Chemical and structural analysis of Eucalyptus globulus and E. camaldulensis leaf cuticles: a lipidized cell wall region

The plant cuticle has traditionally been conceived as an independent hydrophobic layer that covers the external epidermal cell wall. Due to its complexity, the existing relationship between cuticle chemical composition and ultra-structure remains unclear to date. This study aimed to examine the link between chemical composition and structure of isolated, adaxial leaf cuticles of Eucalyptus camaldulensis and E. globulus by the gradual extraction and identification of lipid constituents (cutin and soluble lipids), coupled to spectroscopic and microscopic analyses. The soluble compounds and cutin monomers identified could not be assigned to a concrete internal cuticle ultra-structure. After cutin depolymerization, a cellulose network resembling the cell wall was observed, with different structural patterns in the regions ascribed to the cuticle proper and cuticular layer, respectively. Our results suggest that the current cuticle model should be revised, stressing the presence and major role of cell wall polysaccharides. It is concluded that the cuticle may be interpreted as a modified cell wall region which contains additional lipids. The major heterogeneity of the plant cuticle makes it difficult to establish a direct link between cuticle chemistry and structure with the existing methodologies.

Composición florística y etnobotánica de las diferentes formaciones vegetales de la provincia de Loja, Ecuador

Pese a que Loja (Ecuador) presenta una gran riqueza ecológica y cultural, ni su patrimonio etnobotánico, ni cómo afecta el uso de las plantas al estado de conservación de los ecosistemas es bien conocido. Por ello se ha realizado un estudio etnobotánico y ecológico para conocer el papel que han jugado las plantas en la cultura tradicional de la provincia y comprender qué factores influyen en su uso. Durante 2006 y 2008 se entrevistó a 770 informantes (196 de ellos vendedores en mercados locales) y se realizaron 373 transectos en 118 remanentes boscosos, 51% indicados por la gente como lugares de recolección. Se ha registrado el saber etnobotánico de 717 especies pertenecientes a 143 familias, la mayoría nativas del Ecuador, 31 de ellas endémicas. Ello supone un 15% de la flora total de Loja. Sin embargo especies introducidas como Matricaria recutita o Eucalyptus globulus están entre las más citadas. La mayor riqueza se obtuvo para las plantas medicinales (509 especies), que se usan sobre todo para tratar las llamadas “inflamaciones internas” (210 especies, Matricaria recutita y Melissa officinalis principalmente) y trastornos del sistema digestivo (188, Matricaria recutita y Mentha pulegium). En los mercados se venden 169 especies, la mayoría para elaborar la bebida típica llamada horchata (65) y para curar “inflamaciones internas” (36). Además se emplean 414 con otros fines (161 para leña, 142 para alimentación humana, 133 para construcción de viviendas y muebles, 98 para artesanías, 89 ornamentales, 27 tintóreas y 72 para otros usos). En los remanentes se inventariaron 19.156 individuos correspondientes a 1440 especies de 153 familias, de las cuales 437 son empleadas por la población. La riqueza media de especies y de plantas útiles fue significativamente mayor en los remanentes recolectados lo cual parece demostrar que la explotación no afecta negativamente a la diversidad de especies al generar variabilidad de hábitats. También se vio que la proporción de especies útiles disminuye en los remanentes más diversos. También se observó que la probabilidad de uso de una especie aumenta con la frecuencia y la abundancia y disminuye con la distancia a los remanentes en los que aparece. Este patrón general no se cumple para las especies medicinales y ornamentales. Estos datos demuestran una gran riqueza de conocimientos tradicionales y una gran vitalidad del uso de muchas especies y corroboran la hipótesis de que los recursos vegetales más accesibles y abundantes son, salvo excepciones, los más empleados por la gente. ABSTRACT Although the Loja province (Ecuador) has a great ecological and cultural richness, neither the ethnobotanical heritage nor the incidence of plant use on ecosystems conservation it is well known. We have made an ethnobotanical and ecological study to determine the role played by plants in the traditional culture of the province and to understand what factors influence their use. Between 2006 and 2008 we interviewed 770 informants (196 of them vendors in local markets) and inventoried 373 transects in 118 forest remnants (51% of them indicated by people as collection sites). We recorded the ethnobotanical knowledge for 717 species belonging to 143 families, mostly native of Ecuador, 31 of which are endemic. This represents about a 15% of the total flora of Loja. However introduced species such as Matricaria recutita and Eucalyptus globulus are among the most cited. Medicinal plants (509 species) were the most abundant, and are used primarily to treat so‐called "internal inflammation" (210 species, with Matricaria recutita and Melissa officinalis as the most valued) and disorders of the digestive system (188, Matricaria recutita and Mentha pulegium). We recorded 169 species sold in markets, most of them (65) employed to elaborate a typical drink called horchata and to heal "internal inflammation" (36). In addition 414 are used for other purposes: 161 for firewood, 142 for human consumption, 133 for construction and furniture, 98 for handicrafts, 89 as ornamental, 27 for dye and 72 for other uses. In the forest remnants, 19,156 individuals corresponding to 1440 species of 153 families, 437 of which are used by the population were inventoried. The average species richness (both total and of useful plants) was significantly higher in exploited remnants, which suggest that exploitation does not adversely affect species diversity and instead increases habitat diversity. We also found that the proportion of useful species decreases in decreased in the most diverse remnants. It was also observed that the probability of use of a species increases with its frequency and abundance and decreases as the distance form localities to remnants where it appears increases. This general pattern however does not hold for medicinal and ornamental species. These results show the extraordinary wealth of traditional knowledge and the great vitality in the use of many species and support the hypothesis that most accessible and abundant plant resources are the most used by people.

Respuestas anatómico fisiológicas frente a estrés hídrico en plantaciones de especies de crecimiento rápido para la producción de biomasa

El objetivo general de la presente Tesis es identificar algunas de las características anatómico-fisiológicas que confieren la capacidad de alcanzar una mejor productividad bajo clima mediterráneo a plantas de diversos genotipos de los géneros Populus y Eucalyptus, caracterizados por su carácter pionero, elevado crecimiento y vulnerabilidad a la cavitación. En los dos primeros capítulos se hace un seguimiento de la conductancia estomática a una selección de clones de eucalipto cultivados en invernadero, sometidos a diferentes dotaciones hídricas. Se realizaron además mediciones periódicas del pH de la savia del tallo y de la pérdida de conductividad hidráulica para investigar su implicación en la regulación química e hidraúlica del cierre estomático. Las variaciones en el pH de la savia obtenidas parecen responder a cambios en el déficit de presión de vapor de agua atmosférico y no a diferencias en la disponibilidad de agua en el suelo. La conductancia estomática presentó una correlación positiva significativa con el pH de la savia, pero no con la conductividad hidráulica. La variabilidad de la conductividad hidráulica máxima se discute a la luz de recientes investigaciones sobre los materiales constituyentes de las membranas de las punteaduras. Los clones que mostraron mayores conductancias hidráulicas y estomáticas presentaron valores más altos de producción y supervivencia, poniendo de manifiesto la utilidad del estudio de estas variables. Por el contrario, los valores más bajos de conductancia estomática e hidraúlica se encontraron en clones que han resultado un fracaso en plantaciones comerciales, en particular, fue destacable el mal resultado de un clon procedente de autocruzamiento respecto de otros híbridos. En el tercer capítulo de la tesis se estudian características anatómicas y funcionales del xilema relacionadas con la eficiencia en el transporte de agua a las hojas, y que pueden afectar directa o indirectamente a la transpiración y al crecimiento. Los estudios anatómicos fueron realizados sobre brotes anuales de chopo en una plantación situada en Granada, en condiciones de riego limitante. La combinación de rasgos anatómicos más favorable de cara a la producción de biomasa fue una densidad alta de vasos de diámetro intermedio. Los clones más productivos figuraron entre los más resistentes a la cavitación. Para estudiar el crecimiento de masas arboladas se utilizan frecuentemente parámetros fisiológicos como el índice de area foliar (LAI). La estimación del LAI a partir de fotografías hemisféricas aplicada a tallares de chopo plantados a alta densidad y a turno corto para biomasa se lleva a cabo mediante una metodología reciente empleada y discutida en el cuarto capítulo de la Tesis. Los resultados muestran que las diferencias de producción existentes entre genotipos, localidades de medición con diferentes dosis de riego, y años, pueden predecirse a partir de la determinación del índice de área foliar tanto por métodos directos como indirectos de estimación. Tanto los estudios realizados en eucalipto como en chopo han mostrado que los genotipos con menores producciones de biomasa en campo alcanzaron los menores valores de conductancia estomática en las condiciones más favorables así como el menor número de vasos en el xilema. La estrecha relación entre crecimiento y LAI confirma una vez más la importancia del desarrollo de la copa para sostener un buen crecimiento. El mayor desarrollo de la copa y rendimiento en biomasa se midieron en uno de los clones con un número de vasos más elevado, y menor vulnerabilidad a la cavitación en condiciones de estrés. Estos resultados ponen de manifiesto la importancia de las características anatómicas y funcionales del xilema como condicionantes del patrón de crecimiento de las plantas y el comportamiento de los estomas. ABSTRACT A number of anatomical xylem traits and physiological variables were analyzed in genotypes of both the Populus and Eucalyptus genera with the main aim of identifying traits in the genotypes which confer the ability to produce an acceptable biomass yield under Mediterranean climatic conditions. In the first two chapters of this PhD, the results of two experiments carried out on several clones of the species Eucalyptus globulus Labill. are presented. Chapters three and four include the results of another two trials on four poplar hybrid genotypes. One of the initial plant responses to water stress is stomatal closure, which can be triggered by hydraulic and/or chemical signals. The two first chapters of this PhD deal with trials in which stomatal conductance and percentage loss of hydraulic conductivity were monitored on a set of eucalyptus clones supplied by ENCE (former National Cellulose Company) and currently used in the company’s own commercial plantings. The experimental trials were carried out in greenhouses and the plants were submitted to two different watering regimes. The pH of the stem sap was periodically measured as the greenhouse temperature and humidity changed. The aim of these measurements was to investigate the role of both sap pH and percentage loss of hydraulic conductivity on stomatal regulation. The results obtained suggest that changes in sap pH are a response to vapor pressure deficit changes rather than to differences in soil water availability. We found significant correlation between stomatal conductance and sap pH, although no significant relationship was found between stomatal conductance and hydraulic conductivity. Variability in maximum hydraulic conductivity is discussed based on recent pit membrane constituent research. The study of hydraulic conductivity proved helpful in order to detect the clones with both higher growth and greater chance of survival, since clones displaying the lowest hydraulic conductivities were those that failed in commercial plantings. Anatomical xylem traits define the water transport efficiency to leaves and can therefore limit transpiration and growth. The third chapter of this PhD addresses anatomical xylem traits in poplar. One year old stem samples were taken from a water-stressed trial in Granada. The anatomical xylem study proved useful for detecting the lowest yielding genotypes. Clones with intermediate vessel size and high vessel densities were found to be those with the highest biomass yield. Differences in cavitation resistance depending on the clone tested and the water treatment applied were also found. The clones with the highest biomass yield were found to be among the most cavitation resistant clones in each watering regime. Xylem and physiological traits along with stomatal behavior are useful tools to determine plant growth. In order to study plantings or forests, it is more common to employ other physiological variables such as leaf area index (LAI). LAI estimation from hemispherical photographs applied to short rotation woody crops is a recently developed method that still requires fine tuning through further investigation. In the fourth chapter, data from LAI monitoring over two consecutive years were analyzed in two different locations where different irrigation treatments were applied. The results showed that differences in yield between genotypes, different irrigation regimes and years could be predicted by using the LAI estimates, either through direct or indirect estimation methods. Our studies of poplar and eucalyptus have shown that the field-grown genotypes with the lowest biomass yield displayed the lowest values of stomatal conductance under the most favorable environmental conditions and also had a low number of xylem conduits. The close relationship between LAI and growth highlights the importance of crown development in biomass growth. The highest LAI and biomass yield were recorded in one of the clones with higher vessel density and the lowest vulnerability to cavitation under stress conditions. These results underline the importance of research into anatomical and functional traits as factors influencing plant growth patterns and stomatal behavior.

Metabolomics demonstrates divergent responses of two Eucalyptus species to water stress

Past studies of water stress in Eucalyptus spp. generally highlighted the role of fewer than five “important” metabolites, whereas recent metabolomic studies on other genera have shown tens of compounds are affected. There are currently no metabolite profiling data for responses of stress-tolerant species to water stress. We used GC–MS metabolite profiling to examine the response of leaf metabolites to a long (2 month) and severe (Ψpredawn < −2 MPa) water stress in two species of the perennial tree genus Eucalyptus (the mesic Eucalyptus pauciflora and the semi-arid Eucalyptus dumosa). Polar metabolites in leaves were analysed by GC–MS and inorganic ions by capillary electrophoresis. Pressure–volume curves and metabolite measurements showed that water stress led to more negative osmotic potential and increased total osmotically active solutes in leaves of both species. Water stress affected around 30–40% of measured metabolites in E. dumosa and 10–15% in E. pauciflora. There were many metabolites that were affected in E. dumosa but not E. pauciflora, and some that had opposite responses in the two species. For example, in E. dumosa there were increases in five acyclic sugar alcohols and four low-abundance carbohydrates that were unaffected by water stress in E. pauciflora. Re-watering increased osmotic potential and decreased total osmotically active solutes in E. pauciflora, whereas in E. dumosa re-watering led to further decreases in osmotic potential and increases in total osmotically active solutes. This experiment has added several extra dimensions to previous targeted analyses of water stress responses in Eucalyptus, and highlights that even species that are closely related (e.g. congeners) may respond differently to water stress and re-watering