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- Field performance of seedlings and microproppagated plants of carob treePublication . Osório, Maria Leonor; Osório, Júlio; David, Maria Manuela; Romano, AnabelaMicropropagated plantlets of cv. Mulata and cv. Galhosa growing in pots, after acclimatization in the glasshouse and growth for several months under natural conditions, were transferred to a field trial. Carob seedlings of ‘Mulata’ were also transferred under the same conditions. The field trial has been established with 100% of success in micropropagated plants and 97% in seedlings. Three months after transfer to the field, plants showed good growth but micropropagated plants of ‘Mulata’ exhibited more vigour than ‘Galhosa’ and than seedlings, with the greatest number of branches and a larger length increase in the main stem. Before transference to the field, net photosynthetic rates (PN), water use efficiency (PN/gs) and quantum yield of PSII (fPSII) of the potted plants were higher in seedlings than in micropropagated plants. Three months after transplantation all gas exchange parameters were improved for all types of plants, particularly net photosynthetic rate, and no significant differences were observed between plants either micropropagated or seedlings. On the other hand, fPSII decreased significantly in seedlings. The results suggest that the pre-acclimation, in pots, to external environmental conditions might have improved field survival, plant vigour and enhanced the endurance to adverse conditions of micropropagated carob trees.
- 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.
- Effects of CO2 concentration on acclimatization and physiological responses of two cultivars of carob treePublication . Osório, Maria Leonor; Gonçalves, Sandra; Osório, Júlio; Romano, AnabelaThis study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 μmol mol-1) or elevated (EC; 810 μmol mol-1) CO2 concentration and a photosynthetic photon flux density of 125 μmol m-2 s-1. CO2 enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO2 treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.
- 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.
- The effects of drought on net CO2 uptake, chlorophyll fluorescence and photosynthetic pigments in mature and young leaves of sunflowerPublication . Osório, Maria Leonor; Barrote, Isabel; Osório, Júlio; David, Maria Manuela; Correia, Maria JoãoPhotosynthesis, photosystem II activity and photosynthetic pigments were analysed in mature and Young leaves of sunflower (Helianthus annuus L.) during dehydration and rehydration, in order to determine the influence of leaf age on the sensitivity to drought. Dehydration caused a more pronounced inhibition in photosynthetic rates in mature leaves compared to younger leaves, but 24 h after rewatering the recovery was complete in spite of leaf age. After exposure to drought, mature leaves showed a larger reduction in the actual PSII photochemical efficiency and a higher increase in nonradiative energy dissipation than young leaves. In well-watered plants the de-epoxidation level represented by the ratio A+Z/V+A+Z was higher in young leaves. However drought had opposite effects on both type of leaves: in young leaves A+Z/V+A+Z decreased with water deficit, while in mature leaves increased with soil drying. The results obtained suggest that leaf age influence the “strategy” to avoid irreversible damage of PSII activity under drought and that older leaves are more drought-sensitive than younger leaves of sunflower.
- Effects of water deficit on the activity of nitrate reductase and content of sugars, nitrate and free amino acids in the leaves and roots of sunflower and white lupin plants growing under two nutrient supply regimesPublication . Correia, Maria João; Fonseca, Filomena; Azedo-Silva, João; Dias, Celeste; David, Maria Manuela; Barrote, Isabel; Osório, Maria Leonor; Osório, JúlioThe effects of a two weeks soil drying period on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied on Helianthus annuus L. and non-nodulated Lupinus albus L. plants, growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts, by measuring the activity of the unphosphorylated active form (NRact), the maximal extractable activity (NRmax) and the activation state. To get insight into potential signalling compounds, nitrate, amino acids and soluble sugars concentrations were also quantified. On both species, foliar NRact and NRmax were negatively affected by soil drying and reduced supply of nutrients, the observed changes in NR activity being linearly-correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots NRmax was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well-watered sunflower roots only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80% or even higher. At the leaf level, NR activation state was hardly changed in response to soil drying. The contribution of changes in the concentrations of soluble 5 sugars and amino acids to explain the observed variations in NR activity are discussed.
- Light-dependent photosynthetic characteristics indicated by chlorophyll fluorescence in micropropagated plants of Rhododendron ponticum subsp baeticum during acclimationPublication . Osório, Maria Leonor; Osório, Júlio; Romano, AnabelaMicropropagated plants of Rhododendron ponticum subsp. baeticum after acclimatization were grown in natural conditions at three levels of irradiance (HL = 1000, IL = 300 and LL = 80 mu mol m(-2) s(-1) using permanent shading. Light acclimation of photosynthesis was characterized using chlorophyll a fluorescence of intact leaves. There was a clear association between light acclimation of photosynthetic performance and growth irradiance. In general, the saturated electron transport rate, ETR, was higher for high (HL) and intermediate light (IL) than for low-light (LL) grown plants. The response of q(p), which represents PSII openness to incident light, indicates a ranking of tolerant capacity to photoinhibition of HL>IL>LL. The light acclimation of the non-radiative dissipation of excess energy in the antenna, q(N), was not very clear despite the highest value shown by HL plants. A clear feature of the light response of qp and qN is the gradual replacement of qp by qN with increasing light. However, this photoprotective strategy in LL plants seems to be less efficient than in HL plants. Moreover, after an exposition to strong light environment, the maximal photochemical efficiency of PSII (F-v/F-m) did not recover after 24 hours in IL and LL plants. This great plasticity of photosynthesis in R. ponticum plants indicates that the high light regimes during acclimation in the field can beneficiate the plant growth without photoinhibition effects.
- 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.