CO2-assimilation, sequestration, and storage by urban woody species growing in parks and along streets in two climatic zones.

Using two cities, Rimini (Italy, Cfa climate) and Krakow (Poland, Cfb), as living laboratories, this research aimed at measuring in situ the capacity of 15 woody species to assimilate, sequester, and store CO2. About 1712 trees of the selected species were identified in parks or along streets of the two cities, and their age, DBH, height, and crown radius were measured. The volume of trunk and branches was measured using a terrestrial LiDAR. The true Leaf Area Index was calculated by correcting transmittance measurements conducted using a plant-canopy-analyser for leaf angle distribution, woody area index, and clumping. Dendrometric traits were fitted using age or DBH as independent variable to obtain site- and species-specific allometric equations. Instantaneous and daily net CO2-assimilation per unit leaf area was measured using an infra-red gas-analyser on full-sun and shaded leaves and upscaled to the unit crown-projection area and to the whole tree using both a big-leaf and a multilayer approach. Results showed that species differed for net CO2-assimilation per unit leaf area, leaf area index, and for the contribution of shaded leaves to overall canopy carbon gain, which yielded significant differences among species in net CO2-assimilation per unit crown-projection-area (AcpaML(d)). AcpaML(d) was underestimated by 6-30 % when calculated using the big-leaf, compared to the multilayer model. While maximizing AcpaML(d) can maximize CO2-assimilation for a given canopy cover, species which matched high AcpaML(d) and massive canopy spread, such as mature Platanus x acerifolia and Quercus robur, provided higher CO2-assimilation (Atree) at the individual tree scale. Land use (park or street), did not consistently affect CO2-assimilation per unit leaf or crown-projection area, although Atree can decline in response to specific management practices (e.g. heavy pruning). CO2-storage and sequestration, in general, showed a similar pattern as Atree, although the ratio between CO2-sequestration and CO2-assimilation decreased at increasing DBH.

Photoprotective Role of Photosynthetic and Non-Photosynthetic Pigments in Phillyrea latifolia: Is Their "Antioxidant" Function Prominent in Leaves Exposed to Severe Summer Drought?

Carotenoids and phenylpropanoids play a dual role of limiting and countering photooxidative stress. We hypothesize that their "antioxidant" function is prominent in plants exposed to summer drought, when climatic conditions exacerbate the light stress. To test this, we conducted a field study on Phillyrea latifolia, a Mediterranean evergreen shrub, carrying out daily physiological and biochemical analyses in spring and summer. We also investigated the functional role of the major phenylpropanoids in different leaf tissues. Summer leaves underwent the most severe drought stress concomitantly with a reduction in radiation use efficiency upon being exposed to intense photooxidative stress, particularly during the central hours of the day. In parallel, a significant daily variation in both carotenoids and phenylpropanoids was observed. Our data suggest that the morning-to-midday increase in zeaxanthin derived from the hydroxylation of ß-carotene to sustain non-photochemical quenching and limit lipid peroxidation in thylakoid membranes. We observed substantial spring-to-summer and morning-to-midday increases in quercetin and luteolin derivatives, mostly in the leaf mesophyll. These findings highlight their importance as antioxidants, countering the drought-induced photooxidative stress. We concluded that seasonal and daily changes in photosynthetic and non-photosynthetic pigments may allow P. latifolia leaves to avoid irreversible photodamage and to cope successfully with the Mediterranean harsh climate.

Unveiling the shade nature of cyanic leaves: A view from the "blue absorbing side" of anthocyanins.

Anthocyanins have long been suggested as having great potential in offering photoprotection to plants facing high light irradiance. Nonetheless, their effective ability in protecting the photosynthetic apparatus from supernumerary photons has been questioned by some authors, based upon the inexact belief that anthocyanins almost exclusively absorb green photons, which are poorly absorbed by chlorophylls. Here we focus on the blue light absorbing features of anthocyanins, a neglected issue in anthocyanin research. Anthocyanins effectively absorb blue photons: the absorbance of blue relative to green photons increases from tri- to mono-hydroxy B-ring substituted structures, reaching up to 50% of green photons absorption. We offer a comprehensive picture of the molecular events activated by low blue-light availability, extending our previous analysis in purple and green basil, which we suggest to be responsible for the "shade syndrome" displayed by cyanic leaves. While purple leaves display overexpression of genes promoting chlorophyll biosynthesis and light harvesting, in green leaves it is the genes involved in the stability/repair of photosystems that are largely overexpressed. As a corollary, this adds further support to the view of an effective photoprotective role of anthocyanins. We discuss the profound morpho-anatomical adjustments imposed by the epidermal anthocyanin shield, which reflect adjustments in light harvesting capacity under imposed shade and make complex the analysis of the photosynthetic performance of cyanic versus acyanic leaves.

Are Flavonoids Effective Antioxidants in Plants? Twenty Years of Our Investigation.

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of "a plant antioxidant", which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.

Metabolic plasticity in the hygrophyte Moringa oleifera exposed to water stress.

Over the past decades, introduction of many fast-growing hygrophilic, and economically valuable plants into xeric environments has occurred. However, production and even survival of these species may be threatened by harsh climatic conditions unless an effective physiological and metabolic plasticity is available. Moringa oleifera Lam., a multipurpose tree originating from humid sub-tropical regions of India, is widely cultivated in many arid countries because of its multiple uses. We tested whether M. oleifera can adjust primary and secondary metabolism to efficiently cope with increasing water stress. It is shown that M. oleifera possesses an effective isohydric behavior. Water stress induced a quick and strong stomatal closure, driven by abscisic acid (ABA) accumulation, and leading to photosynthesis inhibition with consequent negative effects on biomass production. However, photochemistry was not impaired and maximal fluorescence and saturating photosynthesis remained unaffected in stressed leaves. We report for the first time that M. oleifera produces isoprene, and show that isoprene emission increased three-fold during stress progression. It is proposed that higher isoprene biosynthesis helps leaves cope with water stress through its antioxidant or membrane stabilizing action, and also indicates a general MEP (methylerythritol 4-phosphate) pathway activation that further helps protect photosynthesis under water stress. Increased concentrations of antioxidant flavonoids were also observed in water stressed leaves, and probably cooperate in limiting irreversible effects of the stress in M. oleifera leaves. The observed metabolic and phenotypic plasticity may facilitate the establishment of M. oleifera in xeric environments, sustaining the economic and environmental value of this plant.

Modulation of Phytohormone Signaling: A Primary Function of Flavonoids in Plant-Environment Interactions.

The old observation that plants preferentially synthesize flavonoids with respect to the wide range of phenylpropanoid structures when exposed to high doses of UV-B radiation has supported the view that flavonoids are primarily involved in absorbing the shortest solar wavelengths in photoprotection. However, there is compelling evidence that the biosynthesis of flavonoids is similarly upregulated in response to high photosynthetically active radiation in the presence or in the absence of UV-radiation, as well as in response to excess metal ions and photosynthetic redox unbalance. This supports the hypothesis that flavonoids may play prominent roles as scavengers of reactive oxygen species (ROS) generated by light excess. These 'antioxidant' functions of flavonoids appears robust, as maintained between different life kingdoms, e.g., plants and animals. The ability of flavonoids to buffer stress-induced large alterations in ROS homeostasis and, hence, to modulate the ROS-signaling cascade, is at the base of well-known functions of flavonoids as developmental regulators in both plants and animals. There is both long and very recent evidence indeed that, in plants, flavonoids may strongly affect phytohormone signaling, e.g., auxin and abscisic acid signaling. This function is served by flavonoids in a very low (nM) concentration range and involves the ability of flavonoids to inhibit the activity of a wide range of protein kinases, including but not limited to mitogen-activated protein kinases, that operate downstream of ROS in the regulation of cell growth and differentiation. For example, flavonoids inhibit the transport of auxin acting on serine-threonine PINOID (PID) kinases that regulate the localization of auxin efflux facilitators PIN-formed (PIN) proteins. Flavonoids may also determine auxin gradients at cellular and tissue levels, and the consequential developmental processes, by reducing auxin catabolism. Recent observations lead to the hypothesis that regulation/modulation of auxin transport/signaling is likely an ancestral function of flavonoids. The antagonistic functions of flavonoids on ABA-induced stomatal closure also offer novel hypotheses on the functional role of flavonoids in plant-environment interactions, in early as well as in modern terrestrial plants. Here, we surmise that the regulation of phytohormone signaling might have represented a primary function served by flavonols for the conquest of land by plants and it is still of major significance for the successful acclimation of modern terrestrial plants to a severe excess of radiant energy.

Isoprene Responses and Functions in Plants Challenged by Environmental Pressures Associated to Climate Change.

The functional reasons for isoprene emission are still a matter of hot debate. It was hypothesized that isoprene biosynthesis evolved as an ancestral mechanism in plants adapted to high water availability, to cope with transient and recurrent oxidative stresses during their water-to-land transition. There is a tight association between isoprene emission and species hygrophily, suggesting that isoprene emission may be a favorable trait to cope with occasional exposure to stresses in mesic environments. The suite of morpho-anatomical traits does not allow a conservative water use in hygrophilic mesophytes challenged by the environmental pressures imposed or exacerbated by drought and heat stress. There is evidence that in stressed plants the biosynthesis of isoprene is uncoupled from photosynthesis. Because the biosynthesis of isoprene is costly, the great investment of carbon and energy into isoprene must have relevant functional reasons. Isoprene is effective in preserving the integrity of thylakoid membranes, not only through direct interaction with their lipid acyl chains, but also by up-regulating proteins associated with photosynthetic complexes and enhancing the biosynthesis of relevant membrane components, such as mono- and di-galactosyl-diacyl glycerols and unsaturated fatty acids. Isoprene may additionally protect photosynthetic membranes by scavenging reactive oxygen species. Here we explore the mode of actions and the potential significance of isoprene in the response of hygrophilic plants when challenged by severe stress conditions associated to rapid climate change in temperate climates, with special emphasis to the concomitant effect of drought and heat. We suggest that isoprene emission may be not a good estimate for its biosynthesis and concentration in severely droughted leaves, being the internal concentration of isoprene the important trait for stress protection.

Dissecting molecular and physiological response mechanisms to high solar radiation in cyanic and acyanic leaves: a case study on red and green basil.

Photosynthetic performance and the expression of genes involved in light signaling and the biosynthesis of isoprenoids and phenylpropanoids were analysed in green ('Tigullio', TIG) and red ('Red Rubin', RR) basil. The aim was to detect the physiological and molecular response mechanisms to high sunlight. The attenuation of blue-green light by epidermal anthocyanins was shown to evoke shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance. Red basil had a lower mesophyll conductance, partially compensated by the less effective control of stomatal movements, in comparison with TIG. Photosynthesis decreased more in TIG than in RR in high sunlight, because of larger stomatal limitations and the transient impairment of PSII photochemistry. The methylerythritol 4-phosphate pathway promoted above all the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This enabled the green leaves to process the excess radiant energy effectively, and the red leaves to optimize light harvesting and photoprotection. The greater stomatal closure observed in TIG than in RR was due to enhanced abscisic acid (ABA) glucose ester deglucosylation and reduced ABA oxidation, rather than to superior de novo ABA synthesis. This study shows a strong competition between anthocyanin and flavonol biosynthesis, which occurs at the level of genes regulating the oxidation of the C2-C3 bond in the dihydro-flavonoid skeleton.

Mesophyll conductance plays a central role in leaf functioning of Oleaceae species exposed to contrasting sunlight irradiance.

The ability to modify mesophyll conductance (gm ) in response to changes in irradiance may be a component of the acclimation of plants to shade-sun transitions, thus influencing species-specific distributions along light-gradients, and the ecological niches for the different species. To test this hypothesis we grew three woody species of the Oleaceae family, the evergreen Phillyrea latifolia (sun-requiring), the deciduous Fraxinus ornus (facultative sun-requiring) and the hemi-deciduous Ligustrum vulgare (shade tolerant) at 30 or 100% sunlight irradiance. We show that neither mesophyll conductance calculated with combined gas exchange and chlorophyll fluorescence techniques (gm) nor CO2 assimilation significantly varied in F. ornus because of sunlight irradiance. This corroborates previous suggestions that species with high plasticity for light requirements, do not need to undertake extensive reorganization of leaf conductances to CO2 diffusion to adapt to different light environments. On the other hand, gm steeply declined in L. vulgare and increased in P. latifolia exposed to full-sun conditions. In these two species, leaf anatomical traits are in part responsible for light-driven changes in gm , as revealed by the correlation between gm and mesophyll conductance estimated by anatomical parameters (gmA). Nonetheless, gm was greatly overestimated by gmA when leaf metabolism was impaired because of severe light stress. We show that gm is maximum at the light intensity at which plant species have evolved and we conclude that gm actually plays a key role in the sun and shade adaptation of Mediterranean species. The limits of gmA in predicting mesophyll conductance are also highlighted.

Isoprenoids and phenylpropanoids are part of the antioxidant defense orchestrated daily by drought-stressed Platanus × acerifolia plants during Mediterranean summers.

The hypothesis was tested that isoprenoids and phenylpropanoids play a prominent role in countering photooxidative stress, following the depletion of antioxidant enzyme activity in plants exposed to severe drought stress under high solar irradiance and high temperatures. Platanus × acerifolia, a high isoprene-emitting species, was drought-stressed during summer (WS) and compared with unstressed controls (WW). Water relations and photosynthetic parameters were measured under mild, moderate, and severe drought stress conditions. Volatile and nonvolatile isoprenoids, antioxidant enzymes, and phenylpropanoids were measured with the same time course, but in four different periods of the day. Drought severely inhibited photosynthesis, whereas it did not markedly affect the photochemical machinery. Isoprene emission and zeaxanthin concentration were higher in WS than in WW leaves, particularly at mild and moderate stresses, and during the hottest hours of the day. The activities of catalase and ascorbate peroxidase steeply declined during the day, while the activity of guaiacol peroxidase and the concentration of quercetin increased during the day, peaking in the hottest hours in both WW and WS plants. Our experiment reveals a sequence of antioxidants that were used daily by plants to orchestrate defense against oxidative stress induced by drought and associated high light and high temperature. Secondary metabolites seem valuable complements of antioxidant enzymes to counter oxidative stress during the hottest daily hours.

RNA-seq analysis of Quercus pubescens Leaves: de novo transcriptome assembly, annotation and functional markers development.

Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG) resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs) were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

Salinity stress constrains photosynthesis in Fraxinus ornus more when growing in partial shading than in full sunlight: consequences for the antioxidant defence system.

BACKGROUND AND AIMS: A major challenge in plant ecophysiology is understanding the effects of multiple sub-optimal environmental conditions on plant performance. In most Mediterranean areas soil salinity builds up during the summer because of low availability of soil water coupled with hot temperatures. Although sunlight and soil salinity may strongly interact in determining a plant's performance, this has received relatively little attention. METHODS: Two-year-old seedlings of Fraxinus ornus were grown outdoors in pots during a Mediterranean summer in either 45 % (shaded plants) or 100 % (sun plants) sunlight irradiance and were supplied with either deionized water or deionized water plus 75 mm NaCl. Morpho-anatomical traits, water and ionic relations, gas exchange and photosystem II performance, concentrations of individual carotenoids, activity of antioxidant enzymes, concentrations of ascorbic acid and individual polyphenols were measured in leaves. Leaf oxidative stress and damage were estimated by in vivo analysis of stable free radicals and ultrastructural analyses. KEY RESULTS: Leaf concentrations of potentially toxic ions did not markedly differ in shaded or sun plants in response to salinity. Leaves of sun plants displayed superior water use efficiency compared with leaves of shaded plants, irrespective of salinity treatment, and had both better stomatal control and higher CO2 carboxylation efficiency than leaves of shaded plants. In the salt-treated groups, the adverse effects of excess midday irradiance were greater in shade than in sun plants. The activity of enzymes responsible for detoxifying hydrogen peroxide decreased in shaded plants and increased in sun plants as a result of salinity stress. In contrast, the activity of guaiacol peroxidase and the concentration of phenylpropanoids increased steeply in response to salinity in shaded plants but were unaffected in sun plants. CONCLUSIONS: It is concluded that salinity may constrain the performance of plants growing under partial shading more severely than that of plants growing under full sun during summer. The results suggest co-ordination within the antioxidant defence network aimed at detoxifying salt-induced generation of reactive oxygen species.

Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: a fluorescence microscopy analysis.

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15 µm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides.

Evaluation of a high throughput starch analysis optimised for wood.

Starch is the most important long-term reserve in trees, and the analysis of starch is therefore useful source of physiological information. Currently published protocols for wood starch analysis impose several limitations, such as long procedures and a neutralization step. The high-throughput standard protocols for starch analysis in food and feed represent a valuable alternative. However, they have not been optimised or tested with woody samples. These have particular chemical and structural characteristics, including the presence of interfering secondary metabolites, low reactivity of starch, and low starch content. In this study, a standard method for starch analysis used for food and feed (AOAC standard method 996.11) was optimised to improve precision and accuracy for the analysis of starch in wood. Key modifications were introduced in the digestion conditions and in the glucose assay. The optimised protocol was then evaluated through 430 starch analyses of standards at known starch content, matrix polysaccharides, and wood collected from three organs (roots, twigs, mature wood) of four species (coniferous and flowering plants). The optimised protocol proved to be remarkably precise and accurate (3%), suitable for a high throughput routine analysis (35 samples a day) of specimens with a starch content between 40 mg and 21 µg. Samples may include lignified organs of coniferous and flowering plants and non-lignified organs, such as leaves, fruits and rhizomes.

Flavonoids as antioxidants and developmental regulators: relative significance in plants and humans.

Phenylpropanoids, particularly flavonoids have been recently suggested as playing primary antioxidant functions in the responses of plants to a wide range of abiotic stresses. Furthermore, flavonoids are effective endogenous regulators of auxin movement, thus behaving as developmental regulators. Flavonoids are capable of controlling the development of individual organs and the whole-plant; and, hence, to contribute to stress-induced morphogenic responses of plants. The significance of flavonoids as scavengers of reactive oxygen species (ROS) in humans has been recently questioned, based on the observation that the flavonoid concentration in plasma and most tissues is too low to effectively reduce ROS. Instead, flavonoids may play key roles as signaling molecules in mammals, through their ability to interact with a wide range of protein kinases, including mitogen-activated protein kinases (MAPK), that supersede key steps of cell growth and differentiation. Here we discuss about the relative significance of flavonoids as reducing agents and signaling molecules in plants and humans. We show that structural features conferring ROS-scavenger ability to flavonoids are also required to effectively control developmental processes in eukaryotic cells.

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO2 assimilation rate at saturating light and daily assimilated CO2 were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (Φ(PSII)) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chl(tot)) exceeded 200 mmol mol⁻¹ Chl(tot) in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H2O2. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H2O2 in excess light-stressed leaves. Nonetheless, the concentration of H2O2 and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H2O2 within a sub-lethal concentration range.

Effect of controlled inoculation with specific mycorrhizal fungi from the urban environment on growth and physiology of containerized shade tree species growing under different water regimes.

The aim of this work was to evaluate the effects of selected mycorrhiza obtained in the urban environment on growth, leaf gas exchange, and drought tolerance of containerized plants growing in the nursery. Two-year-old uniform Acer campestre L., Tilia cordata Mill., and Quercus robur L. were inoculated with a mixture of infected roots and mycelium of selected arbuscular (maple, linden) and/or ectomycorrhiza (linden, oak) fungi and grown in well-watered or water shortage conditions. Plant biomass and leaf area were measured 1 and 2 years after inoculation. Leaf gas exchange, chlorophyll fluorescence, and water relations were measured during the first and second growing seasons after inoculation. Our data suggest that the mycelium-based inoculum used in this experiment was able to colonize the roots of the tree species growing in the nursery. Plant biomass was affected by water shortage, but not by inoculation. Leaf area was affected by water regime and, in oak and linden, by inoculation. Leaf gas exchange was affected by inoculation and water stress. V(cmax) and J(max) were increased by inoculation and decreased by water shortage in all species. F(v)/F(m) was also generally higher in inoculated plants than in control. Changes in PSII photochemistry and photosynthesis may be related to the capacity of inoculated plants to maintain less negative leaf water potential under drought conditions. The overall data suggest that inoculated plants were better able to maintain physiological activity during water stress in comparison to non-inoculated plants.

Stress-induced flavonoid biosynthesis and the antioxidant machinery of plants.

There is a growing body of evidence that flavonoids do not primarily function as UV-B screening pigments in photoprotection. Recent findings support the idea that excess light stress, irrespective of the relative proportions of the solar wavebands reaching the leaf surface, up-regulates the biosynthesis of dihydroxy B-ring-substituted flavonoid glycosides, as a consequence of and aimed at countering the generation of ROS. Intriguingly, the very conditions that lead to the inactivation of antioxidant enzymes can also up-regulate the biosynthesis of antioxidant flavonoids, which suggests flavonoids constituting a secondary ROS-scavenging system in plants exposed to severe/prolonged stress conditions. H2O2 may diffuse out of the chloroplast at considerable rates and be transported to the vacuole, the storing site for flavonoids, by tonoplast intrinsic proteins, under severe excess light conditions. We suggest that the unanticipated key role of the vacuole in the ROS homeostasis might be mediated by flavonoids.