Susanna Pollastri

Functional roles of flavonoids in photoprotection: new evidence, lessons from the past.

We discuss on the relative significance of different functional roles potentially served by flavonoids in photoprotection, with special emphasis to their ability to scavenge reactive oxygen species (ROS) and control the development of individual organs and whole plant. We propose a model in which chloroplast-located flavonoids scavenge H2O2 and singlet oxygen generated under excess light-stress, thus avoiding programmed cell death. We also draw a picture in which vacuolar flavonoids in conjunction with peroxidases and ascorbic acid constitute a secondary antioxidant system aimed at detoxifying H2O2, which may diffuse out of the chloroplast at considerable rates and enter the vacuole following excess light stress-induced depletion of ascorbate peroxidase. We hypothesize for flavonols key roles as developmental regulators in early and current-day land-plants, based on their ability to modulate auxin movement and auxin catabolism. We show that antioxidant flavonoids display the greatest capacity to regulate key steps of cell growth and differentiation in eukaryotes. These regulatory functions of flavonoids, which are shared by plants and animals, are fully accomplished in the nM concentration range, as likely occurred in early land plants. We therefore conclude that functions of flavonoids as antioxidants and/or developmental regulators flavonoids are of great value in photoprotection. We also suggest that UV-B screening was just one of the multiple functions served by flavonoids when early land-plants faced an abrupt increase in sunlight irradiance.

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.

AGD5 is a GTPase-activating protein at the trans-Golgi network

ARF-GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which facilitate the activation or inactivation of ARF-GTPases, respectively. There are 15 predicted proteins that contain an ARF-GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF-GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF-GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans-Golgi network (TGN), where it co-localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post-Golgi structures of unknown nature. Taking advantage of the in vivo AGD5-ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF-GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post-Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN-localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane-localised ADP ribosylation factor B (ARFB), confirming that ARF-GAP specificity for ARF-GTPases within the cell environment may be spatially regulated.

Isoprene improves photochemical efficiency and enhances heat dissipation in plants at physiological temperatures

Summary At physiological temperatures, chloroplasts of isoprene-emitting leaves dissipate less energy as heat than chloroplasts of non-emitting leaves, determining a more efficient electron transfer through PSII.

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.

Impact of two arbuscular mycorrhizal fungi on Arundo donax L. response to salt stress

AbstractMain conclusionAM symbiosis did not strongly affectArundo donax performances under salt stress, although differences in the plants inoculated with two different fungi were recorded. The mechanisms at the basis of the improved tolerance to abiotic stresses by arbuscular mycorrhizal (AM) fungi have been investigated mainly focusing on food crops. In this work, the potential impact of AM symbiosis on the performance of a bioenergy crop, Arundo donax, under saline conditions was considered. Specifically, we tried to understand whether AM symbiosis helps this fast-growing plant, often widespread in marginal soils, withstand salt. A combined approach, involving eco-physiological, morphometric and biochemical measurements, was used and the effects of two different AM fungal species (Funneliformis mosseae and Rhizophagus irregularis) were compared. Results indicate that potted A. donax plants do not suffer permanent damage induced by salt stress, but photosynthesis and growth are considerably reduced. Since A. donax is a high-yield biomass crop, reduction of biomass might be a serious agronomical problem in saline conditions. At least under the presently experienced growth conditions, and plant–AM combinations, the negative effect of salt on plant performance was not rescued by AM fungal colonization. However, some changes in plant metabolisms were observed following AM-inoculation, including a significant increase in proline accumulation and a trend toward higher isoprene emission and higher H2O2, especially in plants colonized by R. irregularis. This suggests that AM fungal symbiosis influences plant metabolism, and plant–AM fungus combination is an important factor for improving plant performance and productivity, in presence or absence of stress conditions.

Applications of Confocal Microscopy in the Study of Root Apparatus

Confocal microscopy can be considered as one of the most important progress in optical microscopy within the last decades and has become a powerful investigation tool for molecular, cellular, and development biologist. In this chapter, the authors analyze the main uses of confocal microscopy and fluorescent dyes for studying many aspects of root physiology. The developments of confocal laser scanning microscopy and fluorescent probes that can be applied, in vivo, to plant root cells, have improved new possibilities for imaging cellular components and activities. Moreover, the opportunity to create transgenic plants and cells permits the visualization of fluorescently labeled components in cells, with minimum invasive manipulation. The combination of all this techniques provides more information for the comprehension of physiological and developmental processes in plant roots. Finally, the use of confocal laser microscopy in these types of studies has several advantages: (1) it is simple, rapid, and accurate; (2) it is nonhazardous; (3) a proper use of all the equipment is helpful to not obtain artifacted images but reliable ones.

The Vibrating Probe Technique in the Study of Root Physiology Under Stress

The present chapter introduces the use of a non-invasive vibrating probe technique to study membrane-transport processes in plant roots. Net ion fluxes and changes in the extracellular concentrations of ions (such as H+, Ca2+, K+, Mg2+, Cl−, NH 4 + , Cu2+) as well as gaseous molecules (such as O2 and NO) and other compounds (IAA) are very important to understand the physiology of plants under stress conditions. Here, we present an overview on the technique, focusing on background theory, main selective electrodes available, and the major outcomes obtained in the study of roots in order to show how in situ and real-time measurements of net ion and gas fluxes from root cells and tissues can provide insights into the functional genomics of plants and will significantly increase our understanding in plant adaptive responses to environmental stresses.

Multi Electrode Arrays (MEAs) and the Electrical Network of the Roots

Electrically excitable cells are present in many multicellular organisms, especially in brains of animals, but also in lower animals such as sponges, which lack central nervous system and in animals having excitable epithelia, which can conduct signals via neuroid conduction. In plants, most cells are electrically excitable and active, releasing and propagating action potentials (APs), which may affect central physiological processes such as photosynthesis and respiration. The first report describing electrical signals in plants was published over 200 years ago on carnivorous plants. Since then, many researchers have made detailed analyses of the electrical activity of single cells by using microelectrodes for intracellular recordings. However, such techniques cannot address integrated issues of how large assembly of cells can combine information both spatially and temporally. This can be possible using a multi electrode array (MEA) approach, intensively used in neuroscience for any electrogenic animal tissues, and here presented as its first application also for plants tissues. The system allows noninvasive, long time and multisite recording and stimulation with high spatiotemporal resolution. After a short description of the MEA technique in terms of hardware and background, the chapter mainly focuses on the application of this technique in plant electrophysiology by showing some recent works concerning the study of both the intense spontaneous electrical activities and the stimulation-elicited bursts of locally propagating electrical signals generated by the root apex.