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- Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plantsPublication . Osório, Maria Leonor; Gonçalves, Sandra; Coelho, N.; Osório, Júlio; Romano, AnabelaTuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (g(s)) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (P-N) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (F-v/F-m) remained constant at 0.78-0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (I center dot(PSII)) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (I center dot(NO)) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H2O2 levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.
- Chlorophyll fluorescence in micropropagated Rhododendron ponticum subsp baeticum plants in response to different irradiancesPublication . Osório, Maria Leonor; Osório, Júlio; Romano, AnabelaThe aim of this study was to investigate acclimation of micropropagated plants of Rhododendron ponticum subsp. baeticum to different irradiances and recovery after exposure to high irradiance. Plants grown under high (HL) or intermediate (IL) irradiances displayed higher values of maximum electron transport rate (ETR(max)) and light saturation coefficient (E(k)) than plants grown under low irradiance (LL). The capacity of tolerance to photoinhibition (as assessed by the response of photochemical quenching, q(p)) varied as follows: HL > IL > LL. Thermal energy dissipation (q(N)) was also affected by growth irradiance, with higher saturating values being observed in HL plants. Light-response curves suggested a gradual replacement of q(p) by q(N) with increasing irradiance. Following exposure to irradiance higher than 1500 mu mol m(-2) s(-1), a prolonged reduction of the maximal photochemical efficiency of PS 2 (F(v)/F(m)) was observed in LL plants, indicating the occurrence of chronic photoinhibition. In contrary, the decrease in F(v)/F(m) was quickly reverted in HL plants, pointing to a reversible photoinhibition.
- Photosynthesis, energy partitioning, and metabolic adjustments of the endangered Cistaceae species Tuberaria major under high temperature and droughtPublication . Osório, Maria Leonor; Osório, Júlio; Romano, AnabelaIn view of predicted climatic changes for the Mediterranean region, study of high temperature and drought impacts on physiological responses of endangered species regains relevance. In this context, micropropagated plants of Tuberaria major, a critically endangered species, endemic of Algarve, were transferred to a controlled-environment cabinet with day/night temperatures set at 25/18A degrees C (Reference) or 32/21A degrees C (HT). After 15 days of HT acclimation, some plants were subjected to progressive drought followed by rewatering. The enhancement of temperature alone did not affect water relations and photosynthetic rates (P (N)) but the stomatal conductance (g (s)) exhibited a 3-fold increase in comparison with reference plants. The maximum quantum yield of photosystem (PS) II (F-v/F-m), the effective quantum yield of PSII photochemistry (I broken vertical bar(PSII)), carotenoid (Car) and anthocyanin content enhanced, whereas the quantum yields of regulated (I broken vertical bar(NPQ)) and nonregulated (I broken vertical bar(NO)) energy dissipation decreased. Drought combined with HT reduced predawn leaf water potential to values of about -1.3 MPa, which had adverse effects on gas exchange and PSII activity. Values of P (N) and g (s) were 71 and 79% lower than those of HT plants. An impairment of photochemical activity was also observed: the decrease in I broken vertical bar(PSII) and the increase of I broken vertical bar(NPQ). However, an irreversible photoinhibitory damage had not occurred. Carotenoid and anthocyanin content remained elevated and soluble sugars (SS) increased twice, whereas proline and MDA accumulation was not detected. On the first 24 h after water-stress relief, g (s), P (N), I broken vertical bar(PSII), and I broken vertical bar(NPQ) did not recover, but SS returned to the reference level. Overall, T. major acquired an adequate capacity for a protection against the development of oxidative stress during drought and water recovery under HT. These findings suggest that T. major is prepared to deal with predicted climate changes.
- How to monitor the acclimatization of micropropagated plants - From in vitro to the field?Publication . Osório, Maria Leonor; Gonçalves, Sandra; Coelho, N.; Romano, Anabela; Osório, JúlioThe high mortality experienced by plants during ex vitro transplantation stage is the major bottleneck in large scale application of micropropagation. The abnormal physiological and anatomical characteristics of micropropagated plantlets require that they should be gradually acclimatized to the environment of the greenhouse or field. Environmental factors, especially relative humidity and irradiance, play a critical role in both physiological and biochemical functions, namely water relations, gas exchange, photosynthetic efficiency and water oxidation, during transition from in vitro to ex vitro conditions. Throughout this period of transition and acclimatization substantial changes in those characteristics are necessary to achieve a successful micropropagation. This adaptation should be accurately monitored using relevant physiological parameters including pigment content, chlorophyll a fluorescence imaging, net photosynthetic rate, transpiration rate, and stomatal conductance. Growth traits and oxidative stress markers, such as electrolyte leakage, lipid peroxidation and hydrogen peroxide content, should be also analyzed. These parameters must be assessed during the course of ex vitro acclimatization in several periods and compared with those of plants growing in their natural habitat. The performance plants of Tuberaria major, an endangered species endemic from the Algarve region (Portugal), during their acclimatization through indoor-to-outdoor conditions was monitored using several physiological and biochemical traits as indicators. In view of the results, we concluded that the transplantation protocol described can be used for restoration purposes, contributing to the preservation of the species. © ISHS 2013.
- Reflectance indices as nondestructive indicators of the physiological status of Ceratonia siliqua seedlings under varying moisture and temperature regimesPublication . Osório, Júlio; Osório, Maria Leonor; Romano, AnabelaWe investigated the use of spectral reflectance techniques to monitor the physiological responses of Ceratonia siliqua L. seedlings exposed to different levels of water availability under normal (25 : 18 degrees C, day : night) and elevated (32 : 21 degrees C, day : night) temperatures. Three spectral reflectance indices (photochemical reflectance index, PRI; water index, WI; red edge position, REP) were measured along with water status, chlorophyll fluorescence and chlorophyll concentration variables in the leaves of well watered, moderately stressed, severely stressed and rehydrated plants under each temperature regime. The PSII effective photochemical efficiency (phi(2)) and the intrinsic efficiency of open PSII centres (F-v'/F-m') correlated significantly with PRI, and these three variables loaded heavily onto the same principal component of a three-factor principal component analysis solution. Water concentration (WC) and the succulence index (SI) were more strongly correlated with WI than either water potential (Psi(PD)) or relative water content (RWC). Accordingly, WI, WC and SI were combined in the second principal component, and Psi(PD) and RWC in the third. Our results provide clear evidence for interaction between water availability and temperature in the WI and the PRI response segments of the reflectance curves. Elevated temperature inhibited the recovery of WI spectral segments more than that of the PRI segments in SS plants. REP showed a strongly positive linear relationship with leaf total chlorophyll concentration across all water and temperature treatment combinations. PRI, WI and REP are therefore reliable markers that can be used to monitor phi(2), WC and total chlorophyll concentration, respectively, in C. siliqua seedlings under drought and temperature stress.
- Differences in Al tolerance between Plantago algarbiensis and P. almogravensis reflect their ability to respond to oxidative stressPublication . Martins, Neusa; Osório, Maria Leonor; Gonçalves, Sandra; Osório, Júlio; Romano, AnabelaWe evaluated the impact of low pH and aluminum (Al) on the leaves and roots of Plantago almogravensis Franco and Plantago algarbiensis Samp., focusing on energy partitioning in photosystem II, H2O2 levels, lipid peroxidation, electrolyte leakage (EL), protein oxidation, total soluble protein content and antioxidant enzyme activities. In both species, Al triggered more changes in oxidative metabolism than low pH alone, particularly in the roots. We found that Al increased the levels of H2O2 in P. algarbiensis roots, but reduced the levels of H2O2 in P. almogravensis leaves and roots. Neither low pH nor Al affected the spatial heterogeneity of chlorophyll fluorescence, the maximum photochemical efficiency of PSII (F-v/F-m), the actual quantum efficiency of PSII (I center dot(PSII)) or the quantum yields of regulated (I center dot(NPQ)) and nonregulated (I center dot(NO)) energy dissipation, and there was no significant change in total soluble protein content and EL. In P. algarbiensis, Al increased the carbonyl content and the activities of superoxide dismutase (SOD) and catalase (CAT) in the roots, and also CAT, ascorbate peroxidase and guaiacol peroxidase activities in the leaves. In P. almogravensis, Al reduced the level of malondialdehyde in the roots as well as SOD activity in the leaves and roots. We found that P. almogravensis plantlets could manage the oxidative stress caused by low pH and Al, whereas the P. algarbiensis antioxidant system was unable to suppress Al toxicity completely, leading to the accumulation of H2O2 and consequential protein oxidation in the roots.
- Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stressPublication . Martins, Neusa; Osório, Maria Leonor; Gonçalves, Sandra; Osório, Júlio; Palma, Tânia; Romano, AnabelaWe investigated the impact of low pH and aluminum (Al) stress on the growth, nutrients concentration, chlorophyll a fluorescence, photosynthetic pigment contents, proline and carbohydrate accumulation in shoots and plantlets (leaves and roots) of Plantago almogravensis and P. algarbiensis. Both species accumulated considerable and similar amounts of Al in their tissues, mainly in the roots. The presence of Al caused a significant reduction on root elongation in P. algarbiensis. Low pH and Al induced significant changes on nutrient accumulation, but no significant alterations on the maximum efficiency of PSII (Fv/Fm), quantum yield of PSII photochemistry (FiPSII), quantum yield of regulated energy dissipation (FiNPQ) and quantum yield of non-regulated energy dissipation (FiNO) were detected in both species in response to these stresses. However, Al increased significantly the nonphotochemical quenching and the hlorophyll b content and decreased the PSII excitation pressure (1 - qp) in P. almogravensis leaves. Both stress treatments induced carbohydrate accumulation in the shoots and roots of this species, but not in leaves. In P. algarbiensis, low pH and Al decreased the photosynthetic pigment contents in the shoots, whereas Al stimulated the carbohydrate accumulation in the leaves. Although our data showed that both species are tolerant to Al3+ and H+, P. almogravensis appeared to be more adapted to maintain cellular physiology and growth under those conditions.
- Influence of enhanced temperature on photosynthesis, photooxidative damage, and antioxidant strategies in Ceratonia siliqua L. seedlings subjected to water deficit and rewateringPublication . Osório, Maria Leonor; Osório, Júlio; Vieira, Ana; Gonçalves, Sandra; Romano, AnabelaPredicted future climatic changes for the Mediterranean region give additional importance to the study of photooxidative stress in local economic species subjected to combined drought and high-temperature conditions. Under this context, the impact of these stresses on photosynthesis, energy partitioning, and membrane lipids, as well as the potential ability to attenuate oxidative damage, were investigated in Ceratonia siliqua L. Two thermal regimes (LT: 25/18ºC; HT: 32/21ºC) and three soil water conditions (control, water stress, and rewetting) were considered. HT exacerbated the adverse effects of water shortage on photosynthetic rates (PN) and PSII function. The decrease in PN was 33% at LT whereas at HT it was 84%. In spite of this, the electron transport rate (ETR) was not affected, which points to an increased allocation of reductants to sinks other than CO2 assimilation. Under LT conditions, water stress had no significant effects on yield of PSII photochemistry (ΦPSII) and yields of regulated (ΦNPQ) and nonregulated (ΦNO) energy dissipation. Conversely, drought induced a significant decrease of ΦPSII and a concomitant increase of ΦNO in HT plants, thereby favouring the overproduction of reactive oxygen species (ROS). Moreover, signs of lipid peroxidation damage were detected in HT plants, in which drought caused an increase of 40% in malondialdehyde (MDA) content. Concurrently, a marked increase in proline content was observed, while the activities of catalase (CAT) and ascorbate peroxidase (APX) were unaffected. Despite the generation of a moderate oxidative stress response, C. siliqua revealed a great capability for photosynthetic recovery 36 h after rewatering, which suggests that the species can cope with predicted climate change.
- Carob trees (Ceratonia siliqua L.) regenerated in vitro can acclimatize successfully to match the field performance of seed-derived plantsPublication . Osório, Maria Leonor; Osório, Júlio; Gonçalves, Sandra; David, Maria Manuela; Romano, Anabela; Correia, Maria JoãoThe use of in vitro regenerated plants in forestry and orchard depends ultimately on the development of efficient transplantation protocols, ensuring high survival rates and successful establishment under field conditions. We tested the performance of micropropagated carob trees (Ceratonia siliqua L.) throughout the acclimatization process in terms of survival, growth and physiological traits, including field comparisons with seed-derived and mother plants. The field trial was 100 %successful, i.e. we found no major differences between micropropagated, seed-derived and mother plants in terms of growth rate, height, number of leaves, photosynthetic efficiency, chlorophyll fluorescence, chlorophyll content and soluble protein content, although these parameters changed significantly during acclimatization. Stomatal conductance (gs) was reduced by fourfold when plants were transferred from in vitro culture to the growth chamber, thus preventing uncontrolled wilting. The photosynthetic rate (PN) was relatively low in vitro, in the growth chamber and the greenhouse, but increased to match seed-derived and mother plants in the field. The chlorophyll a/b ratio in leaves from in vitro and growth chamber plants was typical of shade plants (2.1) but became more characteristic of sun plants in the subsequent acclimatization stages (3.1–3.5). The maximum efficiency of photosystem II (Fv/Fm) remained mostly constant at ~0.80 throughout acclimatization, as is typical for healthy, non-stressed plants. We conclude that our micropropagation and acclimatization protocols provide a suitable alternative to traditional mass propagation techniques for the carob tree.