Ecophysiological responses of olive (Olea europaea L.) to restricted water availability: limitations, damages and drought resistance mechanisms.

Ecophysiological responses of

olive (Olea europaea L.) to restricted

water availability

Este trabalho foi expressamente elaborado como dissertação original para o efeito de obtenção do grau de Doutor em Engenharia Biológica, de acordo com o disposto no Decreto-Lei n.º 216/92, de 13 de Outubro.

To the memory of Professor José Manuel Gaspar Torres Pereira (1939-2004) with whom I was privileged to work

This work would not have been completed without the close cooperation of a number of different people and institutions. To them I would like to express my sincere gratitude.

I wish to express my gratitude to Professor Mascarenhas Ferreira, Rector of the Universidade de Trás-os-Montes e Alto Douro (UTAD), for all the support provided throughout this research.

I am deeply grateful to my supervisor, Professor Carlos Correia, for his consistent support and guidance throughout the work and for careful revision of this thesis.

I am also truly thankful to Professor Dario Santos for accepting to co-advise this dissertation. Thank you for your important suggestions and guidance.

Special thanks to Professor José Moutinho-Pereira for his support and encouragement during my PhD program.

I am also grateful to Professor Timóteo Ferreira, leader of the Project AGRO-INIA No. 175, for giving me the opportunity and the conditions to develop part of the research presented in this thesis.

Financial support by AGRO-INIA program (No. 175) of Portuguese Ministry of Agriculture is gratefully acknowledged.

My warm thanks to my dear colleague and friend MSc Berta Gonçalves, always available to provide useful suggestions, constant encouragement and fruitful collaboration.

I would also like to thank Eng. João Lopes (Direcção Regional de Agricultura de Trás-os-Montes) for the use of the experimental field, and for providing the olive plants to the glasshouse study.

Sincere thanks to all my colleagues of the Department of Engenharia Biológica e Ambiental (DEBA).

I wish to express my gratitude to the members of the staff of the DEBA for excellent technical support and for providing a thoroughly enjoyable atmosphere. In particular I record my thanks to: Helena Ferreira, Ana Fraga, Donzília Costa, Fernando Ferreira, Natália Teixeira, Rui Martins and Mr Magalhães.

I am also indebted to the editors and the anonymous referees for their relevant comments.

I am deeply grateful to my family and friends for never-ending encouragement and support.

Finally, my warmest thanks go to my husband Miguel Bacelar for his encouragement, technical help, and for being always on my side. I also wish to thank my parents for their unconditional love and encouragement.

O stresse hídrico é o factor que mais limita a produção vegetal à escala mundial, reduzindo significativamente os lucros da actividade agrícola. Do ponto de vista ecofisiológico, designa-se por stresse hídrico qualquer limitação ao funcionamento óptimo das plantas imposta por uma insuficiente disponibilidade de água. As respostas das plantas à falta de água são complexas, envolvendo uma série de adaptações/aclimatações e efeitos nefastos, ou ambos. Por outro lado, à secura do solo associa-se uma forte evaporação causada pela secura do ar, elevada temperatura e elevados níveis de radiação durante o período estival, pelo que é frequente classificar este efeito conjunto de stresse estival.

A oliveira (Olea europaea L.) é uma árvore de folhagem persistente, crescimento lento e grande longevidade. Conta-se entre as poucas plantas cultivadas de origem mediterrânea e a sua difusão é muito antiga. Julga-se que foi domesticada por volta de 3000−4000 anos antes de Cristo na Ásia Menor e a partir daí foi introduzida no Norte de África, Península Ibérica e resto do Sul da Europa pelas civilizações que sucessivamente ocuparam a região mediterrânica. Nos últimos 500 anos foi levada para as Américas, África do Sul, Austrália, China e Japão, mas mantém-se principalmente uma cultura da bacia mediterrânica. O seu cultivo prosperou na região, mesmo em solos pobres, porque esta espécie é capaz de produções aceitáveis, apesar de sujeita à característica falta de água prolongada durante o Verão.

A crescente popularidade da oliveira como árvore de fruto relaciona-se com o aumento da procura dos seus produtos. Esta tendência favorável do mercado é parcialmente devida aos estudos científicos que demonstram as vantagens do consumo regular de azeitonas e azeite na saúde humana. De facto, o baixo teor de ácidos gordos saturados e a presença de antioxidantes pode ajudar a prevenir certas doenças. Os benefícios para a saúde induzidos pelo consumo de azeitonas e azeite virgem são devidos à acção sinérgica de diversos constituintes, como vitaminas (por exemplo α-tocopherol), fitoesteróis, pigmentos, ácidos terpénicos e compostos fenólicos.

Segundo a Organização das Nações Unidas para a Agricultura e a Alimentação (FAO), a área mundial de olival é cerca de 7,5 milhões de hectares e os principais produtores são a Espanha, Itália, Grécia, Tunísia, Turquia, Síria, Marrocos e Portugal. A cultura da oliveira é conhecida em Portugal desde o domínio romano, sendo uma das mais importantes culturas do País, particularmente em Trás-os-Montes, onde detém considerável importância económica, social e ambiental. A excelente qualidade do azeite produzido na região e as suas características únicas são responsáveis pela denominação de origem protegida ῾Azeite de Trás-os-Montes

DOP̕. A criação da denominação de origem é relativamente recente e estimulou a economia local, nomeadamente adicionando valor ao azeite produzido na região.

Tal como outras regiões do Sul da Europa, Portugal é um local de clima Mediterrânico temperado, mas vulnerável às alterações climáticas. Todos os modelos, em todos os cenários, prevêem um aumento de temperatura média em Portugal no final do século XXI. Os cenários de precipitação são muito mais incertos. Contudo, quase todos os modelos prevêem uma redução da precipitação em Portugal Continental durante a Primavera, Verão e Outono. As alterações climáticas à escala global irão afectar significativamente as colheitas das culturas da região mediterrânica. No entanto, o impacto na produção das culturas será determinado pela capacidade dos agricultores/técnicos de se adaptarem aos novos cenários.

São várias as práticas culturais que podem auxiliar a oliveira a sobreviver e a produzir em ambientes de baixa disponibilidade hídrica. Estas práticas visam alcançar um equilíbrio entre a necessidade de transpiração e a água disponível, especialmente durante o longo e seco Verão. Decisões estratégicas são a selecção de cultivares, a densidade de plantação e o tamanho da copa, juntamente com a gestão da superfície do solo. Recentemente, a rega foi introduzida para aumentar produtividade do olival. A expansão do cultivo da oliveira devido à crescente procura dos seus produtos levou a que as novas plantações fossem caracterizadas por um maior número de árvores por hectare, comparativamente aos olivais tradicionais. Nestes sistemas de cultivo intensivo, a rega é essencial para garantir a optimização da aplicação de água para o crescimento da árvore e produção de azeitona. Todavia, isso pode ser problemático devido aos limitados recursos hídricos disponíveis na região mediterrânica. Uma gestão prudente da água é, por isso, essencial para uma olivicultura sustentável.

A redução da disponibilidade hídrica antecipada pelos cenários das alterações climáticas à escala global irá inevitavelmente agravar o problema da escassez de água em toda a região mediterrânica. Uma vez que a oliveira é a principal cultura da bacia mediterrânica, é fundamental analisar como esta espécie será afectada por uma reduzida disponibilidade hídrica. A melhoria da performance da oliveira não pode ser conseguida sem que os mecanismos de resistência à seca sejam claramente identificados. Tal não é possível sem que haja uma completa compreensão das limitações e dos danos induzidos por um fornecimento de água insuficiente.

A análise da literatura científica apresentada no capítulo 1 revela que a oliveira possui importantes atributos que lhe permitem sobreviver e produzir em ambientes áridos. No entanto, muitos aspectos importantes da resistência à seca não foram ainda abordados e são poucas as diferenças estabelecidas entre cultivares de O. europaea. Apesar das cultivares com maior

importância na região de Trás-os-Montes serem consideradas bem adaptadas às condições locais, não existem estudos científicos que documentem a sua adaptação ao défice hídrico. Por isso, traçaram-se três grandes objectivos: (1) Identificar, em condições de campo, as adaptações morfo-anatómicas, fisiológicas e bioquímicas de várias cultivares de oliveira; (2) Estudar, em condições controladas, as respostas imediatas e as estratégias adaptativas das cultivares de oliveira mais representativas na região de Trás-os-Montes e avaliar os sintomas de stresse oxidativo induzidos pela baixa disponibilidade hídrica; (3) Estudar num olival comercial a influência de diferentes níveis de rega no comportamento fisiológico de oliveiras adultas e analisar os danos oxidativos e a protecção antioxidante ao nível foliar em árvores regadas e não regadas. Para alcançar tais objectivos, foram realizadas três experiências. A componente experimental desta dissertação resulta dos dados obtidos nessas experiências e está estruturada em cinco capítulos, cujos conteúdos incluem integralmente a informação publicada ou submetida a revistas indexadas na lista do Journal Citation Reports.

A primeira experiência decorreu em 2001 e 2002 num olival da Direcção Regional de Agricultura de Trás-os-Montes, instalado em Mirandela (Terra Quente Transmontana). Foram analisadas oliveiras com dez anos de idade de cinco cultivares com diferentes origens geográficas (Trás-os-Montes e diferentes regiões de Espanha): Arbequina, Blanqueta, Cobrançosa, Manzanilla e Negrinha de Freixo (daqui em diante designada simplificadamente por Negrinha).

No capítulo 2 o objectivo específico foi identificar os mecanismos morfo-anatómicos e as adaptações estruturais para reduzir a perda de água pelas folhas das cinco cultivares de oliveira. Foram analisados diversos parâmetros foliares: a espessura dos diferentes tecidos, a densidade estomática, a área foliar, a massa por unidade de área foliar (LMA), a densidade do tecido foliar (D), o conteúdo relativo em água (RWC), a suculência, o défice de saturação em água (WSD), o conteúdo em água em folhas saturadas (WCS) e a taxa de transpiração cuticular. Foram também efectuadas determinações do potencial hídrico do caule antes do nascer do sol (ΨPD) e ao meio-dia solar (ΨMD). Este estudo indicou que as folhas das cinco

cultivares possuem diferentes mecanismos ao nível morfo-estrutural para ultrapassar o stresse estival. Apesar das folhas de todas as cultivares serem bem adaptadas à luminosidade intensa, foi demonstrado que as cultivares nativas de regiões mais secas, como a Cobrançosa, a Negrinha e a Manzanilla apresentaram folhas mais protegidas contra a perda de água do que as cultivares oriundas de regiões com um clima mais temperado, como a Arbequina e a Blanqueta. Verificou-se que as folhas das cultivares mais resistentes apresentavam maior grau de esclerofilia. A Cobrançosa revelou elevada densidade do tecido foliar (i.e. elevada razão

parênquima clorofilino em paliçada/parênquima clorofilino lacunoso) e espessas camadas de cutícula e de escamas peltadas, enquanto a Manzanilla e a Negrinha aumentaram a esclerofilia ao nível foliar, aumentando a espessura dos tecidos parenquimatosos e de estruturas de protecção, como a cutícula superior (Negrinha) e as epidermes superior e inferior (Manzanilla). As folhas da Arbequina apresentaram a menor espessura total da lâmina e uma camada de escamas peltadas mais fina, o que implica que são menos protegidas contra a perda de água. Para além disso, esta cultivar apresentou os valores mais elevados de transpiração cuticular, em resultado de possuir uma camada cuticular mais fina e/ou devido a diferenças na estrutura e composição da cutícula. Como consequência, e apesar do desenvolvimento de folhas mais pequenas, esta cultivar apresentou menor RWC e ΨMD e maior WSD. Por seu lado, a Blanqueta

apresenta folhas de maiores dimensões, responsáveis por uma maior perda de água.

No capítulo 3 foram estudadas as variações diurnas das trocas gasosas das cinco cultivares e avaliadas as limitações na fotossíntese impostas pelas condições de sequeiro. Foram também analisados mecanismos de tolerância à seca associados à acumulação de açúcares solúveis, proteínas solúveis e prolina nas folhas. A variação diurna da taxa de fotossíntese das cultivares de oliveira seguiu uma tendência típica da vegetação lenhosa mediterrânica, com um máximo de manhã e mínimo ao meio-dia ou durante a tarde. Contudo, o grau de depressão da fotossíntese ao meio-dia foi dependente do genótipo, com um máximo na Arbequina e um mínimo na Negrinha. O controlo estomático da perda de água foi identificado como uma resposta inicial das oliveiras ao défice hídrico, levando a uma limitação da assimilação do carbono pelas folhas. No entanto, verificou-se que as reduções na taxa de assimilação de CO2 também foram atribuídas a factores não estomáticos, particularmente na

cultivar Blanqueta. A acumulação de prolina nas folhas de todas as cultivares, essencialmente na Manzanilla, indica um possível papel deste aminoácido na tolerância à seca da espécie O. Europaea.

A segunda experiência foi efectuada numa estufa situada no campus da UTAD durante o ano de 2002. Este estudo foi desenvolvido em oliveiras envasadas, com um ano de idade, das três cultivares mais representativas na região de Trás-os-Montes (Cobrançosa, Madural e Verdeal Transmontana), submetidas a diferentes regimes hídricos durante a época estival (bem regadas − regadas três vezes por semana com a água necessária para manter o solo à capacidade de campo; baixa disponibilidade hídrica − 1/3 da água aplicada às bem regadas).

No capítulo 4 foram objecto de estudo os efeitos da cultivar e do regime hídrico no crescimento e repartição de biomassa, nas trocas gasosas, nas propriedades hidráulicas do xilema e na eficiência do uso da água. Verificou-se que a baixa disponibilidade de água afectou

negativamente o crescimento e a acumulação de biomassa em todas as cultivares, embora em menor grau na Cobrançosa. Verificou-se também que as plantas reagiram à baixa disponibilidade hídrica desenvolvendo mecanismos para evitar a seca baseados no controlo estomático, alterações das propriedades hidráulicas do xilema e redução da área foliar. O decréscimo na taxa de crescimento relativo (RGR) das plantas sob baixa disponibilidade hídrica foi associado a decréscimos da taxa média de assimilação aparente (NAR) e da razão da área foliar (LAR). A menor LAR foi devida a uma menor translocação de biomassa para as folhas (LWR) e, sobretudo, a uma menor área foliar específica (SLA), em consequência de uma maior densidade do tecido foliar. As plantas da cultivar Verdeal Transmontana foram uma excepção a este comportamento, revelando uma tendência para uma maior LAR sob baixa disponibilidade hídrica, em resultado de uma maior expansão das folhas jovens. Sob stresse hídrico, a área foliar por planta foi drasticamente reduzida em todas as cultivares devido a uma combinação da redução da expansão foliar e da abscisão precoce das folhas mais velhas. Outra constatação importante foi que a baixa disponibilidade hídrica induziu um aumento na frequência de vasos xilémicos em todas as cultivares, proporcionado um maior potencial vascular e uma maior segurança da condução da seiva. As plantas da cultivar Cobrançosa que cresceram em condições de baixa disponibilidade hídrica foram as únicas que revelaram maior condutividade hidráulica relativa que as plantas controlo, mas igual índice de vulnerabilidade à cavitação.

No capítulo 5 foram estudados o efeito da cultivar e do regime hídrico na anatomia e estrutura foliar, composição química e relações pressão-volume das folhas e os sintomas de stresse oxidativo induzidos pela baixa disponibilidade hídrica. Anatomicamente, a Cobrançosa e a Madural mostraram-se capazes de lidar melhor com a baixa disponibilidade hídrica que a Verdeal Transmontana. As folhas da Cobrançosa e da Madural revelaram uma epiderme superior e um parênquima clorofilino em paliçada mais espessos e maior densidade estomática que a Verdeal Transmontana. A disponibilidade hídrica apresentou um papel determinante na modificação da estrutura foliar. Assim, as folhas das plantas que cresceram com baixa disponibilidade de água tiveram uma menor SLA que as plantas controlo, em resultado de um aumento da D, uma vez que não foram observadas diferenças de espessura. Entre cultivares, a Cobrançosa apresentou menor SLA e maior D. A maior D pode ser útil para esta cultivar, uma vez que as folhas com maior D têm mais capacidade de resistir à seca severa, apresentando maior resistência aos danos físicos por dessecação. A Cobrançosa e a Madural também mostraram maior capacidade de ajustamento osmótico e um aumento da rigidez dos tecidos em condições de baixa disponibilidade hídrica. Contrariamente, a Verdeal Transmontana não exibiu ajustamento osmótico, mas foi capaz de aumentar a elasticidade dos tecidos e a concentração

de proteínas solúveis. As folhas que cresceram com baixa disponibilidade hídrica mostraram sinais de stresse oxidativo, com decréscimos nas concentrações de pigmentos fotossintéticos e tióis totais e níveis elevados de peroxidação lipídica. No entanto, essas plantas desenvolveram alguns mecanismos de defesa contra o stresse oxidativo, como o aumento das concentrações de compostos fenólicos totais e de proteínas solúveis totais. O aumento da concentração de substâncias reactivas com o ácido tiobarbitúrico (TBARS) e o decréscimo da concentração de tióis totais em condições de baixa disponibilidade hídrica sugere que os mecanismos de defesa contra o stresse oxidativo foram menos eficazes na Madural. Do comportamento da Cobrançosa, com menor translocação de biomassa para as folhas, uma menor redução de NAR, um transporte de água pelo xilema mais eficiente, uma maior eficiência do uso da água para a produção de biomassa em condições de baixa disponibilidade de água e maior protecção contra o stresse oxidativo, é possível concluir que se trata da cultivar mais adequada para cultivo em condições de baixa disponibilidade hídrica.

A terceira experiência decorreu durante três anos consecutivos, de 2002 a 2004, num olival comercial (cv. Cobrançosa) em Vilarelhos (Vale da Vilariça, Terra Quente Transmontana). As oliveiras com oito anos de idade foram submetidas às situações de sequeiro (T0) e três tratamentos de rega durante o período estival (T1, T2, T3), equivalente a 30, 60 e 100% da evapotranspiração potencial, respectivamente.

No capítulo 6 foi estudada a influência dos diferentes regimes hídricos nas trocas gasosas e fluorescência da clorofila a. Foram também analisados os danos por stresse oxidativo e a protecção antioxidante ao nível foliar em árvores regadas e não regadas. O estudo revelou que todos os níveis de rega melhoraram a taxa de fotossíntese aparente das oliveiras e reduziram a depressão da fotossíntese e da condutância estomática observada durante os períodos do meio-dia e tarde. Verificou-se também a ocorrência de fotoinibição dinâmica ao meio-dia solar nas árvores regadas principalmente nas árvores sujeitas aos tratamentos T2 e T3, o que parece ter sido eficaz para proteger o aparelho fotossintético. As folhas das oliveiras que cresceram em condições de sequeiro revelaram sintomas de stresse oxidativo, como um menor teor clorofilino e níveis elevados de peroxidação lipídica. Observou-se também a redução da actividade da enzima superóxido dismutase nas árvores não regadas. Contrariamente, as baixas concentrações de TBARS nas folhas das plantas regadas, indicam que a rega reduz os danos oxidativos por peroxidação lipídica. Concluiu-se também que a rega aumenta a protecção antioxidante ao nível foliar, visto que as folhas das árvores regadas apresentavam maiores concentrações de tióis totais e maior actividade antioxidante total in vitro. Contudo, a maior actividade da enzima guaiacol peroxidase nas folhas das plantas de sequeiro, associada à

manifestação de danos oxidativos, sugere que esta enzima não tem um papel antioxidante muito importante na oliveira. Adicionalmente, os resultados demonstraram que as árvores regadas com o equivalente a 30% da evapotranspiração potencial tiveram maior eficiência intrínseca do uso da água ao meio-dia e à tarde, permitindo economizar grandes volumes de água, o que justifica o RWC similar às árvores dos tratamentos T2 e T3. Para além disso, este nível de rega pareceu ser suficiente para reduzir os danos oxidativos ao nível foliar. Este resultado é importante, uma vez que uma estratégia de rega deficitária é essencial para uma olivicultura sustentável devido aos limitados recursos hídricos na região mediterrânica.

Olive (Olea europaea L.) is an evergreen tree traditionally cultivated in the Mediterranean basin for oil and table fruit consumption. During the summer months, olive, like other Mediterranean xerophytes, is usually subjected to high solar irradiances, high air temperatures, high vapour pressure deficits and limited water availability in the soil. The reduction of moisture availability anticipated in the climate change scenarios would inevitably add to the problem of water scarcity throughout the Mediterranean region. So, how olive trees are affected by restricted water availability, is of great concern.

A survey of the scientific literature revealed that olive has important attributes that enable survival and production in drought-prone environments (chapter 1), but important areas are still untouched and few differences have been established between O. europaea cultivars. Therefore, the aim of this thesis was to identify the drought resistance mechanisms of olive tree cultivars and the limitations and damages imposed by water shortage. It was also our goal to investigate how irrigation assists the olive tree to withstand Mediterranean field conditions.

The experimental work was divided in five chapters, which include the information published or submitted to journals belonging to the Journal Citation Reports. Three experiments have been conducted. In the first experiment, we investigated the morpho-anatomical, physiological and biochemical adaptations of five field-grown olive cultivars with different geographical origins (Arbequina, Blanqueta, Cobrançosa, Manzanilla and Negrinha) to drought conditions (chapters 2 and 3).

Chapter 2 presents data on the leaf-level morphological and structural adaptations to reduce water loss. Leaf measurements included leaf tissue thickness, stomatal density, leaf area, leaf mass per unit area, density of leaf tissue, relative water content, succulence, water saturation deficit, water content at saturation and cuticular transpiration rate. The results revealed that olive cultivars native to dry regions, such as Cobrançosa, Negrinha, and Manzanilla, have more capability to acclimate to drought conditions than cultivars originated in regions with a more temperate climate, like Arbequina and Blanqueta. Adaptation was effected by an increase in sclerophylly. Cobrançosa avoids water loss with high density of foliar tissue and the presence of the thick cuticle and trichome layers. Manzanilla and Negrinha enhanced their sclerophylly by building parenchymatous tissues and increasing protective structures like the upper cuticle (Negrinha) and both the upper and lower epidermis (Manzanilla).

In chapter 3, gas exchange rates, chlorophyll a fluorescence, photosynthetic pigments, plant water relations, total soluble sugars, starch, soluble proteins and proline concentrations

were investigated in the five olive cultivars. Stomatal control of water loss was identified as an early response of all olive cultivars to water deficit, leading to limitation of carbon uptake by the leaves. However, the degree of midday depression of photosynthesis was genotype dependent, with a maximum in Arbequina and a minimum in Negrinha. Non-stomatal factors also play an important role in limiting photosynthesis when olive cultivars (mainly Blanqueta) are submitted to prolonged drought under field conditions. In all cultivars, but mostly in Manzanilla, free proline was accumulated in the foliage. However, olive trees have other mechanisms of drought resistance in addition to the solely proline accumulation, such as stomatal closure and soluble protein accumulation.

In a subsequent study, a glasshouse experiment was conducted. One-year-old plants of three Portuguese O. europaea cultivars (Cobrançosa, Madural and Verdeal Transmontana, the more representative cultivars in Trás-os-Montes) were submitted to contrasting water availability regimes during the dry season (chapters 4 and 5).

Chapter 4 presents the effect of cultivar and watering regime in the vegetative growth, gas exchange, xylem hydraulic properties and water use efficiency of biomass production. Low water availability (LW) affected growth and biomass accumulation of the three cultivars. However, Cobrançosa plants were the less affected. Under LW conditions, total leaf area was sharply reduced due to a combination of leaf growth reduction and shedding of older leaves, minimising water losses by transpiration. Water stress also caused a marked decline on photosynthetic capacity and stomatal control was the major factor affecting photosynthesis. Under LW, water use efficiency of biomass production was improved in Cobrançosa, whereas it decreased in Madural and Verdeal Transmontana. In all cultivars, water stress induced an increase in xylem vessel frequency, providing a greater vascular potential and a greater security of xylem sap conduction under drought conditions. From the behaviour of Cobrançosa plants, with a lower allocation of assimilates to leaves, smaller leaves, a smaller reduction in net assimilation rate and photosynthetic rate, a more efficient water transport through the xylem, and an enhanced water use efficiency of biomass production under LW, we consider this cultivar the more appropriate for cultivation under restricted water availability.

Chapter 5 presents the effect of cultivar and watering regime in leaf anatomy, sclerophylly, pressure–volume relationships, chemical composition and oxidative stress symptoms. Anatomically, Cobrançosa and Madural were more capable than Verdeal Transmontana to cope with LW availability, with a thicker upper epidermis, a thicker palisade parenchyma and higher stomatal density. Cobrançosa leaves also revealed the lowest specific leaf area and the highest density of the foliar tissue. Under LW conditions, Cobrançosa and

Madural showed greater capability for osmotic adjustment and increased tissue rigidity. By contrast, Verdeal Transmontana did not exhibit osmotic adjustment, but was able to increase tissue elasticity and total soluble protein concentration. Leaves grown under LW conditions revealed signs of oxidative stress, with decreases in chlorophyll, carotenoid and total thiol concentrations and increased levels of lipid peroxidation. Nevertheless, LW plants developed some defence mechanisms against oxidative stress, like the increase in total phenol and total soluble protein concentrations. Comparatively, Cobrançosa revealed more protection against oxidative stress. In opposition, the increased levels of lipid peroxidation and the decreased total thiol concentration under LW conditions suggest that the mechanisms against oxidative stress were less effective in Madural.

In chapter 6, the influence of different irrigation regimes were investigated on an 8-year-old olive (Olea europaea L., cv. Cobrançosa) commercial orchard located in northeast Portugal. Trees were subjected to a rainfed control (T0) and 3 treatments (T1, T2, T3) that received a seasonal water amount equivalent to 30, 60 and 100% of the estimated local evaporative demand by a drip irrigation system. All irrigation levels improved carbon assimilation of olive trees and reduced the midday and afternoon depression of photosynthesis and stomatal conductance. The experiment also revealed the occurrence of a dynamic photoinhibition in irrigated trees, mainly in trees irrigated with 60 and 100% of the estimated local evaporative demand, which seemed to be effective in protecting the photosynthetic apparatus from photodamage. Leaves grown under rainfed conditions revealed symptoms of oxidative stress, like the reduction in chlorophyll concentration and the increased levels of lipid peroxidation. We also found that the scavenging function of superoxide dismutase was impaired in rainfed plants. In contrast, the low thiobarbituric acid reactive substances concentration in irrigated trees indicates that irrigation decreases the oxidative damage by lipid peroxidation. Furthermore, we concluded that irrigation enhanced antioxidant protection at leaf level since leaves of irrigated trees had high levels of –SH compounds and the highest antioxidant potential. Meanwhile, the finding that guaiacol peroxidase activity increased in rainfed plants, associated with the appearance of oxidative damage, suggests that this enzyme has no major antioxidative function in olive. Additionally, this experiment demonstrated that olive trees irrigated with 30% of the estimated local evaporative demand had high intrinsic water use efficiency at midday and afternoon, saving consistent amounts of water and revealing a relative water content similar to trees irrigated with high amounts of water. Moreover, this level of irrigation seemed to be sufficient to reduce oxidative damage at leaf level. This is an important

finding since a sagacious irrigation approach is essential for viable olive industry due to the limited water resources available in the Mediterranean region.

Chapter 7 present the general conclusions from the research work that was undertaken and indicates some directions for future research.

Acknowledgements... VII Resumo... IX Abstract... XVII List of figures...XXV List of tables ... XXVII Abbreviations...XXIX CHAPTER 1 GENERAL INTRODUCTION ...3 1.1. Introduction ...3 1.2. Objectives...5 1.3. Literature review ...6

1.3.1. Drought and related terms...6

1.3.2. Time scales of plant response to drought stress...7

1.3.3. Sensing and signalling drought stress ...8

1.3.4. Drought effects and symptoms...8

1.3.5. Strategies to survive drought ... 11

1.3.5.1. Drought escape... 11

1.3.5.2. Drought avoidance ... 12

1.3.5.3. Drought tolerance ... 14

1.3.6. Oxidative damage induced by drought ... 17

1.3.7. Protection against oxidative stress... 20

1.3.8. Olive capacity to withstand arid environments ... 23

1.4. References... 28

CHAPTER 2 SCLEROPHYLLY AND LEAF ANATOMICAL TRAITS OF FIVE FIELD-GROWN OLIVE CULTIVARS GROWING UNDER DROUGHT CONDITIONS... 41

2.1. Abstract ... 41

2.2. Introduction ... 41

2.3. Materials and methods ... 43

2.3.1. Study site... 43

2.3.2. Plant material, leaf anatomy study and tissue measurements... 44

2.3.3. Morphology, sclerophylly and leaf water relations parameters ... 44

2.3.4. Stem water potential... 45

2.4. Results and discussion ... 45 2.4.1. Leaf anatomy study and tissue measurements ... 45 2.4.2. Morphology, sclerophylly and leaf water status... 47 2.4.3. Stem water potential... 50 2.5. Conclusions... 51 2.6. References... 52

CHAPTER 3

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF FIVE Olea europaea L. CULTIVARS TO WATER STRESS IN THE FIELD... 57

3.1. Abstract... 57 3.2. Introduction... 57 3.3. Materials and methods... 59 3.3.1. Study site ... 59 3.3.2. Plant material... 59 3.3.3. Gas exchange and chlorophyll a fluorescence... 59 3.3.4. Plant water relations ... 60 3.3.5. Photosynthetic pigments and metabolites assays... 60 3.3.6. Statistics ... 61 3.4. Results ... 61 3.4.1. Gas exchange and chlorophyll a fluorescence... 61 3.4.2. Plant water relations ... 64 3.4.3. Photosynthetic pigments and metabolites in leaves... 64 3.5. Discussion ... 65 3.6. References... 70

CHAPTER 4

CHANGES IN GROWTH, GAS EXCHANGE, XYLEM HYDRAULIC PROPERTIES AND WATER USE EFFICIENCY OF THREE OLIVE CULTIVARS UNDER CONTRASTING WATER AVAILABILITY REGIMES... 77

4.1. Abstract... 77 4.2. Introduction... 77 4.3. Materials and methods... 79 4.3.1. Plant material and growth conditions... 79 4.3.2. Biomass production and growth ... 80 4.3.3. Water use efficiency of biomass production... 80 4.3.4. Gas exchange and water potential ... 80 4.3.5. Xylem anatomical analyses ... 81

4.3.6. Statistics ... 81 4.4. Results ... 82 4.4.1. Biomass production and growth ... 82 4.4.2. Water use efficiency of biomass production... 87 4.4.3. Gas exchange and water potential... 87 4.4.4. Xylem anatomy... 88 4.5. Discussion... 89 4.6. References... 94

CHAPTER 5

IMMEDIATE RESPONSES AND ADAPTATIVE STRATEGIES OF THREE OLIVE CULTIVARS UNDER CONTRASTING WATER AVAILABILITY REGIMES: CHANGES ON STRUCTURE AND CHEMICAL COMPOSITION OF FOLIAGE AND OXIDATIVE DAMAGE ... 99

5.1. Abstract ... 99 5.2. Introduction ... 99 5.3. Materials and methods ... 101 5.3.1. Plant material and growth conditions ... 101 5.3.2. Leaf anatomy study and tissue measurements... 101 5.3.3. Foliar metabolic assays and oxidative stress indicators ... 102 5.3.4. Statistics ... 104 5.4. Results ... 104 5.5. Discussion... 110 5.6. References... 116

CHAPTER 6

PHYSIOLOGICAL BEHAVIOUR, OXIDATIVE DAMAGE AND ANTIOXIDATIVE PROTECTION OF OLIVE TREES GROWN UNDER DIFFERENT IRRIGATION REGIMES ... 123

6.1. Abstract ... 123 6.2. Introduction ... 124 6.3. Materials and methods ... 125 6.3.1. Site description, plant material and treatments ... 125 6.3.2. Leaf water status and stem water potential ... 126 6.3.3. Gas exchange and chlorophyll fluorescence measurements... 127 6.3.4. Photosynthetic pigments, soluble sugars and starch ... 127 6.3.5. Lipid peroxidation ... 128 6.3.6. Total phenol, UV-B absorbing compounds, soluble protein and total thiol... 128 6.3.7. Radical scavenging activity ... 128 6.3.8. Antioxidant enzymes ... 129

6.3.9. Statistics ...129 6.4. Results ...129 6.4.1. Leaf water status and stem water potential...129 6.4.2. Gas exchange and chlorophyll fluorescence measurements ...130 6.4.3. Lipid peroxidation ...131 6.4.4. Photosynthetic pigments, soluble sugars and starch...132 6.4.5 Total phenol, UV-B absorbing compounds, soluble protein and total thiol...132 6.4.6. Radical scavenging activity ...133 6.4.7. Antioxidant enzymes...133 6.5. Discussion ...133 6.6. References...138

CHAPTER 7

GENERAL CONCLUSIONS AND PERSPECTIVES OF FUTURE RESEARCH ...145

7.1. General conclusions...145 7.2. Perspectives of future research...148

1.1 Typical time course of plant response to environmental stress ...7 1.2 Influence of water stress on the physiology of plants ... 10 1.3 Reactions producing ROS and organic free radicals. ... 18 1.4 Defence systems (enzymatic and non-enzymatic antioxidants) against ROS ... 21 1.5 Olive protections at leaf level against water loss and excessive irradiance ... 25 2.1 Monthly air temperatures and rainfall at Mirandela... 43 2.2 Stem water potentials at predawn and midday of olive cultivars ... 50 3.1 Diurnal evolution of leaf gas exchange parameters of olive cultivars ... 63 3.2 Stem water potentials at predawn and midday of olive cultivars ... 64 4.1 Relative total, aboveground, and belowground biomass of three olive cultivars under

contrasting water availability regimes... 83 4.2 Water use efficiency of biomass production of three olive cultivars under contrasting water

availability regimes... 87 5.1 Total thiol concentration on foliage of three olive cultivars under contrasting water availability

regimes... 110 5.2 Total thiobarbituric acid reactive substances concentration on foliage of three olive cultivars

under contrasting water availability regimes ... 110 6.1 Rainfall and monthly air temperatures at the study site ... 126 6.2 Diurnal evolution of leaf gas exchange parameters in the four irrigation treatments ... 130 6.3 Diurnal evolution of maximum quantum yield of PSII in the four irrigation treatments... 131 6.4 Effect of irrigation on thiobarbituric acid reactive substances concentration of leaf extracts ... 132

1.1 Reactive oxygen species ... 17 2.1 Leaf tissue thickness and stomatal density of olive cultivars ... 48 2.2 Leaf area, leaf mass per unit area, density of the leaf tissue, relative water content,

succulence, water content at saturation, water saturation deficit and cuticular transpiration rate of olive cultivars ... 48 3.1 Chlorophyll a fluorescence parameters at predawn and midday of olive cultivars ... 63 3.2 Photosynthetic pigments, total soluble sugars, starch, soluble proteins and proline

concentrations of olive cultivars... 65 4.1 Root weight ratio, shoot weight ratio, root-shoot ratio, stem weight ratio and leaf weight

ratio of three olive cultivars under contrasting water availability regimes... 84 4.2 Several morphological plant parameters of three olive cultivars under contrasting water

availability regimes... 85 4.3 Relative growth rate, net assimilation rate, leaf area ratio and specific leaf area of three olive

cultivars under contrasting water availability regimes ... 86 4.4 Gas exchange parameters and leaf water potential at midday of three olive cultivars under

contrasting water availability regimes... 88 4.5 Stem xylem vessel frequency, vessel diameter, relative hydraulic conductivity and

vulnerability index of three olive cultivars under contrasting water availability regimes ... 89 5.1 Leaf tissue thickness and stomatal density of three olive cultivars under contrasting water

availability regimes... 105 5.2 Specific leaf area, density of the leaf tissue, relative water content, succulence, water content

at saturation and water saturation deficit of three olive cultivars under contrasting water availability regimes... 106 5.3 Osmotic potential at full turgor, osmotic potential at turgor loss point, maximum bulk

modulus of elasticity, relative water content at turgor loss point and leaf turgid mass/dry mass of three olive cultivars under contrasting water availability regimes... 107 5.4 Photosynthetic pigments, soluble sugars and starch of three olive cultivars under contrasting

water availability regimes... 108 5.5 Total phenol and total soluble protein concentrations of three olive cultivars under

contrasting water availability regimes... 109 6.1 Relative water content, water content at saturation and stem water potential at predawn in

the four irrigation treatments ... 129 6.2 Photosynthetic pigments, soluble sugars and starch of leaf extracts of T0 and T3 ... 132 6.3 Total phenols, ultraviolet-B absorbing compounds, total soluble protein, total thiol

concentration, radical scavenging activity and antioxidant enzymes superoxide dismutase and guaiacol peroxidase activities of leaf extracts of T0 and T3 ... 133

A ...Net CO2 assimilation rate

ABA ...Abscisic acid

A/gs...Intrinsic water use efficiency

Car ...Total carotenoids D ...Leaf tissue density

Ci...Intercellular CO2 concentration

Ci/Ca...Ratio of intercellular to atmospheric CO2 concentration

Chl(a+b)...Total chlorophyll

E ...Transpiration rate

Ec...Cuticular transpiration rate F0...Minimal fluorescence

Fm...Maximal fluorescence

Fv...Variable fluorescence

Fv/Fm...Maximal photochemical efficiency of PSII

gm...Liquid phase diffusive conductance to CO2

gs...Stomatal conductance

LA...Leaf area LAR...Leaf area ratio

LMA ...Leaf mass per unit area

LW...Plants under low water availability NAR ...Net assimilation rate

OA ...Osmotic adjustment

PPFD...Photosynthetic photon flux density PSII ...Photosystem II

RC ...Relative hydraulic conductivity ROS ...Reactive oxygen species R:S ...root-shoot ratio

RSA...Radical scavenging activity RGR ...Relative growth rate RWC ...Relative water content

RWCTLP...Relative water content at turgor loss point

S...Succulence –SH...Total thiols SLA ...Specific leaf area

SS ... Total soluble sugars SP ... Total soluble proteins St ... Starch

TBARS ... Total thiobarbituric acid reactive substances TM/DM... Leaf turgid mass/dry mass ratio

TP ... Total phenols

UV−BAC... Ultraviolet-B absorbing compounds

VD... Vessel diameter VF ... Vessel frequency VI... Vulnerability index VPD ... Vapour pressure deficit WCS ... Water content at saturation WSD ... Water saturation deficit

WUE ... Water use efficiency of biomass production WW ... Well-watered plants

Ψ ... Water potential

Ψ∏FT... Osmotic potential at full turgor

Ψ ∏TLP... Osmotic potential at turgor loss point

CHAPTER 1

1.

1.1. Introduction

Olive is a perennial, long-lived, evergreen tree of subtropical origin (Bongi and Palliotti, 1994). It was domesticated around 3000 to 4000 B.C. in the eastern Mediterranean and from there was spread widely in northern Africa, the Iberian Peninsula, and the rest of southern Europe by civilizations that successively occupied the region. During the last 500 years, olive has been taken to the Americas, South Africa, Australia, China and Japan, but remains principally a crop of the Mediterranean basin (Connor, 2005). Although growth is possible in other latitudes, olive grows best in areas between 30º-45º north and south latitude (Hagidimitriou and Pontikis, 2005). The recognized tolerance of olive to drought, and its capacity to grow in shallow, poor quality soils, makes the species among the most interesting for cultivation in arid and semiarid areas. This agronomic interest is enhanced by the fact that, despite its tough character, the tree shows a remarkable response to any improvement in the cropping conditions (Fernández and Moreno, 1999).

The olive tree is the only species with edible fruits in the family Oleaceae (Rapoport, 2001). Although there are different systems for the botanical classification of the species, it is generally accepted that the commercial cultivars are included in the subspecies sativa and the wild types belong to the subspecies sylvestris (Lavee, 1996). The recent and growing popularity of the olive as fruit tree is related with the increasing demand of its products, both oil and the fruits. This favourable market trend is partly due to dietetic studies providing the advantages of the regular consumption of olives and olive oil for human health. In fact, the low saturated-to-unsaturated fatty acid ratio and the presence of natural antioxidants could prevent certain human diseases (Saldanha, 1999). In most cases, health and dietary benefits induced by consumption of olives and virgin olive oil are due to the synergistic activity of several constituents, such as vitamins (e.g. α-tocopherol), phytosterols, pigments, terpenic acids, and phenolic compounds (Visioli et al., 2002). In particular, phenolic compounds are strong antioxidants and are also responsible for the astringency and bitterness of olive oils. The presence of some phenolic compounds in olive oil and drupes has been related to the prevention of coronary artery disease and atherosclerosis (Caturla et al., 2005).

The world area of olive is around 7.5 million ha and the primary producers of olive oil are Spain, Italy, Greece, Tunisia, Turkey, Syria, Morocco and Portugal (FAOSTAT, 2006). These countries alone account for more than 90% of world production. Portugal is an important table olives and olive oil producer (11,291 t table olives; 280,000 t olive oil, in 2003) (GPPAA, 2004).

Olive cultivation is known in Portugal since the Roman domination (Ribeiro, 1991) and is one of the most important crops in the country. Olive has traditionally been grown in Trás-os-Montes (northeast Portugal) where it is of considerable economic, social and environmental importance. Its excellent quality and unique characteristics are responsible for the denomination of protected origin ῾Azeite de Trás-os-Montes DOP̕. The creation of the denomination of protected origin is relatively new and stimulated the local economy, namely by adding value to the olive oil produced in the area. As a result of the recent strategy to promote quality, we can see today old olive groves, some with more than one century, and modern ones spread throughout the region, especially in the warmer areas.

During the summer, olive, like other Mediterranean xerophytes, is usually subjected to high solar irradiances, high air temperatures, high air vapour pressure deficits and limited water availability in the soil. The severity of these stresses is predicted to increase in the future as a result of global environmental change (Fischer et al., 2001; Centritto, 2002). Research into plant response to water stress is becoming increasingly important, as most climate change scenarios suggest an increase in aridity in many areas of the globe (Petit et al., 1999). Plant responses to water scarcity are complex, involving adaptive changes or deleterious effects (Chaves et al., 2002), or both. A better understanding of the effects of drought on plants is vital for improved management practices and breeding efforts in agriculture and for predicting the fate of natural vegetation under climate change (Chaves et al., 2003).

As other southern European regions, Portugal is a place of mild Mediterranean climate, but with well known vulnerability to climate variability, namely to drought and desertification (Santos et al., 2002). All models, in all scenarios, predict a significant increase in the mean temperature, in all Portuguese regions, by the end of the 21st century (IPCC, 2001). Precipitation scenarios are much more uncertain. However, almost all models predict a reduction in precipitation over mainland Portugal during spring, summer and autumn (IPCC, 2001). Future changes in climate would significantly affect crop yields in the Mediterranean region. However, the overall impact on crop production will be determined by the ability of farmers to adapt to the new scenarios.

There is a range of management practices that assist olive to survive and produce in water-limited environments. These practices seek to balance transpiration demand with water available, especially during the long, dry summer. Strategic decisions are selection of cultivars, tree density, and canopy size, together with surface management (Connor, 2005). Recently, irrigation has been introduced to increase the low land productivity. The expansion of olive cultivation, due to increased demand for olive oil, has led new plantations to be characterized

by higher number of trees per ha compared to traditional orchards. Under such intensive systems, irrigation is essential to ensure optimum water application for tree growth and olive production (Patumi et al., 1999). However, this can be problematic due to limited water resources available in the Mediterranean regions (Villalobos et al., 2000). Prudent management of water is, therefore, essential for a viable olive industry.

1.2. Objectives

The reduction of moisture availability anticipated in the climate change scenarios would inevitably add to the problem of water scarcity throughout the Mediterranean region. Since olive is a major crop in the Mediterranean basin, how olive trees are affected by restricted water availability at Mediterranean latitudes, is of great concern.

The potential for improvement of olive performance cannot be realised until characteristics and mechanisms of drought resistance are clearly identified. This in turn cannot occur without a through understanding of the restrictions and damages that are induced by an insufficient water supply. A survey of the scientific literature revealed that olive has important attributes that enable survival and production in drought-prone environments, but important areas are still untouched, and few differences have been established between O. europaea cultivars. Although the cultivars most frequently grown in Trás-os-Montes are considered well adapted to drought, there are no studies documenting the drought adaptation of these cultivars. Therefore, the general objectives of this thesis were:

• to identify the morpho-anatomical, physiological and biochemical adaptations that enable olive tree cultivars with different geographical origins (Trás-os-Montes and several regions of Spain) and diverse genetic background to cope with summer stress in the field;

• to study the immediate responses and adaptative strategies of the three more representative O. europaea cultivars in Trás-os-Montes (Cobrançosa, Madural and Verdeal Transmontana) growing in a glasshouse under contrasting water availability regimes during the dry season, and to evaluate the symptoms of oxidative stress induced by low water availability;

• to study the influence of different irrigation regimes on the physiological behaviour of mature olive trees (cv. Cobrançosa) growing in commercial-like field conditions and to analyse the oxidative damage and antioxidative protection at leaf level in irrigated and non-irrigated trees.

1.3.Literature review

1.3.1. Drought and related terms

Water is an important component of the metabolism of all living organisms, facilitating many vital biological reactions by being a solvent, a transport medium and evaporative coolant (Bohnert et al., 1995). In plants and other photoautotrophs, water plays the additional role of providing the energy necessary to drive photosynthesis. Water molecules are split, in a process termed autolysis, to yield the electrons that are used to drive the energy yielding photosystem II reaction centre (Salisbury and Ross, 1992). Thus, any limitation in the availability of water has a great influence on plant life.

Many environmental conditions can lead to water deficit in plants (Bray et al., 2000). Periods of little or no rainfall can lead to meteorological condition called drought. Transient or prolonged drought conditions reduce the amount of water available for plant growth. Other climatic factors such as high temperature, high irradiance, high wind, and low relative humidity are often associated with it in many regions of the world and can significantly aggravate its severity. Drought can be permanent, as in arid regions, or seasonal or even random. The period of time without rainfall actually needed to produce a drought depends mainly on the water holding capacity of the soil and the rate of evapotranspiration (Jones, 1992; Larcher, 1995; Kozlowski and Pallardy, 1997).

Water deficit can also occur in environments in which water is not limiting (Bray et al., 2000). In saline habitats, the presence of high salt concentrations makes it more difficult for plant roots to extract water from the environment. Low soil temperatures reduce soil and plant hydraulic conductance and eventually water uptake (e.g. Kramer and Boyer, 1995) through an increase in water viscosity and a decrease in membrane permeability and in metabolic activity of roots (Kaufmann, 1975), as well as hampering the production of new fine roots. Exposure to freezing temperatures can lead to cellular dehydration as water leaves the cells and forms ice crystals in the intercellular spaces. Occasionally, well-watered plants will show periodic signs of water stress such as transient loss of turgor during some periods of the day. In this case, wilting indicates that the transpirational water loss has exceeded the rate of water absorption.

Drought is considered a multidimensional stress affecting plants at various levels of their organization (Yordanov et al., 2000). The responses of plants to drought stress are highly complex, involving deleterious and/or adaptive changes. The plant response to drought at the whole plant and crop levels reflects the integration of stress effects and responses at all underlying levels of organization over space and time (Blum, 1996).

1.3.2. Time scales of plant response to drought stress

Plant responses to stresses such as drought involve a variety of temporal scales, from a matter of seconds to evolutionary time scales. Lambers et al. (1998) recognize at least three distinct time scales of plant response to stress: stress response, acclimation and adaptation (Figure 1.1). Stress response is the immediate detrimental effect of a stress on a plant process. This usually occurs over a time scale of seconds or days in which the net effect is a decline in plant performance.

Figure 1.1

Typical time course of plant response to environmental stress (Lambers et al., 1998).

Acclimation is the morphological and physiological adjustment by plants to compensate the decline in performance following exposure to stress. Acclimation occurs within the life time of the plant, usually within days or weeks of the stress exposure. During acclimation, an organism alters its homeostasis, its steady-state physiology, to accommodate shifts in its external environment.

Adaptation is the evolutionary response that results from genetic changes in populations leading to morphological and physiological compensation for the decline in performance caused by stress. This may be similar to acclimation, but because it requires genetic changes, it must occur over many generations. Some plants possess adaptations to arid environments, such as the C4 and CAM modes of metabolism (Taiz and Zieger, 1998). In CAM plants the stomata are

open during the night and can be closed in daytime, so that water loss is very low in relation to dry matter production. C4 plants exhibit no photorespiration and can fix CO2 even in hot and dry

conditions, requiring less water as a consequence.

Min Day Month Generation Evolutionary time

Stress response Acclimation Adaptation Homeostatic compensation Ra te o f pr oc ess Stress

Min Day Month Generation Evolutionary time

Stress response Acclimation Adaptation Homeostatic compensation Ra te o f pr oc ess Stress

1.3.3. Sensing and signalling drought stress

A stress response is initiated when the plant recognises a stress at the cellular level (Bray et al., 2002). Stress recognition activates signal transduction pathways that transmit information within individual cells and throughout the plant. It is not perfectly clear how cells perceive cellular water deficit and how cellular water deficit is transduced and transcribed into the various consequences of this stress. Furthermore, it has not been clearly established which of these responses are stress adaptive and which are expressions of system degradation. The nature of the primary mediators of cellular responses to water deficit−water status, turgor, bound water, hormones (e.g. abscisic acid, ABA), alteration in cell membranes and others−are still under debate (Chaves et al., 2003). After the first stress-recognition events, cell-to-organ drought-mediated responses diverge in different pathways according to the involvement or not of ABA (Chaves et al., 2003). In the ABA-dependent pathway, the accumulation of ABA activates numerous ῾stress responsive̕ genes. These gene products are either functional (such as aquaporins or the enzymes of osmoprotectant biosynthesis) or regulatory (such as protein kinases) and they are involved in mediating various cellular responses some of which are recognized as adaptive (Chaves et al., 2003). Several genes are induced by cell dehydration in ABA-deficient and ABA-insensitive mutants, suggesting that such genes do not require ABA for expression (Shinozaki and Yamaguchi-Shinozaki, 1997; Luan, 1998). The ABA independent-pathway so far identified is still poorly understood, but is known to be rapidly induced by water stress. Although the ABA-dependent and ABA-independent pathways are usually considered to function independently from each other, it is certainly possible that some cross-talk exists between them, as supported by Kizis et al. (2001).

1.3.4. Drought effects and symptoms

Many factors can affect the response of a plant to drought stress. The duration, severity, and rate at which a stress is imposed all influence how a plant responds (Gaspar et al., 2002). Stress due to drought, in contrast to many other stressful events, does not occur abruptly, but develops slowly and increases in intensity the longer it lasts (Larcher, 1995). Features of the plant, including organ or tissue identity, developmental age, and genotype, also influence plant response to stress (Bray et al., 2000).

Hsiao (1973) used three degrees of water stress, in relation to a ῾typical mesophyte̕: mild stress, water potential (Ψ) slightly lowered, typically down to −0.5 MPa at most; moderate stress, Ψ in the range −0.5 to −1.2 or 1.5 MPa; and severe stress, Ψ below −1.5 MPa. Lawlor

(1995) has proposed an alternative, but broadly compatible, classification of mesophytes, based on relative water content (RWC): values down 90% are associated with effects on stomata and cell expansion; 80−90% with effects on photosynthesis and respiration; and bellow 80% (corresponding to water potentials of −1.5 MPa or lower) with the cessation of photosynthesis and the disruption of cell metabolism.

The interpretation of the effects of water stress on plant physiology can be complicated by the fact that responses can be evoked at the organ, tissue, cell or molecular level. Figure 1.2 outlines the influence of water stress on the physiology of plants. Some of the earliest responses to water stress appear to be mediated by biophysical events rather than by changes in chemical reactions resulting from dehydration (Taiz and Zieger, 1998). As the water content of the plant decreases, the cells shrink and the cell walls relax. This decrease in cell volume results in lower hydrostatic pressure, or turgor. Cell expansion is a turgor driven process and appears to be one of the plant processes most sensitive to water stress (Hsiao, 1973). Inhibition of cell expansion results in a slowing of leaf expansion early in the development of water deficits. The smaller leaf area transpires less water, effectively conserving a limited supply in the soil for use over a longer period (Taiz and Zieger, 1998). If plants became water stressed after a substantial leaf area has developed, leaves will senesce and eventually fall off. Water stress limits not only the size of individual leaves, but also the number of leaves on an indeterminate plant, because it decreases both the number and growth rate of branches. The process of growth of the other organs is probably affected by the same forces that limit leaf area during stress.

In addition to slowing growth, a lowering of water potential by less than 0.5 MPa is associated with some disruption of biosynthetic activities (Figure 1.2). The inhibition of cell expansion is usually followed closely by a reduction in cell wall synthesis (Fitter and Hay, 2002). Protein synthesis may be almost equally sensitive to water stress (Salisbury and Ross, 1992). At slightly more negative water potentials, protochlorophyll formation is inhibited (Fitter and Hay, 2002). Some studies also indicate that activities of certain enzymes, especially nitrate reductase, phenylalanine ammonia lyase and few others, decrease sharply as water stress increases (Salisbury and Ross, 1992).

Under moderate water stress there is further reduction in turgor, leading to the narrowing of stomatal aperture and a progressive reduction in photosynthetic activity (Figure 1.2). Increased respiration may also play a part in stomatal closure owing to an increase in CO2

Lowering of leaf water potential at which symptom first appears (MPa)

0 −1.0 −2.0

Symptom

Loss of turgor Reduced rate of cell expansion Reduced cell wall synthesis Reduced protein synthesis Reduced protochlorophyll synthesis Reduced levels of nitrate reductase Increased abscisic acid synthesis Stomatal closure Reduced CO2 assimilation

Increased rate of respiration Cavitation of xylem elements Accumulation of organic solutes Disruption of phloem function Reduced inorganic nutrient supplies Leaf wilting

Figure 1.2

Influence of water stress on the physiology of plants. The horizontal bars are guides to the level of stress at which the relevant symptoms first occur. The lowering of leaf water potential is in relation to a well-watered plant under mild evaporative demand (Updated from Hsiao et al., 1976).

Because guard cells are exposed to the atmosphere, they can lose water directly by evaporation and so loose turgor, causing the stomata to close by a mechanism called hydropassive closure. A second mechanism, called hydroactive closure, closes the stomata when the whole leaf or the roots are dehydrated and depends on metabolic processes in the guard cells. A reduction in the solute content of the guard cells results in water loss and decreased turgor, causing the stomata to close (Taiz and Zieger, 1998). Solute loss from guard cells can be triggered by decreasing water status in the rest of the leaf, and there is much evidence that ABA plays an important role in this process. The stomata respond to two sources of ABA: (1) an ῾early warning system̕ involving root ABA, indicating that some roots are drying; and (2) ABA translocation within the leaf, resulting from desiccation of the leaves themselves (Taiz and Zieger, 1998). Nevertheless, stomatal responses to dehydration can vary widely both within and across species.

There are several reports, which mark the stomatal limitation of photosynthesis as a primary event, followed by respective changes of the photosynthetic reactions (Chaves, 1991; Cornic and Massacci, 1996). However, at long-term water deficit the non-stomatal limitation predominates (Yordanov et al., 2001). Non-stomatal limitations has been attributed to reduced

carboxilation efficiency (Wise et al., 1991; Jia and Gray, 2004), reduced ribulose-1,5-biphosphate (RuBP) regeneration (Gimenez et al., 1992; Tezara and Lawlor, 1995), and/or to a reduced amount of functional Rubisco (Kanechi et al., 1995).

With the onset of severe stress, a general disruption of metabolism is signalled by high rates of respiration and the build up of proline and soluble sugars in leaf tissues (Figure 1.2). In plants resistant to drought such accumulation of organic solutes, leading to osmorregulation, can occur at lower stress (see section 1.3.5.3.).

Under moderate and severe water stress (xylem water potential lower than −1.0 MPa), herbaceous plants begin to show symptoms of xylem cavitation and reduced xylem conductance (Tyree et al., 1986). However, cavitation of xylem elements in trees tends to begin at much lower potentials, typically −2.0 to −3.0 MPa (Jones and Sutherland, 1991). There can also be reductions in the supply of mineral nutrients to the leaf via the xylem, and, under severe stress, in rates of flow in the phloem (Schulze, 1991) (Figure 2.2).

In summary, exposure of plants to even mild water stress can affect growth and disturb metabolic processes. Depending on their severity, such effects can reduce the ability of plants to survive and reproduce. Much of the relevant research on drought effects was done with herbaceous plants, but the results probably are equally applicable to woody plants (Kozlowski and Pallardy, 1997).

1.3.5. Strategies to survive drought

The ability of plants to survive the consequences of drought is termed drought resistance. There is no universal way by which this can be achieved and in consequence the different components of drought resistance have been classified in different ways by different authors. Classically, plant resistance to drought has been divided into escape, avoidance and tolerance strategies (Levitt, 1972; Turner, 1986). Nevertheless, these strategies are not mutually exclusive and, in practice, plants may combine a range of response types (Ludlow, 1989).

1.3.5.1. Drought escape

Plants that escape drought, like desert ephemerals and annual crop and pasture plants, exhibit a high degree of developmental plasticity, being able to complete their life cycle before water deficit occur. Escape strategies rely on successful reproduction before the onset of severe stress. Improved reproductive success also includes better partitioning of assimilates to

developing fruits. This is associated with the plant’s ability to store reserves in some organs (stems and roots) and to mobilise them for fruit production, a response well documented in crop plants, such as cereals (Gebbing and Schnyder, 1999; Bruce et al., 2002) and some legumes (Rodrigues et al., 1995; Chaves et al., 2002).

1.3.5.2. Drought avoidance

Plants that tend to avoid drought generally have tissues that are very sensitive to dehydration, and thus they have to avoid water deficits whenever water shortage occurs (Ludlow, 1989). Dehydration avoidance is common to both annuals and perennials and is associated with a variety of adaptative traits. These involve maximising water uptake and minimising water loss (Chaves et al., 2003).

The most effective protection against drought is a deep, extensively branched root system that can absorb water from a large volume of soil (Arndt, 2000). Moreover, roots with low hydraulic conductance or few but long roots can permit a slow but sustainable supply of water to the plant (Passioura, 1983).

Water movement from the roots to the atmosphere is controlled by the conductance of the components of the water pathway (Lovisolo and Schubert, 1998). Traditionally, stomatal conductance and root conductivity have been considered the main controlling factors of water flow in the plant (Jones, 1992). However, the efficiency of the water transport system (the xylem vessels in angiosperms) can also significantly affect water movement by imposing conductivity constrains (Tyree and Ewers, 1991). It has been reported that water stress affects shoot conductivity by inducing embolism in xylem vessels (Schultz and Matthews, 1988; Tyree and Sperry, 1989; Tognetti et al., 1996) or by a reduction in the vessel diameter (Lovisolo and Schubert, 1998). Embolisms are important because they reduce the hydraulic conductivity of the xylem, giving rise to the possibility of ῾runaway̕ reduction in hydraulic conductivity unless transpiration is reduced to relieve tension and prevent further cavitation (Tyree and Ewers, 1991). Adaptation requires a fine balance because features that reduce vulnerability to cavitation, narrow conduits and many inter-conduit connections, also reduce hydraulic conductivity that generates the high xylem tensions that trigger cavitation (Sperry, 2003). In general, vessels with narrow diameters are less susceptible to embolism (Lovisolo and Schubert, 1998). However, variations in xylem conduit diameter can radically affect the function of the conducting system because of the fourth-power relationship between radius and flow through a capillary tube, as described by the Hagen-Poiseuille law (Fahn, 1986).

Leaves growing under conditions of water deficiency develop smaller but more densely distributed stomata. This modification makes a leaf able to reduce transpiration by a quicker onset of stomatal regulation (Larcher, 1995). Stomata are mainly confined to the abaxial surface and are often hidden beneath dense hair or in depressions (grooves or crypts). In this way the boundary layer resistance is increased and the air outside the stomata becomes moister (Larcher, 1995). Other morpho-anatomical traits that help to minimize water loss during drought include leaf rolling (Schwabe and Lionakis, 1996), dense leaf pubescence (Palliotti et al., 1994; Karabourniotis and Bornman, 1999; Liakoura et al., 1999), a thick cuticle and epicuticular wax layer (Leon and Bukovac, 1978; Liakoura et al., 1999; Richardson and Berlyn, 2002), heavily lignified tissue (Richardson and Berlyn, 2002), smaller mesophyll cells and less intercellular spaces (Bongi et al., 1987; Mediavilla et al., 2001). Moreover, leaf movements, such as para-heliotropism, can also prevent damage by high temperatures, dehydration, and photoinhibition (Ludlow, 1989). Another common feature in water stressed plants is the reduction of the canopy leaf area through reduced growth and shedding of older leaves. This usually begins with the oldest leaves and progresses toward stem tips. Although the loss of leaves results also in a reduction of the photosynthetic surface it consequently reduces water loss and prolongs survival (Kozlowski et al.,1991).

Ludlow (1989) outlined the ecophysiological implications of drought avoidance. Plants that avoid water deficits by obtaining access to water deep in the soil may have little penalty in terms of carbon acquisition. On the other hand, in plants where water deficits are avoided by minimising water loss, rather than maximising water uptake, there will be both short- and long-term costs for carbon acquisition, depending upon whether the responses are elastic or plastic. Elastic responses, like stomatal closure or leaf movements, allow leaves to survive short-term stress periods, and so contribute to growth and yield after rainfalls, and so there may be few long-term costs. In contrast, for plastic responses, like reduced leaf area expansion in new leaves or the shedding of older leaves, there will be both short- and long-term costs.

In summary, because of their ability to postpone or avoid tissue water deficits, plants that avoid drought could be said to have good short-term survival. However, they have poor long-term survival, because the avoidance mechanisms eventually fail to prevent dehydration of tissues that are relatively sensitive to desiccation (Ludlow, 1980).