Carob: A Mediterranean Resource for the Future.

For centuries, the carob tree (Ceratonia siliqua L.) has contributed to the economy of the Mediterranean basin, mainly as food for livestock. Nowadays, the value of the carob tree extends far beyond its traditional uses, encompassing a wide range of industries and applications that take advantage of its unique properties and nutritional benefits. Despite its high industrial demand and European indications, there has been a 65% reduction in the area cultivated throughout the Mediterranean area in the 21st century. Given the threats posed by climate change, including reduced water availability and nutrient-depleted soils, there is a growing need to focus on this crop, which is well placed to cope with unpredictable weather. In this review, we use a bibliographic search approach to emphasise the prioritisation of research needs for effective carob tree exploitation. We found enormous gaps in the scientific knowledge of this under-utilised crop species with fruit pulp and seeds of high industrial value. Insufficient understanding of the biology of the species, as well as inadequate agronomic practices, compromise the quantity and the quality of fruits available to the industry. In addition to industrial applications, carob can also be used in reforestation or restoration programmes, providing a valuable crop while promoting biodiversity conservation and soil restoration. The carbon sequestration potential of the trees should be taken into account as a promising alternative in fighting climate change. This bibliographic search has highlighted clusters with different knowledge gaps that require further research and investment. The carob tree has untapped potential for innovation, economic development, and environmental sustainability.

Ammonium as a driving force of plant diversity and ecosystem functioning: observations based on 5 years' manipulation of N dose and form in a Mediterranean ecosystem.

Enhanced nitrogen (N) availability is one of the main drivers of biodiversity loss and degradation of ecosystem functions. However, in very nutrient-poor ecosystems, enhanced N input can, in the short-term, promote diversity. Mediterranean Basin ecosystems are nutrient-limited biodiversity hotspots, but no information is available on their medium- or long-term responses to enhanced N input. Since 2007, we have been manipulating the form and dose of available N in a Mediterranean Basin maquis in south-western Europe that has low ambient N deposition (<4 kg N ha(-1) yr(-1)) and low soil N content (0.1%). N availability was modified by the addition of 40 kg N ha(-1) yr(-1) as a 1∶1 NH4Cl to (NH4)2SO4 mixture, and 40 and 80 kg N ha(-1) yr(-1) as NH4NO3. Over the following 5 years, the impacts on plant composition and diversity (richness and evenness) and some ecosystem characteristics (soil extractable N and organic matter, aboveground biomass and % of bare soil) were assessed. Plant species richness increased with enhanced N input and was more related to ammonium than to nitrate. Exposure to 40 kg NH4+-N ha(-1) yr(-1) (alone and with nitrate) enhanced plant richness, but did not increase aboveground biomass; soil extractable N even increased under 80 kg NH4NO3-N ha(-1) yr(-1) and the % of bare soil increased under 40 kg NH4+-N ha(-1) yr(-1). The treatment containing less ammonium, 40 kg NH4NO3-N ha(-1) yr(-1), did not enhance plant diversity but promoted aboveground biomass and reduced the % of bare soil. Data suggest that enhanced NHy availability affects the structure of the maquis, which may promote soil erosion and N leakage, whereas enhanced NOx availability leads to biomass accumulation which may increase the fire risk. These observations are relevant for land use management in biodiverse and fragmented ecosystems such as the maquis, especially in conservation areas.

Photosynthesis of Quercus suber is affected by atmospheric NH3 generated by multifunctional agrosystems.

Montados are evergreen oak woodlands dominated by Quercus species, which are considered to be key to biodiversity conservation and ecosystem services. This ecosystem is often used for cattle breeding in most regions of the Iberian Peninsula, which causes plants to receive extra nitrogen as ammonia (NH(3)) through the atmosphere. The effect of this atmospheric NH(3) (NH(3atm)) on ecosystems is still under discussion. This study aimed to evaluate the effects of an NH(3atm) concentration gradient downwind of a cattle barn in a Montado area. Leaves from the selected Quercus suber L. trees along the gradient showed a clear influence of the NH(3) on δ(13)C, as a consequence of a strong limitation on the photosynthetic machinery by a reduction of both stomatal and mesophyll conductance. A detailed study of the impact of NH(3atm) on the photosynthetic performance of Q. suber trees is presented, and new mechanisms by which NH(3) affects photosynthesis at the leaf level are suggested.

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids.

BACKGROUND AND AIMS: Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids. METHODS: Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F(1) hybrid individuals were also studied for comparison. KEY RESULTS: Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F(1) hybrids in which N. cavanillesii acted as a mother. CONCLUSIONS: Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.

Carbon allocation in ectomycorrhizal plants at limited optimal N supply: an attempt aat unraveling conflicting theories.

With regard to mycorrhiza, conflicting theories try to explain how the balance between fungal demand for carbohydrates and the plant's needs for nutrients varies, resulting in conflicting predictions. In order to evaluate current concepts, we investigated some metabolic parameters, which are indicative for plant carbon allocation in response to mycorrhization at limited and optimal N supply. Pinus pinaster seedlings were inoculated with living or dead (control) cultures of Pisolithus tinctorius, supplied with ammonium at 4 (limiting) or 7% d−1 (non-limiting) N relative addition rate (RARN), and followed development for 29 days. Mycorrhizal colonization of roots was quantified by the determination of ergosterol. A series of enzymes (sucrose and trehalose metabolism, anaplerosis) and metabolites (soluble carbohydrate, including trehalose; fructose 2,6 bisphosphate, free amino acids) relevant in the C/N exchange between symbionts, and in the carbon allocation and sink strength within the plant were assayed for 2-day-intervals for up to 14 days, and at 5-day-intervals for the rest of the experiment. The first 10 days reflected the establishment of mycorrhizal interaction, and the carbon allocation to the root was higher in M plants independent of N supply. Following this period, carbon allocation became N-related, higher at low, and lower at high N supply. The belowground C investment of M plants was dependent on N availability, but not on N gain. Finally, increased belowground C allocation was accompanied by a shift from plant to fungal metabolism.

Unraveling cryptic reticulate relationships and the origin of orphan hybrid disjunct populations in Narcissus.

Evolutionary consequences of natural hybridization between species may vary so drastically depending on spatial, genetic, and ecological factors that multiple approaches are required to uncover them. To unravel the evolutionary history of a controversial hybrid (Narcissus x perezlarae), here we use four approaches: DNA sequences from five regions (four organellar, one nuclear), cytological studies (chromosome counts and genome size), crossing experiments, and niche modeling. We conclude that (1) it actually consists of two different hybrid taxa, N.xperezlarae s.s. (N. cavanillesii x N. miniatus) and N.xalentejanus (N. cavanillesii x N. serotinus); (2) both have been formed several times independently, that is, polytopically; (3) N. cavanillesii was the mother progenitor in most hybridization events. We also address the origin of orphan hybrid populations of N.xperezlarae in eastern Spain, hundreds of kilometers away from N. cavanillesii. Although long-distance dispersal of already formed hybrids cannot be completely rejected, extirpation of N. cavanillesii via demographic competition is a more likely explanation. Low-reproductive barriers to fertilization by foreign pollen in N. cavanillesii, molecular footprints of the former presence of this species in the area, active asexual propagation by bulbs in N.xperezlarae, and overlapping ecological niches are consistent with the extirpation scenario.

Intracellular and extracellular ammonium (NH4(+)) uptake and its toxic effects on the aquatic biomonitor Fontinalis antipyretica.

The objective of this work is to validate the use of the aquatic moss Fontinalis antipyretica as biomonitor of NH(4)(+) aquatic pollution. In order to achieve this objective we needed to understand the pattern of uptake of NH(4)(+) by the moss and evaluate the impact of high concentrations on its physiological performance. The cellular location of NH(4)(+) in the moss is crucial for understanding its monitoring capacity. We were able to show that a sequential elution technique, based on the use of NiCl(2) as an efficient displacing agent, allowed the quantification of the cellular location of NH(4)(+). This was done along a concentration gradient and time of exposure. The extracellular and intracellular NH(4)(+) concentrations that caused significant physiological impact in membrane permeability of F. antipyretica were the same that caused significant decreasing in the photosynthetic capacity of the same moss. The former NH(4)(+) concentration thresholds were shown to decrease with increasing exposure time. These results are important since under natural conditions lower concentration of NH(4)(+) are present in waters but for very long periods of time. The importance of applying this knowledge in biomonitoring studies to fulfil the requirements of the Water Framework Directive is discussed.

Nitrogen nutrition and antioxidant metabolism in ammonium-tolerant and -sensitive plants.

Ammonium nutrition is of interest as an alternative to that of using nitrate. However, the former has been reported as stressful to many plant species especially to some important crops, as most abiotic stresses may trigger oxidative imbalances in plants. In this work, we investigate the response of oxidative metabolism of two plant species, spinach (Spinacia oleracea L. cv. Gigante de invierno) and pea (Pisum sativum L. cv. Rondo), which have distinct tolerance to ammonium. Plants were grown in the presence of 1.5 and 3.0 mM N as ammonium and compared with equivalent nitrate nutrition. The antioxidant enzymes and metabolites as well as oxidative damage to proteins were determined. Protein and amino acid contents in both types of plants were also analysed. Ammonium nutrition in sensitive spinach or in the tolerant pea plants does not alter the redox status of ascorbate and glutathione or the phenolic contents, while no clear effect is seen in the antioxidant enzymes. The results showed that the stress originated from applying ammonium as the only N source is not an oxidative stress, independent of the ammonium tolerance of the plant species studied. Moreover, ammonium stress diminishes oxidative damage to proteins in the spinach plants. The data of the protein oxidation together with those from N metabolism highlight the relation between the stress induced by ammonium and an increased protein turnover.

Heterogeneity of soil surface ammonium concentration and other characteristics, related to plant specific variability in a Mediterranean-type ecosystem.

Heterogeneity and dynamics of eight soil surface characteristics essential for plants--ammonium and nitrate concentrations, water content, temperature, pH, organic matter, nitrification and ammonification rates--were studied in a Mediterranean-type ecosystem on four occasions over a year. Soil properties varied seasonally and were influenced by plant species. Nitrate and ammonium were present in the soil at similar concentrations throughout the year. The positive correlation between them at the time of greatest plant development indicates that ammonium is a readily available nitrogen source in Mediterranean-type ecosystems. The results presented here suggest that plant cover significantly affects soil surface characteristics.

Enzymatic evidence for the key role of arginine in nitrogen translocation by arbuscular mycorrhizal fungi.

Key enzymes of the urea cycle and (15)N-labeling patterns of arginine (Arg) were measured to elucidate the involvement of Arg in nitrogen translocation by arbuscular mycorrhizal (AM) fungi. Mycorrhiza was established between transformed carrot (Daucus carota) roots and Glomus intraradices in two-compartment petri dishes and three ammonium levels were supplied to the compartment containing the extraradical mycelium (ERM), but no roots. Time courses of specific enzyme activity were obtained for glutamine synthetase, argininosuccinate synthetase, arginase, and urease in the ERM and AM roots. (15)NH(4)(+) was used to follow the dynamics of nitrogen incorporation into and turnover of Arg. Both the absence of external nitrogen and the presence of L-norvaline, an inhibitor of Arg synthesis, prevented the synthesis of Arg in the ERM and resulted in decreased activity of arginase and urease in the AM root. The catabolic activity of the urea cycle in the roots therefore depends on Arg translocation from the ERM. (15)N labeling of Arg in the ERM was very fast and analysis of its time course and isotopomer pattern allowed estimation of the translocation rate of Arg along the mycelium as 0.13 microg Arg mg(-1) fresh weight h(-1). The results highlight the synchronization of the spatially separated reactions involved in the anabolic and catabolic arms of the urea cycle. This synchronization is a prerequisite for Arg to be a key component in nitrogen translocation in the AM mycelium.

Revisiting the plant hyperaccumulation criteria to rare plants and earth abundant elements.

The several established criteria to define a hyperaccumulator plant were applied to a rare and endangered species, Plantago almogravensis, and to the 3rd most abundant element in the earth crust, Al. Using the most common criteria, P. almogravensis undoubtedly is an Al hyperaccumulator plant. If the recent proposed requirements were considered, most of them matching those for a plant to be used in phytoextraction, it can only be considered an unusual accumulator of Al. A discussion is made concerning the several criteria of a hyperaccumulator plant in order to include rare and endemic ones and abundant elements. In ecological terms, the enrichment in Al and Fe observed may account for the differences in the vegetation pattern. Due to the rarity and endangered nature of this plant, the contribution of this work is also relevant for the ecological understanding and the development of conservation options of this endemic species.

Nitrogen uptake in relation to excess supply and its effects on the lichens Evernia prunastri (L.) Ach and Xanthoria parietina (L.) Th. Fr.

The aim of this study was to compare the physiological responses to increased nitrogen (N) supply between the nitrophytic lichen Xanthoria parietina (L.) Th. Fr. and the acidophytic lichen Evernia prunastri (L.) Ach. The two lichens were exposed to a weekly dosage of 0.05, 0.1, 0.2, 0.6 or 2.4 g N m(-2) for 2 months, administered as NH(4)NO(3) dissolved in artificial rainwater (1 l m(-2)). After the treatments, in vivo chlorophyll a fluorescence was determined to assess vitality; concentrations of total N, ammonium, nitrate and dominant amino acids, including glutamate, glutamine and arginine, were quantified in order to follow changes in N status; and the polyols ribitol, arabitol and mannitol were quantified to follow changes in the lichens' carbon (C) status. The uptake of N was quantified by labelling the fertiliser with (15)N in the ammonium position; chlorophyll a was used as an indirect marker for algal activity, and ergosterol as an indirect marker of fungal activity. Nitrogen uptake was higher in E. prunastri than in X. parietina, although the latter species may have used the mannitol reserves to obtain C skeletons and energy for N assimilation. Chlorophyll a and ergosterol concentrations remained unaltered in X. parietina irrespective of N dosage while ergosterol decreased with increasing N uptake in E. prunastri. The latter species had accumulated a large pool of ammonium at the highest N dosage, whilst in X. parietina a significant nitrate pool was instead observed. Taken together, these short-term responses to high N supply observed in the two lichens, and the differences between them, can partly explain the higher tolerance of X. parietina towards increased atmospheric N levels.

Functional aspects of root architecture and mycorrhizal inoculation with respect to nutrient uptake capacity.

The aim of this research was to investigate the effect of arbuscular mycorrhizal (AM) colonisation on root morphology and nitrogen uptake capacity of carob (Ceratonia siliquaL.) under high and low nutrient conditions. The experimental design was a factorial arrangement of presence/absence of mycorrhizal fungus inoculation (Glomus intraradices) and high/low nutrient status. Percent AM colonisation, nitrate and ammonium uptake capacity, and nitrogen and phosphorus contents were determined in 3-month-old seedlings. Grayscale and colour images were used to study root morphology and topology, and to assess the relation between root pigmentation and physiological activities. AM colonisation lead to a higher allocation of biomass to white and yellow parts of the root. Inorganic nitrogen uptake capacity per unit root length and nitrogen content were greatest in AM colonised plants grown under low nutrient conditions. A better match was found between plant nitrogen content and biomass accumulation, than between plant phosphorus content and biomass accumulation. It is suggested that the increase in nutrient uptake capacity of AM colonised roots is dependent both on changes in root morphology and physiological uptake potential. This study contributes to an understanding of the role of AM fungi and root morphology in plant nutrient uptake and shows that AM colonisation improves the nitrogen nutrition of plants, mainly when growing at low levels of nutrients.

Nitrogen use efficiency by a slow-growing species as affected by CO2 levels, root temperature, N source and availability.

This study examines the importance of N source and concentration on plant response to distinct CO2 concentrations and root temperatures. The experimental design of this work was a factorial combination of: CO2 concentration, nitrogen concentration, nitrogen source and root temperature. Carob (Ceratonia siliqua L.) was assessed as a potential model of a slow growing Mediterranean species. The results showed that: 1) biomass increment under high CO2 varied between 13 and 100% in relation to plants grown under the same conditions but at ambient CO2 concentrations, depending on the root temperature and nitrogen source; 2) nitrate-fed plants attained a larger increase in biomass production compared to ammonium-fed ones. This performance seems to be linked to the co-ordinated regulation of the activities of glutamine synthetase and sucrose phosphate synthase. The variations in the magnitude and nature of growth responses to elevated CO2 observed resulted in substantial changes in the chemical composition of the plant material and consequently in plant nitrogen use efficiency. Although performed with seedlings and under controlled conditions, this work emphasizes the importance of the nitrogen source used by the plants, a factor rarely taken into consideration when forecasting plant responses to global changes. Particularly, the results presented here, highlight the potential for uncoupling biomass accumulation from increment of air CO2 concentration and show that more than nitrogen availability N source may offset positive plant growth responses under elevated CO2 and root temperature.