Alessio Fini

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.

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.

Flavonoids as Antioxidants in Plants Under Abiotic Stresses

Flavonoids make a relevant contribution to the response mechanisms of higher plants to a plethora of abiotic stresses. In addition to the long-reported functions as screeners of damaging short-wave solar radiation, flavonoids have been suggested as playing key functions as antioxidants in stressed plants, by inhibiting the generation and reducing reactive oxygen species (ROS) once formed. The ROS-scavenging properties of flavonoids are restricted to few structures, namely, the dihydroxy B-ring-substituted flavonoid glycosides. This structure–activity relationship conforms to the well-known stress-induced preferential biosynthesis of dihydroxy B-ring-substituted both flavones and flavonols. These flavonoids, especially the derivatives of quercetin, have been shown to greatly affect the movement of auxin at intra- and intercellular levels, and hence to tightly regulate the development of individual organs and the whole plant. The effectiveness of flavonoids to inhibit the activity of the auxin efflux facilitator proteins tightly depends on the chemical features that confer the antioxidant potential. In this review article, we discuss about (1) the effect of different abiotic stresses on the accumulation of individual flavonoids, (2) the potential role served by antioxidant flavonoids in the antioxidant machinery of plants exposed to severe stress conditions, and (3) the function of flavonoids as developmental regulators.

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.

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.

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.

Nature based solutions to mitigate soil sealing in urban areas: Results from a 4-year study comparing permeable, porous, and impermeable pavements

ABSTRACT Soil sealing is one of the most pervasive forms of soil degradation that follows urbanization and, despite innovative pavements (i.e. pervious) are being installed in urban areas to mitigate it, there is little research on the effects of pervious pavements on soil water and carbon cycle and on the physiology of urban trees. The aim of this 4‐year experiment was to assess the effects of three pavements, differing in permeability to water and gases, on some soil physical parameters, and on growth and physiology of newly planted Celtis australis and Fraxinus ornus. Treatments were: 1) impermeable pavement (asphalt on concrete sub‐base); 2) permeable pavement (pavers on crushed rock sub‐base); 3) porous design (porous pavement on crushed rock sub‐base); 4) control (unpaved soil, kept free of weed by chemical control). Soil (temperature, moisture, oxygen content and CO2 efflux) and plant (above‐ and below‐ground growth, leaf gas exchange, chlorophyll fluorescence, water relations) parameters were measured. All types of pavements altered the water cycle compared to unpaved soil plots, but this disturbance was less intense in porous pavements than in other soil cover types. Porous pavements allowed both higher infiltration and evaporation of water than both pavers and asphalt. Reduction of evaporative cooling from soil paved with permeable and impermeable pavements contributed to significant soil warming: at 20 cm depth, soils under concrete pavers and asphalt were 4 and 5 °C warmer than soil covered by porous pavements and unpaved soils, respectively. Thus, enhancing evaporation from paved soil by the use of porous pavements may contribute to mitigating urban heat islands. CO2 greatly accumulated under impermeable and permeable pavements, but not under porous pavements, which showed CO2 efflux rates similar to control. Soil oxygen slightly decreased only beneath asphalt. Growth of newly planted C. australis and F. ornus was little affected by pavement type. Tree transpiration rapidly depleted soil moisture compared to the not‐planted scenario, but soil moisture did not fall below wilting point (particularly in the deeper soil layers, i.e. 40–50 cm) in any treatment. While C. australis showed similar leaf gas exchange and water relations in all treatments, F. ornus showed a depression in CO2 assimilation and slight signs of stress of the photosynthetic apparatus when planted in soil covered with impermeable pavement. The effects of soil cover with different materials on tree growth and physiology were little, because newly planted trees have most of their roots still confined in the unpaved planting pit. Still, the reduction of soil sealing around the planting pit triggered the establishment of sensitive species such as ash. Further research is needed to assess the effects of different pavement types on established, larger trees. HighlightsSoil sealing depresses water and carbon exchange between soil and the atmosphere.As a consequence of reduced evaporation, warming occurs in sealed soils.Disturbance to element cycling can be mitigated by the use of porous pavements.Tree responses to pavements is species‐specific, but generally little for establishing trees.

Physiological Responses to Different Irrigation Regimes for Shade Trees Grown in Container

The aim of this work was to investigate the effects of two different irrigation regimes on growth and physiology of some widely used shade tree species grown in containers. Trees were irrigated with 1200 ml/day (normal irrigation, NI), according to the standard water volumes distributed in the nurseries, or 600 ml/day (reduced irrigation, RI). Among the species investigated, only Tilia cordata showed greater growth under normal irrigation if compared to reduced irrigation. The other species included in this experiment (Acer pseudoplatanus, A. platanoides, T. tomentosa, and T. platyphyllos) had no benefits from normal irrigation and, under reduced irrigation, showed no symptoms of water stress. Irrigation had no effect on leaf gas exchange of the species considered. Therefore, for these species, water consumption can be reduced by a half without affecting nursery stock quality. This study demonstrated that irrigation volumes usually distributed in nurseries are generally too high if compared to the real needs of the crops and that they can be reduced without decreasing product quality. This will lead to greater sustainability of nursery management practices and water savings.