Lutein/ß-carotene ratio in extra virgin olive oil: An easy and rapid quantification method by Raman spectroscopy.

Carotenoids play an important role in the stability, freshness, and nutritional value of extra-virgin olive oil (EVOO). However, the carotenoid content in EVOO changes over time as a function of olive ripening and degrading events. A reliable quality marker is the ratio between the two most abundant carotenoids, namely lutein and ß-carotene, since the second degrades more rapidly. Thus, to obtain a fast quantification of the lutein/ß-carotene ratio in olive oil could deserve a certain interest. Resonant Raman spectroscopy is a rapid and non-destructive technique, widely applied for food chemical characterization. In this work, using high-performance liquid chromatography and UV-vis absorption spectroscopy as calibration techniques, we present a reliable method to assess the lutein/ß-carotene ratio in EVOO using a single Raman spectrum. The novel approach deserves several methodological and applicative interests, since it would allow rapid, on-site screening of EVOO quality and authenticity, especially if implemented as a portable system.

Consequences of Grazing Cessation for Soil Environment and Vegetation in a Subalpine Grassland Ecosystem.

Areas covered by seminatural grasslands have been in constant decline for decades in Europe. This trend is particularly strong for mountain territories, where such traditional agricultural practices as cattle grazing are no longer economically feasible. This study was conducted in the subalpine pasture of Cinte Tesino (TN, Italy), where local farmers have applied the following different management strategies: shorter and longer grazing durations during the season and a complete abandonment for the last 15 years. We aimed to study how these different management strategies impact the functioning and diversity of vegetation and the chemical and biological characteristics of the soil. Species richness was higher in plots subjected to longer grazing with a prevalence of D. caespitosa in terms of biomass share. A decline in species richness in abandoned plots was accompanied by an increase in the share of other graminoids in collected biomass. A concomitant increase in leaf N concentration and light availability in grazed plots resulted in higher photosynthetic efficiency in some species, as revealed by the δ13C of plant tissues. Soils under grazing were characterised by a higher concentration of total and extractable N, almost doubled microbial biomass C and increased extracellular enzymes activity, evidencing nutrient cycling mobilization. While the microbial pool was characterised by lower mineralization rates, C was lost from the soil with 15 years of abandonment. The longer grazing season demonstrated to be the most beneficial, promoting species richness, C accumulation and better soil microbial functioning. A change in soil pH from strongly acidic to moderately acidic with longer grazing is likely one of the important factors adding to the success in the functioning of primary producers and decomposers in this site.

Carbon isotope discrimination and water use efficiency in interspecific Prunus hybrids subjected to drought stress.

In C3 plants, carbon isotope composition (δ13C) is influenced by isotopic effects during diffusion from the atmosphere to the chloroplasts and carboxylation reactions. This work aimed to demonstrate if δ13C of leaf soluble carbohydrates (δ13Cleaves) and of dry matter from new-growth shoots (δ13Cshoots) of Prunus plants subjected to a period of water deficit was related to water use efficiency (WUE). For this purpose, three interspecific Prunus hybrids rootstocks (6-5, 7-7 and G × N) were gradually subjected to drought and then rewatered. Soil water content (SWC) decreased from 26.1 to 9.4% after 70 days of water shortage, when plants reached values of predawn leaf water potential (LWP) ranging from -3.12 to -4.00 MPa. Gas exchange, particularly net photosynthetic and transpiration rates, differed among the three hybrids, leading to different values of WUE. After 70 days of drought, a significant δ13C increase of 5.86, 4.28 and 4.99‰ was observed in 6-5, 7-7 and G × N, respectively. Significant correlations between δ13C and other parameters (substomatal CO2/atmospheric CO2 ratio, stomatal conductance and stem water potential) were found in all hybrids. The rewatering phase caused a recovery of the physiological status of the plants. The isotope composition of δ13Cshoots was correlated with the average WUE measured during the whole experiment. δ13Cleaves and δ13Cshoots were positively related (r = 0.87; p < 0.001). The isotopic signature was a reliable screening tool to identify Prunus genotypes tolerant to drought stress. The results suggest the possibility of using δ13C as an integrated indicator of level of drought stress in plants subjected to prolonged stress conditions.

Carbon allocation strategies and water uptake in young grafted and own-rooted hazelnut (Corylus avellana L.) cultivars.

In this study, grafted and own-rooted young hazelnut plants of three high-quality cultivars were cultivated in Central Italy to investigate possible differences in growth, fruit and flower production, and physiological processes encompassing water uptake, photosynthetic variables and non-structural carbohydrate allocation. Stable isotopes and photosynthetic measurements were used to study carbon and water fluxes in plants. For the first time, an ecophysiological study was carried out to understand the seasonal growth dynamics of grafted plants in comparison with own-rooted plants. The own-rooted hazelnuts showed rapid above-ground development with large canopy volume, high amount of sprouts and earlier yield. The grafted plants showed greater below-ground development with lower canopy volumes and lower yield. However, later, the higher growth rates of the canopy led these plants to achieve the same size as that of the own-rooted hazelnuts and to enter the fruit production phase. Different seasonal behaviour in root water uptake and leaf photosynthesis-related variables was detected between the two types of plants. The grafted plants showed root development that allowed deeper water uptake than that of the own-rooted hazelnuts. Moreover, the grafted plants were characterized by a higher accumulation of carbohydrate reserves in their root tissues and by higher stomatal reactivity, determining significant plasticity in response to seasonal thermal variations.

Effects of rain shortage on carbon allocation, pools and fluxes in a Mediterranean shrub ecosystem - a 13C labelling field study.

Hydrological cycle is expected to become the primary cause of ecosystem's degradation in near future under changing climate. Rain manipulation experiments under field conditions provide accurate picture on the responses of biotic processes to changed water availability for plants. A field experiment, mimicking expected changes in rain patterns, was established in a Mediterranean shrub community at Porto Conte, Italy, in 2001. In November 2011 Cistus monspeliensis, one of the dominating shrub species in the Mediterranean basin, was 13C labelled on plots subjected to extended rain shortage period and on control non manipulated plots. Carbon (C) allocation was traced by 13C dynamics in shoots, shoot-respired CO2, roots, microbial biomass, K2SO4-extractable C and CO2 respired from soil. Most of the recovered 13C (60%) was respired by shoots within 2weeks in control plots. In rain shortage treatment, 13C remained incorporated in aboveground plant parts. Residence time of 13C in leaves was longer under the rain shortage because less 13C was lost by shoot respiration and because 13C was re-allocated to leaves from woody tissues. The belowground C sink was weak (3-4% of recovered 13C) and independent on rain manipulation. Extended rain shortage promoted C exudation into rhizosphere soil in expense of roots. Together with lowered photosynthesis, this "save" economy of new C metabolites reduces the growing season under rain shortage resulting in decrease of shrub cover and C losses from the system on the long-term.

Carbon and nitrogen allocation strategy in Posidonia oceanica is altered by seawater acidification.

Rising atmospheric CO2 causes ocean acidification that represents one of the major ecological threats for marine biota. We tested the hypothesis that long-term exposure to increased CO2 level and acidification in a natural CO2 vent system alters carbon (C) and nitrogen (N) metabolism in Posidonia oceanica L. (Delile), affecting its resilience, or capability to restore the physiological homeostasis, and the nutritional quality of organic matter available for grazers. Seawater acidification decreased the C to N ratio in P. oceanica tissues and increased grazing rate, shoot density, leaf proteins and asparagine accumulation in rhizomes, while the maximum photochemical efficiency of photosystem II was unaffected. The 13C-dilution in both structural and non-structural C metabolites in the acidified site indicated quali-quantitative changes of C source and/or increased isotopic fractionation during C uptake and carboxylation associated with the higher CO2 level. The decreased C:N ratio in the acidified site suggests an increased N availability, leading to a greater storage of 15N-enriched compounds in rhizomes. The amount of the more dynamic C storage form, sucrose, decreased in rhizomes of the acidified site in response to the enhanced energy demand due to higher shoot recruitment and N compound synthesis, without affecting starch reserves. The ability to modulate the balance between stable and dynamic C reserves could represent a key ecophysiological mechanism for P. oceanica resilience under environmental perturbation. Finally, alteration in C and N dynamics promoted a positive contribution of this seagrass to the local food web.

Isoscapes of carbon and oxygen stable isotope compositions in tracing authenticity and geographical origin of Italian extra-virgin olive oils.

The authentication and verification of the geographical origin of food commodities are important topics in the food sector. This study shows the spatial variability in δ(13)C and δ(18)O of 387 samples of Italian extra-virgin olive oil (EVOO) collected from 2009 to 2011. EVOOs' δ(13)C and δ(18)O values were related to GIS (Geographic Information System) layers of source water δ(18)O and climate data (mean monthly temperature and precipitation, altitude, xerothermic index) to evaluate the impact of the most significant large-scale drivers for the isotopic composition of Italian EVOOs. A geospatial model of δ(18)O and δ(13)C was developed for the authentication and verification of the geographical origin of EVOOs. The geospatial model identified EVOOs from four distinct areas: north, south-central Tyrrhenian, central Adriatic and islands, highlighting the zonation of the expected isotopic signatures. This geospatial approach can be used to define a protocol for analyzing the isotopic composition of EVOOs in order to certify their origin and prevent food fraud. Limits and perspectives of the model are discussed.

Combining stable isotope and carbohydrate analyses in phloem sap and fine roots to study seasonal changes of source-sink relationships in a Mediterranean beech forest.

Carbon isotope composition (δ(13)C) and carbohydrate content of phloem sap and fine roots were measured in a Mediterranean beech (Fagus sylvatica L.) forest throughout the growing season to study seasonal changes of source-sink relationships. Seasonal variations of δ(13)C and content of phloem sap sugars, collected during the daylight period, reflected the changes in soil and plant water status. The correlation between δ(13)C and content of phloem sap sugars, collected from plants belonging to different social classes, was significantly positive only during the driest month of July. In this month, δ(13)C of phloem sap sugars was inversely related to the increment of trunk radial growth and positively related to δ(13)C of fine roots. We conclude that the relationship between δ(13)C and the amount of phloem sap sugars is affected by a combination of causes, such as sink strength, tree social class, changes in phloem anatomy and transport capacity, and phloem loading of sugars to restore sieve tube turgor following the reduced plant water potential under drought conditions. However, δ(13)C and sugar composition of fine roots suggested that phloem transport of leaf sucrose to this belowground component was not impaired by mild drought and that sucrose was in a large part allocated towards fine roots in July, depending on tree social class. Hence, fine roots could represent a functional carbon sink during the dry seasonal periods, when transport and use of assimilates in other sink tissues are reduced. These results indicate a strict link between above- and belowground processes and highlight a rapid response of this Mediterranean forest to changes in environmental drivers to regulate source-sink relationships and carbon sink capacity.

Comparing integrated stable isotope and eddy covariance estimates of water-use efficiency on a Mediterranean successional sequence.

Water-use efficiency (WUE), thought to be a relevant trait for productivity and adaptation to water-limited environments, was estimated for three different ecosystems on the Mediterranean island of Pianosa: Mediterranean macchia (SMM), transition (S(TR)) and abandoned agricultural (SAA) ecosystems, representing a successional series. Three independent approaches were used to study WUE: eddy covariance measurements, C isotope composition of ecosystem respired CO2, and C isotope discrimination (Δ) of leaf material (dry matter and soluble sugars). Seasonal variations in C-water relations and energy fluxes, compared in S(MM) and in SAA, were primarily dependent on the specific composition of each plant community. WUE of gross primary productivity was higher in SMM than in SAA at the beginning of the dry season. Both structural and fast-turnover leaf material were, on average, more enriched in (13)C in S(MM) than SAA, indicating relatively higher stomatal control and WUE for the long-lived macchia species. This pattern corresponded to (13)C-enriched respired CO2 in SMM compared to the other ecosystems. Conversely, most of the annual herbaceous SAA species (terophytes) showed a drought-escaping strategy, with relatively high stomatal conductance and low WUE. An ecosystem-integrated Δ value was weighted for each ecosystem on the abundance of different life forms, classified according to Raunkiar's system. Agreement was found between ecosystem WUE calculated using eddy covariance and those estimated using integrated Δ approaches. Comparing the isotopic methods, Δ of leaf soluble sugars provided the most reliable proxy for short-term changes in photosynthetic discrimination and associated shifts in integrated canopy-level WUE along the successional series.

Seasonal and inter-annual dynamics of growth, non-structural carbohydrates and C stable isotopes in a Mediterranean beech forest.

Seasonal and inter-annual dynamics of growth, non-structural carbohydrates (NSC) and carbon isotope composition (δ(13)C) of NSC were studied in a beech forest of Central Italy over a 2-year period characterized by different environmental conditions. The net C assimilated by forest trees was mainly used to sustain growth early in the season and to accumulate storage carbohydrates in trunk and root wood in the later part of the season, before leaf shedding. Growth and NSC concentration dynamics were only slightly affected by the reduced soil water content (SWC) during the drier year. Conversely, the carbon isotope analysis on NSC revealed seasonal and inter-annual variations of photosynthetic and post-carboxylation fractionation processes, with a significant increase in δ(13)C of wood and leaf soluble sugars in the drier summer year than in the wetter one. The highly significant correlation between δ(13)C of leaf soluble sugars and SWC suggests a decrease of the canopy C isotope discrimination and, hence, an increased water-use efficiency with decreasing soil water availability. This may be a relevant trait for maintaining an acceptable plant water status and a relatively high C sink capacity during dry seasonal periods. Our results suggest a short- to medium-term homeostatic response of the Collelongo beech stand to variations in water availability and solar radiation, indicating that this Mediterranean forest was able to adjust carbon-water balance in order to prevent C depletion and to sustain plant growth and reserve accumulation during relatively dry seasons.

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Estimating mesophyll conductance to CO2: methodology, potential errors, and recommendations.

The three most commonly used methods for estimating mesophyll conductance (g(m)) are described. They are based on gas exchange measurements either (i) by themselves; (ii) in combination with chlorophyll fluorescence quenching analysis; or (iii) in combination with discrimination against (13)CO(2). To obtain reliable estimates of g(m), the highest possible accuracy of gas exchange is required, particularly when using small leaf chambers. While there may be problems in achieving a high accuracy with leaf chambers that clamp onto a leaf with gaskets, guidelines are provided for making necessary corrections that increase reliability. All methods also rely on models for the calculation of g(m) and are sensitive to variation in the values of the model parameters. The sensitivity to these factors and to measurement error is analysed and ways to obtain the most reliable g(m) values are discussed. Small leaf areas can best be measured using one of the fluorescence methods. When larger leaf areas can be measured in larger chambers, the online isotopic methods are preferred. Using the large CO(2) draw-down provided by big chambers, and the isotopic method, is particularly important when measuring leaves with high g(m) that have a small difference in [CO(2)] between the substomatal cavity and the site of carboxylation in the chloroplast (C(i)-C(c) gradient). However, equipment for the fluorescence methods is more easily accessible. Carbon isotope discrimination can also be measured in recently synthesized carbohydrates, which has its advantages under field conditions when large number of samples must be processed. The curve-fitting method that uses gas exchange measurements only is not preferred and should only be used when no alternative is available. Since all methods have their weaknesses, the use of two methods for the estimation of g(m), which are as independent as possible, is recommended.

Photosynthesis research in Italy: a review.

This historical review was compiled and edited by Giorgio Forti, whereas the other authors of the different sections are listed alphabetically after his name, below the title of the paper; they are also listed in the individual sections. This review deals with the research on photosynthesis performed in several Italian laboratories during the last 50 years; it includes research done, in collaboration, at several international laboratories, particularly USA, UK, Switzerland, Hungary, Germany, France, Finland, Denmark, and Austria. Wherever pertinent, references are provided, especially to other historical papers in Govindjee et al. [Govindjee, Beatty JT, Gest H, Allen JF (eds) (2005) Discoveries in Photosynthesis. Springer, Dordrecht]. This paper covers the physical and chemical events starting with the absorption of a quantum of light by a pigment molecule to the conversion of the radiation energy into the stable chemical forms of the reducing power and of ATP. It describes the work done on the structure, function and regulation of the photosynthetic apparatus in higher plants, unicellular algae and in photosynthetic bacteria. Phenomena such as photoinhibition and the protection from it are also included. Research in biophysics of photosynthesis in Padova (Italy) is discussed by G.M. Giacometti and G. Giacometti (2006).

Precipitation pulse use by an invasive woody legume: the role of soil texture and pulse size.

Plant metabolic activity in arid and semi-arid environments is largely tied to episodic precipitation events or "pulses". The ability of plants to take up and utilize rain pulses during the growing season in these water-limited ecosystems is determined in part by pulse timing, intensity and amount, and by hydrological properties of the soil that translate precipitation into plant-available soil moisture. We assessed the sensitivity of an invasive woody plant, velvet mesquite (Prosopis velutina Woot.), to large (35 mm) and small (10 mm) isotopically labeled irrigation pulses on two contrasting soil textures (sandy-loam vs. loamy-clay) in semi-desert grassland in southeastern Arizona, USA. Predawn leaf water potential (psi(pd)), the isotopic abundance of deuterium in stem water (deltaD), the abundance of 13C in soluble leaf sugar (delta13C), and percent volumetric soil water content (theta(v)) were measured prior to irrigation and repeatedly for 2 weeks following irrigation. Plant water potential and the percent of pulse water present in the stem xylem indicated that although mesquite trees on both coarse- and fine-textured soils quickly responded to the large irrigation pulse, the magnitude and duration of this response substantially differed between soil textures. After reaching a maximum 4 days after the irrigation, the fraction of pulse water in stem xylem decreased more rapidly on the loamy-clay soil than the sandy-loam soil. Similarly, on both soil textures mesquite significantly responded to the 10-mm pulse. However, the magnitude of this response was substantially greater for mesquite on the sandy-loam soil compared to loamy-clay soil. The relationship between psi(pd) and delta13C of leaf-soluble carbohydrates over the pulse period did not differ between plants at the two sites, indicating that differences in photosynthetic response of mesquite trees to the moisture pulses was a function of soil water availability within the rooting zone rather than differences in plant biochemical or physiological constraints. Patterns of resource acquisition by mesquite during the dynamic wetting-drying cycle following rainfall pulses is controlled by a complex interaction between pulse size and soil hydraulic properties. A better understanding of how this interaction affects plant water availability and photosynthetic response is needed to predict how grassland structure and function will respond to climate change.

Constitutive expression of pea Lhcb 1-2 in tobacco affects plant development, morphology and photosynthetic capacity.

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

Comparisons of delta13C of photosynthetic products and ecosystem respiratory CO2 and their responses to seasonal climate variability.

This study investigated the relationship between delta13C of ecosystem components, soluble plant carbohydrates and the isotopic signature of ecosystem respired CO2 (delta13CR) during seasonal changes in soil and atmospheric moisture in a beech (Fagus sylvatica L.) forest in the central Apennine mountains, Italy. Decrease in soil moisture and increase in air vapour pressure deficit during summer correlated with substantial increase in delta13C of leaf and phloem sap soluble sugars. Increases in delta13C of ecosystem respired CO2 were linearly related to increases in phloem sugar delta13C (r2=0.99, P