A range of plant species typical of semi-natural grasslands were tested for their sensitivity to short-term ozone injury under normal and reduced irrigation, and in relationship to air vapour pressure deficit. Potted specimens of 24 herbs, legumes and grasses were exposed during two seasons to four O3 treatments in open-top chambers. The ozone treatments were: (a) charcoal-filtered air; (b) charcoal-filtered air plus ozone to match ambient levels; (c) charcoal-filtered air plus O3 to ambient levels 1.5 and (d) charcoal-filtered air with ozone added to twice ambient levels during selected episodes of 7–13 d. During these ozone episodes, half of the plants in each ozone treatment received reduced irrigation (dry treatment) while the rest was kept under full irrigation (wet treatment). Type and date of first occurrence of leaf injury were noted during individual growth periods. Plants were harvested three times per year, and the percentage of injured leaves was recorded. Depending on species, injury symptoms were expressed as flecking (O3-specific injury), leaf yellowing or anthocyanin formation. Carum carvi and most species of the Fabaceae family (Onobrychis sativa, Trifolium repens, Trifolium pratense) were found to be most responsive to O3, injury occurring after only a few days of exposure in treatment (b). An episodic reduction in irrigation tended to reduce the expression of O3-specific symptoms, but only in species for which a reduction in soil moisture potential and an associated reduction in stomatal conductance during the dry episodes were observed. In other species, the protection from O3 injury seemed to be of little importance. Using artificial neural networks the injury response of nine species was analysed in relation to Species, stomatal conductance, ozone as AOT40 (accumulated exposure above a threshold of 0.04 ppm for periods with global radiation [ges ]50 W m−2 (Fuhrer et al., 1997)), mean relative growth rate, air vapour pressure deficit and global radiation. In the model with all factors, Species was most important, and when Species was omitted, stomatal conductance was the most important determinant for leaf injury to occur, whereas mean relative growth rate was less important. With no plant-related factors included, air vapour pressure deficit and AOT40 were of highest importance. Only in eight species was a positive relationship found between these two factors during the five days before the onset of injury, indicating increasing protection from ozone with increasing air vapour pressure deficit in some but not all species. These data show that across a range of grassland species, leaf injury caused by elevated levels of ozone is most likely to occur in species with high stomatal conductance and that protection from ozone during dry periods is species-specific and depends on a reduction in stomatal conductance due to a decrease in soil moisture potential. Protection under increased vapour pressure deficit can occur in some but not all species, depending on the relationship between stomatal conductance and air vapour pressure deficit.

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis, one of the Rhamnaceae which also includes Ceanothus. The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.

The use of genetics in plant biology aims at the physiological and molecular genetical characterization of the phenotypic variation for the trait under study. Efficient mutant and gene isolation procedures have been developed for a number of plant models such as Arabidopsis thaliana. For this, the map position of the genes and insertion mutagenesis are used. The latter also allows the characterization of genes that are not easily recognized in mutant approaches, by using enhancer or gene-trapping procedures and reverse genetics. In addition to mutants, natural variation present among wild and cultivated varieties of a species provides an important source of genetic variation. The use of molecular markers, advanced mapping populations and specific cytogenetic stocks in case of polyploids, enables a detailed characterization of such natural variation even when it is of a quantitative and polygenic nature. Examples of the various genetic approaches are given.

Natural flooding is one of the major factors affecting vegetation dynamics in many regions of the world. The Flooding Pampa Grasslands (Argentina) are frequently exposed to flooding events of diverse intensity and duration, some of which Leontodon taraxacoides, an exotic dicot. frequent in these grasslands, seems to survive. Its responses to four different water depths (0, 1, 7 and 13 cm) were studied. The results indicate that plants in conditions of total submergence (depth of 13 cm) did not survive. In less severe flood conditions, increases in the leaf insertion angle resulted in the maintenance of a large proportion of the total leaf area above the water. Differences in leaf length and a decrease in the width and the proportion of lobes per leaf were also found under partial submergence conditions (depth of 7 cm). Root and leaf aerenchyma, present in unflooded plants, showed a significant increase in flood conditions. In spite of the anatomical and morphological responses, total biomass and leaf area were severely affected by water depth. Control plants allocated more biomass to reproductive organs, while partly submerged plants allocated more to leaves and less to reproductive organs. Mature L. taraxacoides plants presented a wide range of plastic adjustment as a survival strategy in soil anaerobiosis, and appear to be able to survive short spring floods in a vegetative state; in contrast, they might not tolerate total submergence conditions imposed by more intense and long-lasting floods.

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F1 hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn5-luxAB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn5[ratio ][ratio ]luxAB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux-tagged strain DF57-40E7, which carries a constitutively expressed luxAB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.

Man's activities pose a number of threats to the functioning, structure and diversity of natural and semi-natural ecosystems. One of the main threats is the increase in concentrations in air pollutants in this century (Wellburn, 1988; Tamm, 1991). This paper is a commentary on the effects of tropospheric ozone (O3) and airborne nitrogen deposition (both oxidized (NOx) and reduced (NHy) compounds) on natural and semi-natural ecosystems, based upon the oral presentations and the discussions during the Symposium, extended with a personal overview and some suggestions about future challenges for research. The most important effects of these air pollutants on natural and semi-natural vegetation are summarized and evaluated in ecological terms, with respect to the functioning and structure of unaffected systems. Air pollutants are transported over both short and long distances (as far as a few thousand km) before being deposited on surface water, vegetation or soil. In this way, vegetation over a large area or in remote regions can be influenced by airborne pollutants (see Fowler et al. (1998); Asman, Sutton & Schjørring (1998)).

Plant species typical of semi-natural grasslands were tested for their sensitivity to long-term ozone effects on growth under two irrigation regimes, and the relationship between ozone sensitivity, relative growth rate, and plant strategy was analysed. Individuals of 24 grasses, herbs and legumes were grown in pots and exposed for two seasons to four levels of ozone in open-top chambers: (a) CF, charcoal-filtered air, (b) CFAA, charcoal-filtered air plus ozone to match ambient levels, (c) CF+, charcoal-filtered air plus ozone added to match 1.5 times ambient levels and (d) CF++, charcoal-filtered air with ozone added to match twice ambient levels during selected episodes of 7–13 d. During the ozone episodes, one half of the plants received reduced irrigation (dry treatment) while the rest was kept well watered (wet treatment). The effect of ozone on the relative growth rate was determined during the initial growth period during the first season. Above-ground plant dry weight was determined three times during each season, and stubble dry weight at the end of the experiment. In most species, the effect of ozone on relative growth rate was not significant, except in Centaurea jacea and Lychnis flos-cuculi for which a significant stimulation of relative growth rate was found. There was a significant negative relationship between percentage relative growth rate and relative growth rate measured in the CF treatment, indicating that in species growing faster in clean air relative growth rate tended to be more sensitive to ozone than in slower-growing species. After the first re-growth period during the first season, a significant effect of ozone on above-ground biomass was found in five species, i.e. a decrease in Chrysanthemum leucanthemum, Trifolium pratense and T. repens, and an increase in Silene dioïca. During the second season no significant ozone effect was detected, except for an increase in stubble mass in C. leucanthemum. In 10 species, the dry treatment caused a significant decrease in biomass, but in Plantago lanceolata during the first year, and in Knautia arvensis and T. repens during the second year, an ozone×irrigation interaction was observed. Exposure–response patterns in the wet treatment varied between species. In some species growth tended to be increased in charcoal-filtered air plus ozone to ambient levels, in others either a continuous increase or decrease in biomass was observed with increasing ozone. Trifolium repens showed the lowest AOT40 (ozone accumulated above a threshold of 40 ppb) corresponding to a 10% change in biomass. The comparison between exposure–response pattern between the wet and dry treatments revealed reduced, increased, or unchanged ozone sensitivity. The shift was related to plant strategy and not to the specific sensitivity to the dry treatment. The specific response to both ozone and dry treatment tended to be related to Grime's triangular classification of plant strategies, i.e. the C-S-R model. In species with a large component of C (competitor strategy), and/or R (ruderal strategy), biomass was more sensitive to both stresses, compared with species with a high component of S (stress tolerators), and ozone×drought interactions were more pronounced. The results indicate that specific relative growth rate is an important determinant for the potential sensitivity of grassland species to long-term effects of ozone, and that reduced irrigation causes no general protection from the effects of ozone on growth.

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O3) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 70 nmol mol−1 O3 for 7 h d−1 over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c. 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O3 resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain ‘unspecific’ peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O3 fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O3 with plant age. By contrast, changes in ‘unspecific’ peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O3 resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O3.

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea (Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.

Spore capsules of four Sphagnum species were buried at different depths in peat on a bog. Spore viability was determined after 0, 1, 2 and 3 yr. Viability generally declined with time, but viable spores were still found at all depths after 3 yr. The light-coloured spores of S. balticum and S. tenellum retained their viability better than the darker spores of S. fuscum and S. lindbergii. Survival was highest under wet but aerobic conditions, but was also high under humid or periodically desiccated conditions. By contrast, most spores stored under wet, anaerobic conditions died within 2–3 yr. These results, and predictions from them, are not consistent with earlier results for spores of long-lived and dominant bryophytes, or for seeds of phanerogams of undisturbed wetlands and forests. There was no correlation between spore size and longevity across species, but the small spores from small capsules of S. balticum and S. tenellum generally showed higher viability than those from the medium-sized and large capsules of the same species. This suggests a positive intraspecific relationship between longevity and dispersal distance. There was an indication of conditional dormancy, controlled by weather, in Sphagnum spores. The experiments indicate that Sphagnum spores can form a long-term persistent spore bank under suitable conditions, with a half-life of between 1 and 20 yr (mean across species of 2.6 and 5.0 yr at two depths studied), and with potential values in individual spore capsules of several decades, or even of centuries. Sphagnum spores kept refrigerated showed 15–35% viable spores after 13 yr. The capacity to form a persistent spore bank that can be activated whenever favourable conditions occur might help explain the wide geographical distribution of many Sphagnum species in the boreal and temperate zones, where they have managed to colonize almost every suitable patch of acidic, nutrient-poor wetland.

The concept of the ‘nucleotype’ embraces aspects of the nuclear DNA that affect the phenotype other than through the coding sequences. In this context, we consider one particular situation, namely DNA variation due to the presence of supernumerary B chromosomes (Bs); these Bs, which are additional to the basic complement of A chromosomes, generate a spectrum of DNA amounts, and thus nucleotypes, within many species. We consider the physiological consequences of carrying extra chromosomes that appear by and large to lack coding sequences, and focus on two species with contrasting case histories: rye (Secale cereale L.) and chives (Allium schoenoprasum L.), which illustrate ‘selfish’ and ‘adaptive’ explanations of the persistence of B chromosomes.

The extent of arbuscular mycorrhizal colonization was assessed in 10 field-collected plant species, representing three annual forbs, three perennial forbs, three perennial grasses and one annual grass. Each root system of each plant was split into four portions, and for each portion, mycorrhizal structures were revealed with epifluorescence microscopy (under which only arbuscules are generally visible) and three commonly used stains (Chlorazol Black E, Acid Fuchsin and Trypan Blue). The aim of the study was not to evaluate the efficacy of each method, but to compare results obtained by each under standard laboratory conditions. The recorded colonization levels of arbuscules, total arbuscular mycorrhizal fungal material and total fungal (arbuscular mycorrhizal+non-arbuscular mycorrhizal) material differed significantly between visualization methods in a number of species. However, there were also interactions between stain and plant species, indicating that the performance of a stain is dependent on the plant species being examined. In some cases (e.g. Plantago lanceolata), each visualization method produced the same colonization level, while in others (e.g. Dactylis glomerata), each method gave a different result. These data therefore suggest that the level of mycorrhizal colonization recorded in any particular plant species at a particular time is dependent on the technique employed.

Measurements of the chitin content of the rooting horizons of a typical mor-humus heathland soil, indicate that chitin can contain in excess of 20% of the total nitrogen in the litter (L) horizon and 30% in the fermentation (F) horizon. Much of this chitin-nitrogen is thought to be contained in the mycelial walls of soil fungi.

Experiments were therefore designed to test the hypothesis that such sources of N could be rendered accessible to the ericaceous plants by their fungal endophytes. Mycelium of the ericoid endophyte Hymenoscyphus ericae (Read) Korf & Kernan and of the ectomycorrhizal fungus Suillus bovinus (Fr.) O. Krantze were grown in liquid culture before being killed and added either in the intact condition, or after fractionation, as sole sources of N to sterile media upon which were grown H. ericae in pure culture, or mycorrhizal and non-mycorrhizal plants of Vaccinium macrocarpon Ait. and Calluna vulgaris L. The abilities of the test organisms to utilize the nitrogen contained in the intact mycelial necromass, or in its fractions, were assessed by determining their yields and nitrogen concentrations of their tissues.

It was revealed that H. ericae was able to produce significantly higher yield when grown on intact fungal necromass than when provided with equivalent concentrations of N in the form of ammonium. Its yields on mycelial fractions were lower, but still significantly greater than those obtained in the controls lacking N. Significantly greater yields and N contents were also found in the ericaceous plants grown with these nitrogenous substrates in the mycorrhizal condition. Without H. ericae they had no access to the substrates. The possible ecological implications of these results are discussed.

We measured the flow of methane in Typha latifolia L. (cattail)-dominated wetlands from microbial production in anoxic sediment into, through, and out of emergent T. latifolia shoots (i.e. plant transport). The purpose was to identify key environmental and plant factors that might affect rates of methane efflux from wetlands to the Earth's atmosphere. Methane accumulated in leafy T. latifolia shoots overnight, reaching concentrations up to 10000 μl l−1 (vs. atmospheric concentrations <4 μl l−1), suggesting that lower stomatal conductance at night limits methane efflux from the plant into ambient air. Daytime light and (or) lower atmospheric humidity that induce convective gas flow through the plant coincided with (a) an increase in the rate of methane efflux from T. latifolia leaves to ambient air (from <0·1 to >2·0 μmol m−2 (leaf) s−1) and (b) a decrease in shoot methane concentration to <70 μl l−1. Very short fluctuation in stomatal conductance during the day did not affect the methane efflux rate unless, possibly, the rate of photosynthesis decreased. A strong relationship between the maximum daily rate of methane efflux and shoot methane concentration (measured before the onset of convective gas flow) suggests T. latifolia plants behave like a capacitor (filling with methane at night, emitting the stored methane during the day). Experimentally cutting leaves (to prevent pressurization) reduced plant capacitance for methane.

Although mat-forming cyanobacteria dominate many freshwater ecosystems in the Arctic and Antarctic, their optimal temperature for growth (Topt) is usually much higher than the temperature range of their native habitat. The present study compared the temperature dependence of growth, pigment composition and absorbance, photosynthesis and photosynthate partitioning for two strains of cyanobacteria with contrasting Topt values; Phormidium subfuscum, isolated from McMurdo Ice Shelf, Antarctica, and Phormidium tenue, collected from the Kuparuk River in the tundra region of northern Alaska. Phormidium subfuscum grew between 5 and 20°C with a Topt of 15°C whereas P. tenue showed detectable growth from 10 to 40°C and a Topt of 30°C. Light utilization efficiency, photosynthetic capacity and the irradiance at the onset of light saturation increased with increasing temperature up to Topt in both strains. The cellular concentrations of chlorophyll a (Chl a) and carotenoid (CAR) and the in vivo absorbance maxima for Chl a, CAR, C-phycocyanin and allophycocyanin changed little for P. subfuscum but all these variables increased across the temperature range up to Topt for P. tenue. Neither P. subfuscum nor P. tenue showed changes in relative carbon allocation with varying temperature, suggesting that gross biochemical alterations are not a characteristic of temperature acclimation in these cyanobacteria. We conclude that the eurythermal cyanobacterium P. tenue optimizes growth over a wide range of temperatures by adjusting its light-capturing as well as carbon fixation characteristics, whereas stenothermal P. subfuscum relies on changes in carbon fixation without concomitant shifts in pigment content.

Photosynthetic rates of plants grown in natural systems exhibit diurnal patterns often characterized by an afternoon decline, even when measured under constant light and temperature conditions. Since we thought changes in the carbohydrate status could cause this pattern through feedback from starch and sucrose synthesis, we studied the natural fluctuations in photosynthesis rates of plants grown at 36 and 56 Pa CO2 at a FACE (free-air-CO2-enrichment) research site. Light-saturated photosynthesis varied by 40% during the day and was independent of the light-limited quantum yield of photosynthesis, which varied little through the day. Photosynthesis did not correspond with xylem water potential or leaf carbohydrate build-up, but rather with diurnal changes in air vapor-pressure deficit and light. The afternoon decline in photosynthesis also corresponded with decreased stomatal conductance and decreased Rubisco carboxylation efficiency which in turn allowed leaf-airspace CO2 partial pressure to remain constant. Growth at elevated CO2 did not affect the afternoon decline in photosynthesis, but did stimulate early-morning photosynthesis rates relative to the rest of the day. Plants grown at 56 Pa CO2 had higher light-limited quantum yields than those at 36 Pa CO2 but, there was no growth–CO2 effect on quantum yield when measured at 2 kPa O2. Therefore, understory plants have a high light-limited quantum yield that does not vary through the day. Thus, the major diurnal changes in photosynthesis occur under light-saturated conditions which may help understory saplings maximize their sunfleck-use-efficiency.

The lower part (460–650 cm) of a lacustrine sequence from Lac de Creno, Corsica (1310 m) is analysed on the basis of 68 pollen spectra and with the support of 10 14C dates (including nine A.M.S. dates). This sequence, which extends from the end of the Würm to the beginning of the Postglacial, reveals a complete late-glacial. The absence of forest dynamics during the late glacial Interstadial in Corsica is a real mystery. Pollen data clearly suggest that Pinus laricio ( = Pinus nigra Arnold ssp. laricio Maire) and perhaps other tree species did not exist in Corsica at that time.

The coupling between leaf structure and function is illustrated with reference to two examples, the C4 photosynthetic pathway and leaf pubescence. A distinction is made between function and functional significance. The latter is defined as the role, significance or consequence of a structure, whereas the former is more simply the action that a structure is capable of performing. Using the two examples, four generalizations are made concerning the relationships between structure, function and functional significance: the functional significance of leaf structure is environment-dependent; the relationship between functional significance and structure is sometimes non-intuitive; functional equivalency means that there is often more than one ‘solution’ to the same ‘constraint’; and the consequences of leaf structure can exert profound effects at levels of organization beyond those of the individual organism and may play a critical role in determining community structure and function, through interactions with other species and trophic levels. The importance of understanding the consequences in variation in leaf structure at the global scale is illustrated with reference to the issue of global climate change.

Environmental effects on the concentration of photosynthetic pigments in micro-algae can be explained by dynamics of photosystem synthesis and deactivation. A model that couples photosystem losses to the relative cellular rates of energy harvesting (light absorption) and assimilation predicts optimal concentrations of light-harvesting pigments and balanced energy flow under environmental conditions that affect light availability and metabolic rates. Effects of light intensity, nutrient supply and temperature on growth rate and pigment levels were similar to general patterns observed across diverse micro-algal taxa. Results imply that dynamic behaviour associated with photophysical stress, and independent of gene regulation, might constitute one mechanism for photo-acclimation of photosynthesis.

Leaf anatomical parameters such as leaf mass per area (LMA) and biochemical composition can be used as indicators of leaf photosynthetic capacity. The aims of this study are to evaluate the potential of reflectance spectroscopy of fresh leaves for assessing and predicting various parameters, anatomical (LMA and tissue thickness) and biochemical (nitrogen concentration). This paper describes results obtained with fresh leaves of holm oak (Quercus ilex), an evergreen oak that is widely distributed from mesic to xeric habitats in the Mediterranean. Fresh leaves (560) were collected over 3 yr at six different sites, from the top to the bottom of the canopy. The reflectance of each leaf was obtained within 1 h of sampling with an NIRSystems 6500 spectrophotometer over the range 400–2500 nm. LMA was determined for all samples; biochemical and anatomical measurements were conducted over representative subsample populations of 92 and 87 leaves, respectively. Stepwise regression calibrations and partial least squares (PLS) calibrations were developed and compared with different spectral regions and mathematical treatments. Calibration equations had high coefficients of determination (r2 ranging from 0.94 for nitrogen to 0.98 for LMA and tissue thickness). The PLS regressions gave better results than stepwise regressions for all parameters studied. Compared with regressions calculated on raw spectral data, calculations on second derivatives of spectra improved results in all cases. The use of scatter corrections also improved results. These results show that visible and near-infra red reflectance can be used for accurately predicting anatomical parameters and the nitrogen concentration of fresh holm oak leaves. The results support the suggestion that high spectral resolution imaging spectrometry can be a useful tool for assessing functional processes in forest ecosystems.