família Lamiaceae, Mentha x piperita, Ocimum basilicum e Salvia deserta produtoras de metabólitos secundários. Assim, como em outras famílias de espécies medicinais, existe crescente busca por espécies pouco exploradas e produtoras de metabólitos secundários, cuja bioprospecção revele compostos com atividades biológicas ou ainda estudos que abordem o cultivo de plantas medicinais visando melhorar a produção e qualidade de metabólitos de óleos essenciais. Um estudo sobre o estresse osmótico induzido pelo PEG em Mentha x piperita L.: aspectos estruturais e respostas metabólicas é apresentado no capítulo I. O capítulo II refere-se a efeitos da fertilização orgânica versus convencional em manjericão doce (Ocimum basilicum L.) cultivado em casa de vegetação. No capítulo III o estudo realizado aborda atividades antimicrobianas e antileishimania de diterpenóides isolados de raízes de Salvia deserta Shang. __________________________ _ _ _Introdução e Revisão Bibliográfica 5 Revisão Bibliográfica Família Lamiaceae A família Lamiaceae ou Labiatae pertencente a ordem Lamiales, que também inclui outras famílias como Verbenaceae, Scrophulariaceae e Acanthaceae (TUCKER; NACZI, 2006), é conhecida como família da menta (JUDD et al., 2009) e representada por 252 gêneros, com 6.800 a 7.200 espécies (JUDD et al,. 2009; RAJA, 2012). Recentes estudos sobre morfologia química e filogenia molecular têm identificado alterações na classificação da família, resultando na adição de gêneros anteriormente incluídos na família Verbenaceae (JUDD et al., 2009; HARLEY et al., 2012). Essa família possui distribuição cosmopolita (JUDD et al., 2009), suas espécies são nativas principalmente do Mediterrâneo, embora algumas tenham origem na Austrália, Sudoeste da Ásia e América do Sul. Atualmente ocorrem em regiões tropicais e temperadas, em todo o mundo, exceto na Antártida (HARLEY et al., 2012). As espécies podem ser ervas, arbustos ou árvores e apresentam importância econômica por conterem óleos essenciais ou também pela utilização como especiarias, incluindo gêneros como Mentha, Lavandula, Rosmarinus, Salvia, Ocimum, Tymus e Origanum (JUDD et al., 2009). As principais espécies estão alocadas na subfamília Nepetoideae que representa 47% das espécies (TUCKER; NACZI, 2006). Entre elas, Salvia officinalis L., Rosmarinus officinalis L., Mentha L. spp., Thymus vulgaris L., Origanum L. spp., Satureja hortensis L., Monarda L. spp., Melissa officinalis L., Lavandula L. spp., Aeollanthus suaveolens Mart. ex Spreng., Ocimum spp. e Plectranthus L’Hér. spp. e são conhecidas por produzirem óleos aromáticos com grande importância comercial ou medicinal (HARLEY et al., 2012). Atualmente o conhecimento dos inúmeros compostos químicos provenientes de espécies da família Lamiaceae é muito extenso, sendo dominado por óleos essenciais, especialmente por monoterpenos e sesquiterpenos encontrados em gêneros de importância econômica (WU et al., 2012). No entanto, constituintes químicos, como diterpenos, triterpenos, fenólicos, e outros compostos podem também ser de grande importância como caracteres taxonômicos, auxiliando na identificação de espécies e compostos biologicamente ativos, com papel ecológico, na busca de novos compostos (WU et al., 2012) com fins terapêuticos. __________________________ _ _ _Introdução e Revisão Bibliográfica 6 Mentha x piperita L. O gênero Mentha apresenta cerca de 19 espécies e 13 híbridos naturais pertencentes a família Lamiaceae (TELES et al., 2013). O gênero inclui muitas espécies produtoras de óleo essencial utilizadas na perfumaria, cosméticos e indústrias farmacêuticas (TELCI et al., 2011) e é responsável por aproximadamente 2000 toneladas de óleo essencial, representando o segundo óleo mais importante depois do gênero Citrus (MUCCIARELLI et al., 2001). Além da utilização de espécies do gênero Mentha como base de alimentos e preparações de chá, os usos medicinais que remotam aos tempos antigos, incluem aplicações carminativas, antinflamatórias, antiespasmódicas, analgésicas e estimulantes (HENDRIKS, 1998; COWAN, 1999). As espécies de menta também são utilizadas contra náuseas, bronquite, flatulência, úlcera e problemas no fígado (FOSTER, 1990; TYLER, 1993; HENDRIKS, 1998; COWAN, 1999). Os óleos essenciais e extratos vegetais das espécies de menta apresentam atividades antimicrobianas (ISCAN et al., 2002), antioxidantes, citotóxicas (YADEGARINIA et al., 2006; GULLUCE et al., 2007; HUSSAIN et al., 2010; SINGH et al., 2011) e também revelaram propriedades inseticidas sendo considerados fontes para pesticidas ecologicamente correto (KUMAR et al., 2011). A Mentha x piperita L., híbrido natural resultante do cruzamento de M. aquatica e M. spicata, espécie explorada na produção de terpenóides (MAFFEI et al., 1999) é conhecida como hortelã pimenta, menta e hortelã-apimentada. A espécie é considerada perene, apresenta forte odor pungente, semelhante ao da pimenta (pepper) de onde originou o nome específico ‘piperita’ (CHAUDHRY et al., 1957). A planta é nativa da Europa no Mediterrâneo, naturalizada nos norte dos Estados Unidos e Canadá, sendo cultivada em muitas partes do mundo (MCKAY; BLUMBERG, 2006). A espécie é cultivada principalmente em áreas de clima temperado e tropical para a produção de óleo essencial nas folhas (MAFFEI; SACCO, 1987) que ocorre em órgãos especializados, os tricomas glandulares (TURNER et al., 2000). Na indústria alimentícia, as espécies M. x piperita, M. arvensis e M. spicata, são utilizadas como agentes flavorizantes e produção de alimentos e bebidas (ZHELJAZKOV et al., 2010) sendo consideradas espécies de maior importância econômica (TELCI et al., 2011). Seu óleo essencial, líquido de cor amarelo claro, odor forte e agradável, possui sabor aromático (CHAUDHRY et al., 1957), sendo um dos óleos essenciais mais produzidos e consumidos no mundo (ISCAN et al., 2002). As folhas de hortelã pimenta são utilizadas pela população na forma de chá, em casos de má digestão, náuseas e problemas intestinais no aparelho digestivo (LORENZI; MATOS, __________________________ _ _ _Introdução e Revisão Bibliográfica 7 2002). A espécie M. x piperita tem sido utilizada como planta modelo para o estudo do metabolismo de terpenos (CROTEAU et al., 2005; RIOS-ESTEPA et al., 2008). Os principais monoterpenos encontrados no óleo essencial da M. x piperita são mentol, mentona, pulegona, limoneno, eucaliptol e acetato de mentila (MAFFEI; SACCO, 1987). O óleo essencial de hortelã pimenta apresenta mais de 200 componentes (LAWRENCE, 1988) sendo a maioria monoterpenos e a via biossintética inclui reações enzimáticas que conduz a formação do principal componente, o mentol, (RIOS-ESTEPA et al., 2008) considerado constituinte majoritário (ANSARI et al., 2000). Estudos com o óleo essencial da M. x piperita tem demonstrado apresentar atividade contra microorganismos, sendo sugerido o mentol como responsável pela bioatividade (ISCAN et al., 2002; AZUMA et al., 2003). O óleo essencial de M. x piperita também apresenta atividade larvicida e forte ação repelente contra mosquitos adultos (ANSARI et al., 2000). Além disso, o óleo essencial de hortelã pimenta também demonstra atividade genotóxica (LAZUTKA et al., 2001) e antiviral (MINAMI et al., 2003; SCHUHMACHER et al., 2003). A proporção dos principais componentes voláteis identificados no óleo essencial da M. x piperita são mentol (33-60%), mentona (15-32%), isomentona (2-8%), eucaliptol (5-13%), acetato de mentila (2-11%), mentofurano (1-10%), limoneno (1.7%), β-mirceno (0.1–1.7%), β-cariofileno (2–4%), pulegona (0.5–1.6%) e carvona (1%) (SANG, 1982; PITTLER; ERNST, 1998; DIMANDJA et al., 2000; GHERMAN et al., 2000). O rendimento do óleo essencial das folhas da M. x piperita ocorre em torno de 1.2 - 3.9% em volume por massa de material seco (PICURIC-JOVANOVIC et al., 1997; BLUMENTHAL et al., 1998). A composição química das plantas é conhecida por ser influenciada por fatores externos e alguns compostos podem ser acumulados em resposta à alterações ambientais (KOENEN, 2001) e condições de crescimento (FIGUEIREDO et al., 2008). A composição química do óleo essencial da M. x piperita varia com a maturidade, região geográfica, condições de processamento do óleo e variedade das espécies (CLARK; MENARY, 1981; ROHLOFF, 1999; GHERMAN et al., 2000; BLANCO et al., 2002; PINO et al., 2002; RUIZ DEL CASTILLO et al., 2003; XU et al., 2003). Diversos trabalhos na literatura demonstram a variação no conteúdo e composição química do óleo essencial da M. x piperita em diferentes condições de cultivo. Esses trabalhos correlacionam a otimização do cultivo da M. x piperita em diferentes condições de temperatura (BURBOTT; LOOMIS, 1967; FAHLEN et al., 1997), água (CHARLES, et al. 1990; MARCUM; HANSON, 2006; PEGORARO et al. 2010), luz (BURBOTT; LOOMIS, 1967; PEGORARO et al. 2010), nutrição (MAROTTI et al. 1994) e sazonalidade (MAROTTI et al. 1993), com a produtividade de biomassa vegetal e a biossíntese de compostos voláteis de elevado valor comercial. Entre as condições abióticas __________________________ _ _ _Introdução e Revisão Bibliográfica 8 que interefem no crescimento e metabolismo do vegetal a baixa diponibilidade de água causa o déficit hídrico e afeta o funcionamento normal, alterando o estado fisiológico e o equilíbrio da planta (GIMENEZ et al., 2005; XU et al., 2010; SHAO et al., 2008). Nas plantas medicinais e aromáticas, a menor disponibilidade hídrica no solo pode causar significantes alterações no acúmulo e composição do óleo essencial (PETROPOULOS et al., 2008) e influenciar a rota metabólica de biossíntese do óleo essencial promovendo reações de oxidação ou redução e assim, interferir no acúmulo e concentração de compostos específicos (CHARLES, et al. 1990). Existe grande interesse em entender os fatores que afetam a biossíntese do óleo essencial de hortelã pimenta, a fim de aumentar o conteúdo de compostos comercialmente importantes, como mentol e mentona, e diminuir a presença de indesejáveis, pulegona e mentofurano (MAHMOUD; CROTEAU, 2002). Pulegona e metonfurano participam da rota metabólica de biossíntese dos monoterpenos e o composto pulegona é convertido a mentofurano ou mentona e então à mentol (MARCUM; HANSON, 2006). De acordo com estudos anteriores, a redução da mentona a mentol aumenta a qualidade do óleo essencial de hortelã pimenta (CLARK; MENARY, 1980). Assim deve ser considerado que a composição química dos óleos essenciais das plantas é subordinada a variações quantitativas e qualitativas (HUSSAIN et al., 2010). Essas variações podem interferir nas atividades biológicas que são dependentes da composição química dos óleos essenciais, uma vez que, o material vegetal coletado em diferentes condições ambientais pode conter diferentes compostos ou novos compostos com outras bioatividades (ELOFF, 1999). Ocimum basilicum L. O gênero Ocimum L., apresenta cerca de 150 espécies (JAVANMARDI et al., 2003), entre elas o manjericão doce (Ocimum basilicum L.). A espécie também é conhecida como manjericão, alfavaca, alfavaca-cheirosa, ou basílico sendo considerada uma das mais cultivadas em muitos países, pois representa fonte de matéria-prima para a indústria de óleos essenciais (JAVANMARDI et al., 2003; HUSSAIN et al., 2008). Seus extratos também são utilizados na produção de cosméticos e produtos farmacêuticos e pesticidas (UMERIE et al., 1998; KEITA et al., 2001; PASCUAL-VILLALOBOS; BALLESTA-ACOSTA, 2003). O manjericão é uma erva anual, amplamente utilizada em muitos tipos de preparações culinárias em países do Mediterrâneo (SIFOLA; BARBIERI, 2006), contem flores roxas ou brancas, originalmente nativo da Índia e outras regiões da Ásia (KLIMANKOVA et al., 2008). De acordo com o aroma, o manjericão pode ser classificado em doce, limão, canela, __________________________ _ _ _Introdução e Revisão Bibliográfica 9 cânfora, anis e cravo (BLANK et al., 2004). As plantas de manjericão são utilizadas na culinária, como planta ornamental, medicinal e aromática, sendo o óleo essencial valorizado no mercado internacional pelo elevado teor do composto linalol (BLANK et al., 2004). Suas folhas são utilizadas secas ou frescas, como agente aromatizante em alimentos, produtos de confeitaria e bebidas (KOPSELL et al., 2005). Tradicionalmente a medicina popular tem empregado o uso da espécie em propriedades carminativas, estimulante e antiespasmódica (MAROTTI et al., 1996). O óleo essencial é utilizado nas indústrias alimentícias e perfumaria (PRASAD et al., 1986) e alguns dos seus componentes, como eucaliptol, linalol e cânfora, são conhecidos por apresentarem atividade biológica (MORRIS et al., 1979) como antibacteriana (PRASAD et al., 1985; ELGAYYAR et al., 2001) e inseticida (BOWERS; NISHIDA, 1980). Além disso, cânfora e eucaliptol sao compostos também relatados como agentes alelopáticos (RICE, 1979). O elevado valor agregado ao óleo essencial de manjericão é devido a presença de fenilpropanóides, como eugenol, chavicol e seus derivados, ou terpenóides, como os monoterpenos linalol, metil cinamato e limoneno (SIFOLA; BARBIERI, 2006). Os componentes do óleo essencial de manjericão, eugenol, metil chavicol e linalol acumulam-se em estruturas especializadas, os tricomas glandulares (GANG et al., 2001). Os fenilpropanóides e terpenóides presentes no óleo essencial são sintetizados por rotas metabólicas diferentes. Os compostos chavicol, metil chavicol e eugenol são sintetizados a partir da rota do chiquimato, que produz o precursor aminoácido aromático fenilalanina (GANG et al., 2001; SANGWAN et al., 2001), enquanto os terpenóides são derivados da rota do mevalonato e/ou da deoxilulose (DXP) (MAHMOUD; CROTEAU, 2002). O gênero Ocimum pertencente à família Lamiaceae é caracterizado por uma grande variabilidade de morfologia e quimiotipos (LAWRENCE, 1988). A facilidade de polinização cruzada levou ao surgimento de um grande número de subespécies, variedades e formas (GUENTHER, 1949). Diferentes quimiotipos tem identificado acessos de O. basilicum baseado na composição química do óleo essencial. Grayer et al. (1996) classificou cinco perfis de óleos dependentes da abundância dos seguintes compostos: (i) linalol; (ii) metil cavicol; (iii) eugenol; (iv) linalol e eugenol; (v) metil chavicol e metil eugenol. No mercado internacional os quimiotipos de O. basilicum são relatados com base na composição química do óleo essencial e localização geográfica (BOWES; ZHELJAZKOV, 2004). O manjericão Europeu tem como componentes principais, o linalol e metil chavicol, frequentemente considerado por conter sabor mais fino. O manjericão Reunion é o quimiotipo que possui maior nível de metil chavicol e o principal componente do quimiotipo tropical é o metil __________________________ _ _ _Introdução e Revisão Bibliográfica 10 cinamato. Outro tipo que possui elevado teor de eugenol é cultivado no Norte da África, Rússia e Europa Oriental e partes da Ásia (MAROTTI et al., 1996). A literatura também mostra grande diversidade genética de O. basilicum quanto as características bioquímicas como antocianinas (SIMON et al., 1999) e compostos voláteis terpenóides e fenilpropanóides (IIJIMA et al., 2004). Além disso, também são consideradas diferenças nos cultivares quanto a morfologia relacionadas à altura das plantas, cor, tamanho e textura das folhas. Além das características morfológicas e aromáticas determinadas pelo genótipo, a composição química das plantas é muito influenciada pelas condições ambientais e técnicas agronômicas (PICCAGLIA et al., 1991; MAROTTI et al., 1996). A nutrição mineral é um exemplo a ser considerado, uma vez que, aplicações de nitrogênio mostraram aumentar a produção do óleo essencial em plantas aromáticas (RAM et al., 1995) assim como as diferentes fontes de nitrogênio, orgânico e inorgânico (ADLER et al., 1989; KANDEEL et al., 2002) que podem interferir no perfil do óleo essencial. Os principais componentes do óleo essencial de O. balisicum variam em relação a quantidades dependendo do quimiotipo da planta e também pelas técnicas de processamento do óleo essencial (PINO et al., 1994; GILL; RANDHAWA, 1996; MAROTTI et al., 1996). O método de secagem de plantas de manjericão para extração de óleo essencial e o cultivar (LACHOWIEZ et al., 1997) podem afetar o conteúdo e a qualidade do óleo essencial (GRAYER et al., 1996; YOUSIF et al., 1999) Salvia deserta Shang Salvia, maior gênero na família Lamiaceae, apresenta cerca de 900 espécies, amplamente distribuídas no mundo, na região do Mediterrâneo, África do Sul, América Central, América do Sul e Sudeste da Ásia. O nome Salvia (sage) é derivado de ‘salvare’ originário do latin que significa ‘cura’ (TOPCU, 2006). Na Europa, particularmente em países do Mediterrâneo, infusões de várias espécies de Salvia são geralmente utilizadas na medicina popular, sendo que comercialmente as espécies mais utilizadas são S. officinalis e S. triloba (ULUBELEN; TOPCU, 1998; ULUBELEN, 2000). Algumas espécies de Salvia possuem importância econômica por serem utilizadas como agentes aromatizantes em perfumaria e cosméticos (WU et al., 2012) como S. sclare e S. pratensis (SALEHI et al., 2008) e outras são cultivadas para fins culinários (LU; FOO, 2002). As espécies de Salvia demonstram atividades antioxidantes (TEPE, et al., 2006), antisséptica (DANIELA, 1993), antibacteriana (ULUBELEN, 2003), antifúngica (KOBAYASHI, 1987), antiviral (TADA, et al., 1994), citotóxica (LIN, 1989; ULUBELEN, __________________________ _ _ _Introdução e Revisão Bibliográfica 11 1999), carminativa, diurética e hipoglicemica (JIMENEZ, 1986). Keller (1978) listou mais de 60 doenças diferentes a qual espécies de Salvia possuem finalidade terapêutica. As várias espécies de Salvia, conhecidas por possuir propriedades antimicrobianas, antioxidantes e anticâncer, são consideradas plantas populares utilizadas na medicina desde os tempos antigos (COWAN, 1999; GALI-MUHTASIB et al., 2000; BADISA et al., 2005;). As espécies de Salvia estudadas têm produzido uma série de metabolitos secundários, que têm atraído considerável atenção das comunidades científicas por apresentar um amplo espectro de atividades biológicas e novas estruturas (WU et al., 2012). Essas espécies são fontes ricas em compostos fitoquímicos incluindo flavonóides, sesquiterpenóides, diterpenóides, sesterpenes e triterpenes (LU; FOO, 2002; HASSANZADEH et al. 2011). A parte área dessas plantas contém flavonóides, triterpenóides e monoterpenos particularmente nas flores e folhas, enquanto os diterpenos ocorrem frequentemente nas raízes (TOPCU, 2006). No entanto, há registros na literatura de algumas espécies de Salvia Americana que contêm diterpenos na parte aérea e triterpenos e flavonas nas raízes (TOPCU, 2006). Os diterpenos são metabólitos secundários estruturalmente mais diversificados isolados de espécies de Salvia. Vários compostos ativos como tanshinones, D (+) ácido láctico 3,4-dihidroxifenol, ácidos salvianólico (A-F) e ácido rosmarínico (LI, 1997; WANG et al., 2010) foram isolados e identificados nos últimos anos (TAYARANI-NAJARAN et al., 2013). Os diterpenos isolados a partir das espécies de Salvia demonstram atividades antioxidante, antibacteriana, antimutagênica, antiinflamatória e propriedades citotóxicas (AMARO-LUIZ et al., 1998) como por exemplo, o taxol, o cafestol, e kahweol substâncias que apresentam propriedades anticâncer (WU et al., 2012). A Salvia deserta Shang é considerada planta herbácea, perene, geralmente encontrada em áreas de estepes, bordas de matas e margens de rios (ABDULINA, 1999). As plantas dessa espécie possuem 35-90 cm de altura, pequenas folhas e inflorescências roxas (PAVLOV et al., 1964). A espécie está distribuída na Ásia Central em regiões montanhosas, Cáucaso e Sibéria Ocidental (SOKOLOV, 1991) e a composição química de S. deserta contém ácidos orgânicos, alcalóides, vitamina C, taninos, flavonóides e quinonas (PAVLOV et al., 1964). Nas raízes, são identificadas quinonas e a parte aérea contém pequenas quantidades de óleo essencial (0,02%), com ação antimicrobiana (PAVLOV, 1964). As sementes secas contêm aproximadamente 23% de ácido graxo, utilizados na fabricação de vernizes. Na medicina popular, as partes aéreas são utilizadas para o tratamento de infecções intestinais e febre, enquanto as folhas e flores são utilizadas para problemas do coração. Seu fruto contém óleo, que é utilizado para cicatrização de feridas, taquicardia e disenteria. (PAVLOV, 1964). __________________________ _ _ _Introdução e Revisão Bibliográfica 12 A parte aérea de S. deserta contém triterpenóides como ursane, oleanano e derivados de lupine (SAVONA et al., 1987), enquanto diterpenos (royleanone, ferruginol, taxodione, etc.), derivados do ácido cafeico (ácido rosmarínico, ácido litospermico B, etc.) e o esteróide daucosterol (TEZUKA et al., 1998) são encontrados principalmente nas raízes. Referências Bibliográficas1 ABDULINA S.A. List of vascular plants of Kazakhstan. Almaty, 1999. - p.112. ADLER, P.R.; SIMON, J.E.; WILCOX, G.E. Nitrogen form alters basil growth and essential oil content and composition. Hortscience. v.24, p.789–790, 1989. AMARO-LUIS, J. M.; HERRERA, J. R.; LUIS, J. G. Abietane diterpenoids from Salvia chinopeplica. Phytochemistry. v.47, p. 895-897, 1998. ANSARI, M.A.; VASUDEVAN, P.; TANDON, M.; RAZDAN, R.K. Larvicidal and mosquito repellent action of peppermint (Mentha piperita) oil. 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Para tal, foram seguidas as normas de formatação de periódicos científicos internacionais (A2, B1 – Comitê de Biodiversidade da Capes). CCaappííttuulloo II –– MMaannuussccrriittoo II** *De acordo com as normas da revista Environmental and Experimental Botany _________ ___ _ Manuscrito I 20 PEG-induced osmotic stress in Mentha x piperita L.: structural features and metabolic responses Jennifer Búfaloa*, Tatiane Maria Rodriguesa, Luiz Fernando Rolim de Almeidaa, Luiz Ricardo dos Santos Tozina, Marcia Ortiz Mayo Marquesb, Carmen Silvia Fernandes Boaroa aDepartment of Botany, Institute of Biosciences (IB), UNESP - Univ. Estadual Paulista, Botucatu, São Paulo, 18618-970, P.O. Box: 510, Brazil bCampinas Agronomic Institute, Campinas, SP, Brazil *Corresponding author: email address: jenniferbufalo@yahoo.com.br; phone number: (+55) 15 981464967; (+55) 14 38800124 Abstract The present study investigated whether osmotic stress induced by two polyethyleneglycol levels in a short time in peppermint (Mentha x piperita L.) changes the physiological parameters, leaf anatomy and ultrastructure and essential oil content and composition. Evaluations of water potential, relative water content, anatomy, ultrastructural features, gas exchange, chlorophyll fluorescence, biochemistry and essential oil content and composition were performed under two polyethyleneglycol levels (PEG 50 and PEG 100) in a hydroponic experiment. None of the tested morphological and physiological parameters of M. x piperita showed significant changes in plants exposed to PEG 50. These plants activated antioxidant defense systems and showed smaller morphological changes but no ultrastructural changes. By contrast, the osmotic stress caused by PEG 100 inhibited leaf gas exchange, reduced the essential oil content and changed the oil composition in the plants, including a decrease in menthone and an increase in menthofuran. However, PEG 100 increased the total soluble sugar content in plants, which indicates osmotic adjustments for preventing water loss. These plants also showed an increase in peroxidase activity, but this change was not sufficient to decrease the lipoperoxide level responsible for damaging the membranes of organelles. No significant changes were found in chlorophyll fluorescence, intercellular CO2 concentration or water use efficiency in plants exposed to osmotic stress. Morphological changes were correlated with the physiological features evaluated, as plants exposed to PEG 100 showed collapsed cell areas, an increase in intercellular space, mesophyll thickening, and cuticle shrinkage. In addition, plants exposed to PEG 100 exhibited signs of cytoplasm degeneration, including smaller nuclei, morphological changes in plastids, and lysis of mitochondria and _________ ___ _ Manuscrito I 21 organelles. In summary, our results reveal that PEG-induced osmotic stress in M. x piperita depends on the exposure time and intensity of osmotic stress applied. Plants exposed to higher PEG levels exhibited changes in physiological parameters, morphology and ultrastructural features, which modified essential oil content and quality and potentially also influenced growth. Keywords: water deficit, Mentha, ultrastructure, enzymes antioxidant, gas exchange 1. Introduction Plant growth and productivity are influenced by abiotic and biotic stresses. Plants are often exposed to stress conditions caused by temperature, salinity, water and nutrient availability and heavy metal toxicity (Shao et al., 2008). High temperature, light intensity and drought are among the most important environmental stresses affecting plant survival and crop productivity (Boyer, 1982). These environmental stresses trigger a wide variety of plant responses, ranging from changes in gene expression to altered cellular metabolism (Shao et al., 2008). The use of polyethyleneglycol (PEG) is known to reduce the water potential (Michel, 1983) and induce plant water deficits (Perez-Alfocea et al., 1993; O'Donnell et al., 2013), causing physiological disorders and resulting in a lower water uptake and loss of cell turgor (Munoz- Mayor et al., 2012). Tissue dehydration affects plants at various levels of their organization (Yordanov et al., 2000), causing changes in water relations, biochemical and physiological processes, membrane structure and organelle morphology (Gaff, 1989; Stevanovic et al., 1992; Tuba et al., 1993; Sarafis, 1998). The reaction of a plant to water stress depend on the its intensity and duration of the stress as well as the plant species and its stage of development (Holtman et al., 1994; Jaleel et al., 2007; Jayakumar et al., 2007). Certain plant responses may provide degrees of tolerance to osmotic stress (Amaya et al., 1999). Tolerant plants have developed strategies to cope with water deficits, including anatomical, morphological and metabolic mechanisms (Pereyra et al., 2012) that adjust their physiology and metabolism to accommodate osmotic stress (Bohnert and Sheveleva, 1998). One such strategy is the control of stomatal closure, the initial cell response against desiccation (Yordanov et al., 2000), which promotes transpiration reduction and water-saving by plants (Kaiser, 1987; Chaves, 1991). However, stomatal closure caused by osmotic stress reduces the CO2/O2 ratio in leaves and inhibits photosynthesis (Moussa, 2006), leading to further reductions in the photosynthetic electron chain and increased production of reactive oxygen species (ROS) (Candan and Tarhan, 2012). Consequently, to protect their cellular and _________ ___ _ Manuscrito I 22 sub-cellular systems from the cytotoxic effects of ROS, plants activate antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) (Candan and Tarhan, 2012), to minimize oxidative stress. Another tolerance response to water deficit is the accumulation of compounds, such as soluble sugars, proline and betaine (McCue and Hanson, 1990), that perform osmotic adjustments in cells and help plants resist drought to maintain sufficient turgor for growth (Carvajal et al., 1999) and tissue hydration. Anatomical and ultrastructural changes are indicators of water deficit (Ciamporova, 1976; Shao et al., 2008). Low water conditions usually cause a reduction in cell volume (Guerfel et al., 2006) and increases in cell wall thickness (Guerfel et al., 2009) and cuticle thickness (Liakoura et al., 1999). Drought stress may also result in changes in the nuclei, cytoplasmic membranes, endoplasmic reticulum, mitochondria, dictyossomes, ribosomes (Ciamporova, 1976) and chloroplasts (Da Silvia et al., 1974). Several studies have been conducted to elucidate plant tolerance to osmotic stress in response to water deficit and to identify the mechanisms that allow plants to adapt to stress and maintain their growth, development and productivity; such studies also aid in the identification of resistant plants (Candan and Tarhan, 2012). Mentha x piperita L. (peppermint), an important medicinal and aromatic plant belonging to the Lamiaceae family, is a natural hybrid resulting from crossing M. aquatica and M. spicata (Maffei et al., 1999). This specie is exploited for its production of terpenoids (Maffei et al., 1999) and is grown mainly for essential oil extraction (Maffei and Sacco, 1987). However, like most cultivated plants, its growth and yield can be affected by environmental constraints, such as water stress (Candan and Tarhan, 2012), salt stress (Oueslati et al., 2010) and osmotic stress. In the case of osmotic stress, different polyethyleneglycol levels alter the osmotic potential of a solution, generating a water deficit in plants. Osmotic stress may influence the growth and modify the content and quality of M. x piperita’s essential oil. We found no studies in the literature that characterize the influence of osmotic stress on primary metabolism and essential oil in association with morphology and ultrastructure in this specie. Thus, in the present study, we evaluated whether osmotic stress induced via two polyethyleneglycol levels in a short time (1) interferes with plant physiological parameters, (2) changes the anatomy and leaf ultrastructure, and (3) modifies essential oil content and composition. To this end, we evaluated the following variables: water potential and relative leaf water content, anatomical and ultrastructural features of the leaves, gas exchange, chlorophyll fluorescence, antioxidant enzymes levels, lipoperoxide levels, total soluble sugar content, and content and composition of essential oils. _________ ___ _ Manuscrito I 23 2. Materials and methods 2.1. Plant material and location Fertile branches were collected from adult plants grown in a bed at the Department of Botany, Biosciences Institute of UNESP, Botucatu City, Sao Paulo State, Brazil (22◦ 52’ 20” S 48◦ 26’37” W). Vouchers were deposited in the Herbarium Irina Delanova Gemtchujnicov (BOTU) under number 027610. For cuttings, stem fragments of peppermint (M. x piperita) measuring approximately 10 cm were placed in trays containing commercial substrate (Bioplant®, Nova Ponte, Minas Gerais, Brazil) and were maintained under humid conditions until rooting. After 25 days, the peppermint saplings were transferred to 5.0 L pots filled with complete Hoagland and Arnon’s (1950) No. 2 nutrient solution. The pots were maintained in a greenhouse under mean maximum and minimum air temperatures of 31.5°C and 21.2°C, respectively, and a mean relative humidity of 75% until the harvest. The solutions were prepared using deionized water and were permanently aerated and renewed every week to minimize a pH shift and nutrient depletion. 2.2. Osmotic stress treatments At 59 days after transplanting (DAT) to a hydroponic system, the plants were subjected to polyethyleneglycol (PEG-6000) treatments as an osmotic stimulator. Treatments consisted of a control (without PEG [PEG 0]) and two levels of osmotic stress. PEG 6000 was dissolved in the Hoagland solution at two levels: PEG 50 (50 g of PEG per 1000 mL) and PEG 100 (100 g of PEG per 1000 mL). The pots were arranged in a randomized design in a greenhouse with eight pots exposed to each of three treatments. 2.3. Leaf water status The leaf water potential (Ψw) and relative water content (RWC) were measured before plants were exposed to osmotic stress (control condition) and then again 72 hours after PEG-induced stress. The Ψw was measured at 5:00 am (predawn) and 12:00 pm (midday) with a Dewpoint Potentiometer WP4-T (Decagon Devices Inc., Pullman, WA, US) and was expressed in ‘MPa.’. For RWC measurement, the mature leaves were subjected to measurements of fresh weight (FW), dry weight (DW) and turgid weight (TW). RWC (%) was computed using the formula RWC (%) = [(FW - DW)/(TW - DW)] x 100. 2.4. Structural studies 2.4.1 Light microscopy (LM) _________ ___ _ Manuscrito I 24 A fully expanded leaf was collected from each individual in each treatment (n=8) 24 hours after PEG administration. For analyses of glandular density, leaves were observed with a Leica M205C stereomicroscope, and the number of glandular trichomes in 1 mm2 was calculated using the Leica Application Suite software (LAS). For anatomical studies, leaf blade samples were fixed in FAA 50, dehydrated in alcoholic series and embedded in methacrylate resin (Gerrits, 1991). Cross sections (6 μm thick) were obtained with a rotatory microtome and stained with toluidine blue 0.05%, pH 4.7 (O’Brien, 1964). Permanent slides were mounted with Permount and examined with an Olympus BX 41 photomicroscopy equipped with a digital camera. Measurements were performed using the CellB Olympus-Imaging Software for Life Science Microscopy. 2.4.2 Scanning electron microscopy (SEM) Leaf blade samples were collected 24 hours after PEG administration and fixed in 2.5% glutaraldehyde solution with 0.1 M phosphate buffer (pH 7.3) overnight at 4°C; they were then dehydrated in a graduated acetone series, critical-point dried, mounted on aluminum stubs, gold coated (Robards, 1978), and examined with a Fei Quanta scanning electron microscope at 12.5 kV. 2.4.3 Transmission electron microscopy (TEM) Leaves were collected 24 hours after the treatments were administered and fixed in Karnovsky 2.5% in phosphate buffer 0.1 M (pH 7.3) for 24 hours at 5°C. The samples were post-fixed with 1% osmium tetroxide aqueous solution in the same buffer for 1 hour at 25°C and then dehydrated in a graduated series of acetone and embedded in Araldite resin (Machado and Rodrigues, 2004). Ultra-thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). The samples were examined with a FeiTecnaiTM transmission electron microscope at 80 kV. 2.5. Measurement of leaf gas exchange characteristics and chlorophyll fluorescence After 48 hours of exposure to osmotic stress, the net photosynthetic rate (Pn, in µmol m-2 s-1), stomatal conductance (gs in mol m-2 s-1), intercellular CO2 concentration (Ci µmol CO2 mol air- 1), transpiration rate (E mmol m-2 s-1) and water use efficiency (WUE µmol CO2 [mmol H2O]- 1) of the plants’ third fully expanded leaf were measured using an infrared gas analyzer (IRGA), the Li-Cor 6400 photosynthesis system (Li-Cor Inc., Lincoln, NE, US), between 09:00 am and 11:00 am. The CO2 concentration reference used during evaluations was the level present in the environment, ranging from 380 to 400 μmol CO2 mol-1. The maximum quantum efficiency of PSII (Fv/Fm) was also determined after placing samples in darkness for _________ ___ _ Manuscrito I 25 30 minutes with a PAM fluorometer Junior (Walz, Effeltrich, Germany). Leaf gas exchange and chlorophyll fluorescence were measured on the same day and under the same environmental conditions (25°C and PPFD of 1500 μmol m-2 s-1). 2.6. Measurement of lipoperoxide levels and total soluble sugar content Leaves from each treatment were collected 72 hours after treatment for lipid peroxidation (LPO) and total soluble sugars (TSS) analysis. The LPO assay was assessed according to the method described by Heath and Packer (1968). Samples were homogenized in a 5 mL solution containing thiobarbituric acid (TBA) 0.25% and trichloroacetic acid (TCA) 10% and incubated in a water bath at 90°C for 1 hour. After cooling, the homogenate was centrifuged at 10,000 g for 15 minutes at room temperature. Then, the supernatant collected from each sample was subjected to absorbance readings in a UV-visible spectrophotometer at 560 and 600 nm. For calculations, the malondialdehyde (MDA) molar extinction coefficient (155 mM cm-1) was used. The TSS extraction was performed according to an adapted methodology from Garcia et al. (2006) using three replicates with 100 mg of leaf per treatment. The TSS was estimated colorimetrically using the phenol-sulfuric method (Dubois et al., 1956) with glucose (100 µg mL-1) as a standard and expressed as milligrams per gram of fresh mass (mg g-1 FM). 2.7. Analysis of enzymatic antioxidant system Seventy-two hours after the treatments were administered, leaves were collected for enzymatic antioxidant system analysis. Enzymatic extracts were obtained according to the method described by Kar and Mishra (1976). The assay of superoxide dismutase activity, (SOD [EC 1.15.1.1]), was conducted according to the method described by Beauchamp and Fridovich (1971). The reaction mixture was composed of 30 µL enzymatic extract, 50 mM sodium phosphate buffer pH 7.8, 33 µM nitroblue tetrazolium (NBT) + 0.66 mM EDTA (5:4), and 10 mM L-methionine + 3.3 M riboflavin (1:1), totaling 3.0 mL. Tubes were illuminated for ten minutes at 25°C, and NBT reduction to blue formazan was measured through absorbance readings in a UV–visible spectrophotometer at 560 nm. SOD activity was expressed as U mg-1 protein. In this case, one unit (U) represents the quantity of enzyme needed to inhibit the NBT reduction ratio by 50%. Peroxidase activity, (POD [EC 1.11.1.7]), was assayed according to the methods described by Teisseire and Guy (2000). The reaction mixture was composed of 30 µL diluted enzymatic extract (1:10 in the extraction buffer), 50 mM potassium phosphate buffer pH 6.5, 20 mM pyrogallol (benzene-1,2,3-triol) and 5 mM hydrogen peroxide (H2O2), totaling 1.0 mL. The reaction was carried out at room temperature for 5 minutes. Purpurogallin formation was measured in a UV–visible spectrophotometer at _________ ___ _ Manuscrito I 26 430 nm, and its molar extinction coefficient (2.5 mM cm-1) was used to calculate the specific activity, expressed as µmol purpurogallin.minute-1 mg-1 protein. The catalase activity (CAT [EC 1.11.1.6]) assay was composed of 50 µL enzymatic extract, 950 µL 0.05 M sodium phosphate buffer pH 7.0 containing 12.5 mM H2O2, totaling 1 mL. After absorbance readings at 240 nm, the molar extinction coefficient of H2O2 (39.4 mM cm-1) was used. The reaction was carried out at room temperature for 80 s. Readings were taken in a UV-visible spectrophotometer at 240 nm at 0 and 80 s. The enzyme activity was expressed as nmol consumed H2O2 minute-1 mg-1 protein (Peixoto et al., 1999). The assessment of soluble protein levels from enzymatic extracts, necessary for calculating the specific activity of the studied enzymes, was performed according to the method described by Bradford (1976). Absorbance readings were conducted in a UV-visible spectrophotometer at 595 nm by using casein as the standard. 2.8. Analysis of essential oils The aerial parts collected 72 hours after treatment were subjected to hydrodistillation in a Clevenger-type apparatus for 2 hours. The qualitative analysis of the essential oil compounds was performed on a gas chromatograph (GC) coupled to a mass spectrometer (MS) (GC–MS; Shimadzu QP5000) operating at an MS ionization voltage of 70 eV. The chromatography was equipped with a fused silica capillary column DB-5 (J and W Scientific; 30 m × 0.25 mm × 0.25 μm), and helium was used as the carrier gas. The following chromatograph conditions were used: injector at 240°C, detector at 230°C, gas flow 1.0 mL/minute, split 1/20, initial column temperature of 60–240°C at a rate of 3°C/minute, and a 1 μL injection of solution (1 mg of essential oil and 1 mL of ethyl acetate). The compounds were identified based on a comparative analysis of the acquired mass spectra with those stored in the GC–MS database of the system (Nist 62.Lib), in a previous study (McLafferty and Stauffer, 1989) and in retention indices (Adams, 2007), which were obtained from the injection of a mixture of n- alcanes (C9H20–C25H52, Sigma Aldrich, 99%) employing a column temperature program as follows: 60–240°C at a rate of 3°C/minute. Quantification (normalization area method) of the substances was carried out with a GC (Shimadzu GC-2010) equipped with flame ionization (GC–FID) and using a DB-5 (J and W Scientific; 30 m × 0.25 mm × 0.25 mm × 0.25 μm) capillary column. Helium was used as the carrier gas, and the temperature injector was at 240°C, the detector at 230°C, and gas flow at 1.0 mL/minute, split 1/20. The following chromatography conditions were used: 60–135°C at a rate of 5°C/minute, then 135–240°C at a rate of 8°C/minute and 60–240°C at a rate of 3°C/minute; 1 μL of solution was injected (1 mg of essential oil and 1 mL of ethyl acetate). _________ ___ _ Manuscrito I 27 2.9. Statistical analysis The overall effects of the treatments were determined by means of a one-way ANOVA followed by Tukey’s test (p<0.05). Data were tested for normality and homogeneity of variances prior to analysis. 3. Results 3.1. Leaf water status To observe the effect of osmotic stress in M. x piperita plants, we evaluated Ψw and RWC in control conditions (PEG 0) and 72 hours after the administration of PEG 50 and PEG 100 in the nutrient solution at predawn and midday. There was no difference in Ψw at predawn (Ψw pd) in plants subjected to PEG 50 and PEG 100 treatments, whereas the plants subjected to PEG 100 showed a reduction in Ψw at midday (Ψw md) of approximately 50% (Fig. 01). There was no difference in RWC in plants under PEG 50, but it was higher in PEG 100 plants compared to the control (Fig. 01). 3.2. Structural analysis 3.2.1 Leaf morphology M. x piperita leaves are bifacial, amphistomatic and homobaric with glandular and non- glandular trichomes on both sides of the leaf surfaces (Fig. 02- A-E). The epidermis is uniseriate (Fig. 02- A, B) and consists of common cells that superficially exhibit a sinuous pattern (Fig. 02- C, D). On both sides of the leaf blade, the epidermal cells are covered with a thin cuticle. Two morphotypes of glandular trichomes were observed. The first shows a basal cell among the other cells of the epidermis and a large secretory head (Fig. 02- A, C). The second morphotype is composed of a basal cell, an unicellular short stalk and an oval head (Fig. 02- D, E). A higher density of glandular trichomes was observed on the abaxial surface of the leaf blade (Table 01). The stomata are arranged at the same level as epidermal cells or are slightly protruding (Fig. 02- C, D). The mesophyll is composed of a parenchyma palisade and three to four layers of spongy parenchyma (Fig. 02- A, B). Collateral bundles surrounded by well-defined endoderm were observed immersed in the mesophyll (Fig. 02- B). In the region of the midrib, the cortex is composed of one or two collenchyma layers on the abaxial surface, two to three layers on the adaxial surface (Fig. 02-F), and three to five layers of voluminous parenchyma cells with regular contours (Fig. 02- F, G.); the vascular system consists of xylem and phloem with the beginning of a cambial installation (Fig. 02- F, G). _________ ___ _ Manuscrito I 28 Plants subjected to PEG 50 showed epidermal cells with more sinuous contours and slight cuticle shrinkage (Fig. 03- A, B) compared with control plants. However, no structural changes were observed under light microscopy (Fig. 03- C). Under SEM, plants exposed to PEG 100 showed intense cuticle retraction and epidermal cell delimitation, which was less obvious when superficially viewed (Fig. 04- A, B). These leaves showed a higher trend of mesophyll thickness (146.19 μm) compared with the PEG 50 plants (139.85 μm) and control treatment plants (138.98 μm) (Table 1). In plants subjected to PEG 100, the area occupied by the intercellular spaces in the mesophyll was larger (592.18 μm2) (Fig. 04- C) than in control plants (256.56 μm2) (Fig. 01- A) (Table 1). Cell collapse regions were observed in several areas along the leaf mesophyll (Fig. 04- D). In plants subjected to higher osmotic stress, we observed more irregular contours (Fig. 04- E) in the parenchyma cortical cells of the midrib region compared with other treatments. 3.2.2 Subcellular features of leaf blade In the leaf blade of control plants, the palisade and spongy parenchyma cells showed regular contours, smaller cytoplasms, developed vacuoles and large nuclei with evident nucleoli (Fig. 05- AC). In the cytoplasms, mitochondria, endoplasmic reticulum, chloroplasts, and dictyosomes were observed (Fig. 05- D). Chloroplasts were lenticular and ellipsoidal (Fig. 05- A, C) and had dense stroma, well-structured grana and widespread plastoglobules (Fig. 05- D); plastids exhibited starch grains (Fig. 05- B, C). These organelles were distributed mainly along the cell periphery (Fig. 05- A, B). In smaller caliber veins, the xylem consists of parenchyma cells and vessel elements; the phloem is composed of sieve tube elements and companion cells (Fig. 05- E) with thin walls, primary pit fields rich in plasmodesmata, and dense cytoplasms and mitochondria, endoplasmic reticulum, dictyosomes, polyribosomes and vesicles (Fig. 05- E, F). Changes in subcellular features were not observed in plants exposed to PEG 50. In plants subjected to PEG 100, the parenchyma palisade and spongy cells of non-collapsed mesophyll regions showed changes in sinuous contours and the wall-folding regions (Fig. 06- A-C). Cytoplasm showed signs of degeneration, and the nuclei were decreased (Fig. 06- C). Dispersed oil droplets were observed in cytoplasm (Fig. 06- D). Mitochondria showed swelled cristae (Fig. 06- E); some of them showed signs of lysis (Fig. 06- E-G). Some chloroplasts exhibited an anomalous format (Fig. 06- F). Within chloroplasts, we observed denser stroma and a loss of the internal organization of thylakoid membranes (Fig. 06- E, F); starch grains and the presence of several plastoglobules were observed (Fig. 06-E, F). Within the vacuoles, flocculated content was observed (Fig. 06-C, G). _________ ___ _ Manuscrito I 29 3.3. Measurement of gas exchange characteristics and chlorophyll fluorescence Plants subjected to PEG 100 exhibited lower stomatal conductance (gs) and transpiration (E) and photosynthetic rates (Pn) compared with control plants and PEG 50 plants (Fig. 07). These plants showed a 40% reduction in photosynthetic rate, whereas plants exposed to PEG 50 showed a 16% reduction, compared with control plants (Fig. 07). No difference was observed between the osmotic stress treatments in the intercellular CO2 concentration (Ci) and water use efficiency (WUE) (Fig. 07). The values of Fv/Fm from plants exposed to osmotic stress (average 0.78 - 0.75 and PEG 50 - PEG 100) showed no significant differences compared with control plants (average 0.78) (Fig. 07) 3.4. Measurement of lipoperoxide levels and total soluble sugar content LPO levels were higher in the leaves of plants exposed to osmotic stress (Fig. 08). The highest LPO level was observed in plants subjected to PEG 100, with an increase of 53% compared with the control. We observed an increase of 41% in TSS content in PEG 100 plants compared with control and PEG 50 plants (Fig. 08). 3.5. Analysis of enzymatic antioxidant system Plants exposed to PEG 50 showed higher SOD and CAT activities when evaluated at 72 hours after the administration of PEG (Fig. 09). Plants subjected to PEG 100 showed no differences in SOD or CAT activities compared with control, but POD activity increased by approximately three-fold (Fig. 09). 3.6. Analysis of essential oils Control and PEG 50 plants showed higher essential oil content (1.32% and 1.30%, respectively) compared with PEG 100 plants (0.87%) (Table 2). Twenty compounds identified represent 99% of the essential oil (data not shown). The major compounds found were menthone (39.4%), menthofuran (32.6%), menthol (15.3%) and pulegone (5.07%) (Table 2). Eucalyptol and limonene (data not shown) were also detected (2.6% each). There was a 36% reduction in menthone in plants subjected to PEG 50 and a 53% reduction in plants treated with PEG 100. The osmotic stress caused by PEG 100 increased the menthofuran percentage by 25%, whereas menthol and pulegone were not affected by treatments. 4. Discussion _________ ___ _ Manuscrito I 30 In the present study, M. x piperita plants exposed for 72 hours to osmotic stress induced by two levels of PEG 6000 showed structural, cellular and physiological changes compared with plants grown in control conditions. The results indicated that osmotic stress responses were dose dependent, as plants subjected to PEG 50 maintained structural features and metabolic functions similar to those of control plants. Plants exposed to PEG 100 showed anatomical changes and ultrastructural damage, which are consistent with the low leaf water potential, gas exchange reduction, and increases in total sugars and the activity of antioxidant enzymes. In addition, we observed that the increased antioxidant enzyme activity was not sufficient to prevent the degradation of the membranes. Our results also indicate that osmotic stress caused by PEG 100 influenced the essential oil content and composition of M. x piperita. Plants subjected to PEG 50 were more tolerant to exposure to osmotic stress because they were able to maintain the photosynthesis rate and stomatal conductance, showing high intercellular CO2 concentrations and transpiration rates, which are indicative of a normal flow of water and root uptake, as observed in control plants. In plants exposed to PEG 100, the leaf water potential showed a higher reduction in response to osmotic stress during midday. Under control conditions, with increasing temperature and decreasing relative humidity, the transpiration rate exceeds the water uptake by the roots and causes a water deficit in plants; thus, water potential during midday is more negative, and this effect is accentuated under stress conditions (Gimenez et al., 2005; Kudoyarova et al., 2013). These plants recovered from the deficit produced by transpiration during daylight hours, showing an increase in leaf water potential during predawn and maintaining tissue hydration, as observed in RWC. Predawn is characterized by a lower water loss by transpiration, as stomata are partially closed and the plant continues to uptake water until complete rehydration, an equilibrium state are achieved overnight (Gimenez et al., 2005; Kudoyarova et al., 2013). M. x piperita plants subjected to PEG 100 showed a 40% reduction in photosynthetic rate and lower transpiration, which is likely attributable to stomatal closure partially in response to osmotic stress. According to Kudoyarova et al. (2013), plants in water deficit conditions perform osmotic adjustment mechanisms to maintain the water content in tissues, such as the partial stomatal closure and decreased transpiration observed in our study. According to Chaves et al. (2003), plants in water deficit conditions show short-term responses such as stomatal closure, decreased carbon assimilation and osmotic adjustments. Additionally, we showed that osmotic stress did not affect photochemical activity, as PEG- treated plants showed similar Fv/Fm compared with control plants, confirming that the photochemical apparatus is resistant to applied osmotic stress. Similar results were found by _________ ___ _ Manuscrito I 31 Guóth et al. (2008) and Silva et al. (2010), who also did not observe damage to the photochemical apparatuses in plants exposed to PEG. Plants subjected to PEG 100 showed a higher TSS content than PEG 50 and control plants. Solute accumulation in the cytoplasm is a mechanism that plants use during water deficit to adjust to low water availability (Bohnert and Jensen, 1996; Bacelar et al., 2009; Kudoyarova et al., 2013), avoiding dehydration and tolerating a low tissue water potential (Chaves et al., 2003). We observed this response in RWC. Plants exposed to PEG 50 had a similar photosynthetic rate to that of the control, which may be related to the activity of antioxidant enzymes. The SOD and CAT activity levels were higher in the PEG 50 plants than in PEG 100 plants. This response demonstrates that plants exposed to PEG 50 activated protective mechanisms against the presence of free radicals in an attempt to maintain normal metabolic functions. Among the antioxidant enzymes, SOD is the primary line of defense against ROS (Blower et al., 1992) and eliminates superoxide radicals, producing O2 and H2O2. H2O2 is harmful to chloroplasts, nucleic acids and proteins and is subsequently eliminated by the action of CAT and POD enzymes and other non-enzymatic antioxidants (Fatima and Ahmad 2004; Srivastava et al., 2010). The POD enzyme, which removes H2O2, also contributes to the defense system of M. x piperita against osmotic stress. Plants subjected to PEG 100 showed higher POD activity. Similar results observed by Oueslati et al. (2010) observed an increase in POD activity among plants exposed to osmotic stress, and Lechno et al. (1997) reported an increase in the activites of the enzymes, CAT and glutathione reductase in plants subjected to salt stress. The same authors did not verify an increase in SOD activity, a result also found in plants subjected to PEG 100. Activation of SOD and CAT may have occurred before the evaluation period, as POD activity was higher in these plants. We also suggest that SOD activity may have been impaired in these plants because damages were observed in the chloroplasts and mitochondria, which are SOD isoform reaction centers (Zelko et al., 2002; Munoz et al., 2005). Candan and Tarhan (2012) demonstrated that SOD activity in M. pulegium plant was positively correlated with the severity of osmotic stress and exhibited maximal activity at the end of evaluation period. According to Shao et al. (2008), reactions of plants to drought depend on the intensity and duration as well as plant species and stage of development. Moreover, Candan and Tarhan (2012) emphasized that a positive antioxidant response to abiotic stress is a symptom of tolerance, and the opposite behavior is therefore evidence of sensitivity. In our study, M. x piperita showed tolerance depending on the osmotic stress level applied. _________ ___ _ Manuscrito I 32 In addition, the antioxidant enzymes activity showed a positive correlation with the intensity of applied stress, as PEG 50 plants showed higher SOD and CAT activities and lower LPO levels. Lipid peroxidation is represented by malondialdehyde accumulation (MDA) and is indicative of oxidative damage in cells (Ennanjeh et al., 2009; Zhang et al., 2011). Low concentrations of MDA have been associated with drought tolerance in some species (Moran et al., 1994; Sairam et al., 2000). Moreover, the membranes of plants exposed to PEG 100 showed oxidative damage, as demonstrated by the increase in LPO levels. The higher POD activity was not sufficient to control damage caused by oxidative stress. M. pulegium subjected to osmotic stress for 8 days showed an increased LPO level with time, which was higher in plants subjected to severe stress than those subjected to moderate stress (Candan and Tarhan, 2012). The increase in LPO levels affects the growth and productivity of plants exposed to abiotic stresses (Ennanjeh et al., 2009; Zhang et al., 2011). Changes in the structural characteristics of the leaves and other anatomical and subcellular changes caused by osmotic stress were observed in M. x piperita exposed to PEG and were more evident in plants subjected to PEG 100. We observed immediate changes, such as cell collapse, cuticle shrinkage and the degeneration of cytoplasmic organelles. The time of plants’ exposure to PEG treatment was not sufficient to ensure the formation of cells and tissues under osmotic stress conditions and thus enable the identification of ontogenetic changes in leaves. Different effects of stress caused by water deficit in the anatomy and ultrastructure of leaves and other tissues in several plant species have been reported (Paakkonen et al., 1998; Chartzoulakis et al., 1999; Dekov et al., 2000; Guerfel et al., 2009; Zaharah and Razi, 2009; Ennajeh et al., 2010). Plants subjected to PEG 50 exhibited smaller changes in epidermal cells, which showed mild shrinkage and thickening of the cuticle and increased cell contour sinuosity. No changes in subcellular features were observed. In plants exposed to PEG 100, anatomical and ultrastructural changes were more intense. The cuticle was found to be retracted, the area occupied by intercellular spaces increased and consequently increased the mesophyll thickness, and collapse cell areas occurred on the leaf mesophyll. Sam et al. (2003) reported that salt stress affected the organization of parenchymatic cells, increased intercellular spaces and changed the format of cells from tomato plants. These authors concluded that such changes depended on the NaCl level used and were associated with the tolerance of cultivars to salt stress. Changes in the thickness of mesophyll in plants exposed to osmotic stress may have been caused by the increased area of intercellular spaces of spongy parenchyma found in PEG 100 _________ ___ _ Manuscrito I 33 plants. Similar results were observed by Rajabpoor et al. (2014), who also noted that the length of palisade parenchyma cells remained unchanged. According to Bosabalidis and Kofidis (2002), another anatomical change that occurs in plants subjected to water deficit is a size reduction of epidermal and mesophilic cells. Small size and straight, not sinuous, walls contribute to resistance against cell collapse in response to water deficit (Oertli et al., 1990; Bosabalidis and Kofidis, 2002). However, although parenchymatic cells showed sinuous contours in M. x piperita plants subjected to PEG 100, there was no significant reduction in cell size, which may be associated with the formation of collapsed areas in the mesophyll. The presence of these collapsed areas in the leaf mesophyll may have influenced M. x piperita metabolic processes, causing a decrease in CO2 uptake area and interfering with gas exchange and consequently with the growth of the plants. Regarding subcellular features, the parenchyma cells of M. x piperita mesophyll exposed to PEG 100 showed signs of cytoplasm degeneration, including smaller nuclei, morphological changes in plastids and lysis of mitochondria and organelles. We observed that plants exposed to PEG 100 exhibited swelled cristae. Morphological changes in mitochondria appear to be a common feature in plants under different