Rodolfo Bernardi

Changes in free amino acid content and protein pattern of maize seedlings under water stress

Abstract The water status of maize seedlings stressed by water shortage was characterized by estimating the leaf water potential and the pressure and osmotic potential by a pressure/volume method. The protein pattern of water-stressed and -unstressed maize leaves and the content of free amino acids were examined. A decrease in water potential caused by water stress was not accompanied by a corresponding reduction in turgor pressure, which remained at the same level of the well-watered plants. An active osmotic adjustment apparently occurs in maize grown under water stress. Drought treatment caused an increase in total free amino acid content and a consistent rearrangement of the amino acid pool, with an accumulation of alanine, arginine, valine, aspartate, serine, threonine and tyrosine, and a decrease in glutamic acid, glycine and methionine. The results from native gel electrophoresis and electrophoresis on dodecylsulfate polyacrylamide gels of soluble proteins from leaves of maize plants grown under wet and drought conditions were compared.

Antioxidant enzyme isoforms on gels in two poplar clones differing in sensitivity after exposure to ozone

The effect of acute ozone (O3) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O3-sensitivity was analysed. Untreated leaves of both the O3-sensitive (O3-S) clone Eridano of Populus deltoides×P. maximowiczii and the O3-resistant (O3-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (Rf) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O3-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O3-R plants seven POD isoforms (Rf= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (Rf= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O3-S plants were resolved. The electrophoretic pattern of POD in O3-S leaves was greatly modified by acute O3-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (Rf= 0.13) in YL. Additionally, O3-exposure induced the appearance of two APX isoforms in YL (Rf= 0.66 and 0.70), and one isoform in ML (Rf= 0.70) of the O3-S clone. By contrast, the activity of the three APX isoformes (Rf= 0.64, 0.70 and 0.76) detected in O3-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O3 stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O3-sensitivity.

Cerato-Platanin Induces Resistance in Arabidopsis Leaves through Stomatal Perception, Overexpression of Salicylic Acid- and Ethylene-Signalling Genes and Camalexin Biosynthesis

Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1–5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

Acetyl-l-carnitine up-regulates expression of voltage-dependent anion channel in the rat brain

Acetyl-L-carnitine (ALC) exerts unique neuroprotective, neuromodulatory, and neurotrophic properties, which play an important role in counteracting various pathological processes, and have antioxidative properties, protecting cells against lipid peroxidation. In this study, suppression subtractive hybridization (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with ALC. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated after ALC treatment. In the present paper, we report the identification of the gene of mitochondrial voltage-dependent anion channel (VDAC) protein which is positively modulated by the ALC treatment. VDAC is a small pore-forming protein of the mitochondrial outer membrane. It represents an interesting tool for Ca(2+) homeostasis, and it plays a central role in apoptosis. In addition, VDAC seems to have a relevant role in the synaptic plasticity.

Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψβ-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.

Cerato-Populin and Cerato-Platanin, Two Non-Catalytic Proteins from Phytopathogenic Fungi, Interact with Hydrophobic Inanimate Surfaces and Leaves

Based on sequence homology, several fungal Cys-rich secreted proteins have been grouped in the cerato-platanin (CP) family, which comprises at least 40 proteins involved mainly in eliciting defense-related responses. The core member of this family is cerato-platanin, a moderately hydrophobic protein with a double ψ–β barrel fold. CP and the recently identified orthologous cerato-populin (Pop1) are involved in host–fungus interaction, and can be considered non-catalytic fungal PAMPs. CP is more active in inducing defense when in an aggregated conformation than in its native form, but little is known about other CP-orthologous proteins. Here, we cloned, expressed, and purified recombinant Pop1, which was used to characterize the protein aggregates. Our results suggest that the unfolded, self-assembled Pop1 is more active in inducing defense, and that the unfolding process can be induced by interaction with hydrophobic inanimate surfaces such as Teflon, treated mica, and gold sheets. In vivo, we found that both CP and Pop1 interact with the hydrophobic cuticle of leaves. Therefore, we propose that the interaction of these proteins with host cuticle waxes could induce unfolding and consequently trigger their PAMP-like activity.

In the Rat Brain Acetyl-l-carnitine Treatment Modulates the Expression of Genes Involved in Neuronal Ceroid Lipofuscinosis

Acetyl-l-carnitine (ALC) is a naturally occurring substance that, when administered at supraphysiological concentration, is neuroprotective. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer’s disease and painful neuropathies. Suppression subtractive hybridization methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after ALC treatment. The method generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes which are regulated by ALC. We report that ALC treatment: (1) upregulates lysosomal H+/ATPase gene expression and (2) downregulates myelin basic protein gene expression. The expression of these genes is altered in some forms of neuronal ceroid lipofuscinosis (NCL) pathologies. In this case, ALC might rebalance the disorders underlying NCL disease represented by a disturbance in pH homeostasis affecting the acidification of vesicles transported to lysosomal compartment for degradation. This study provides evidence that ALC controls genes involved in these serious neurological pathologies and provides insights into the ways in which ALC might exert its therapeutic benefits.

Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil

In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3′-hydroxylase isoform 1 (CS3′H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3′H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.

Xenon up-regulates several genes that are not up-regulated by nitrous oxide

Xenon and other inhalational agents induce cell and organ effects through different and only partially elucidated molecular mechanisms. In this study, we explored the gene transcription consequences of xenon exposure compared with nitrogen or nitrous oxide exposure in rat brain. Seven-day-old Sprague Dawley rats (n=24, 8 for each group) were exposed for 120 minutes to 75% xenon and 25% oxygen, 75% nitrogen and 25% oxygen (air), or 75% nitrous oxide and 25% oxygen. Using suppression subtractive hybridization, relative real-time polymerase chain reaction, and northern blot analyses of on/off gene expression, we were able to identify a set of genes that are significantly up-regulated by xenon exposure. These genes may help explain some of the molecular mechanisms that account for the neuropreconditioning effects exerted by xenon relative to nitrous oxide and air.

Ozone damage and tolerance in leaves of two poplar genotypes.

Abstract The effects induced by an acute ozone exposure were investigated in two poplar hybrids differen- tially O3 susceptible in terms of leaf injuries: Populus deltoides x maximowiczii, Eridano clone and Populus x euramericana, I-214 clone, the sensitive and the tolerant respectively. Both the leaf anatomy and the responses induced by ozone in the leaves were analysed, using a cyto-histochemical approach. Morphoanatomical characters, such as amphistomatous lamina, higher stomatal density and relaxed mesophyll cell packing (evaluated by the palisadeness coefficient), observed in the sensitive clone leaves, may favour a greater O3 uptake in the apoplast and increase the cumulative dose of pollutant per mesophyll cell, with respect to tolerant clone leaves. Mesophyll cells of sensitive plants were the main targets for O3. After an acute ozone treatment, the palisade parenchyma cells showed a decrease in chloroplast number and size, resulting best suited both to perceive the stress by O3 or reactive oxygen species and to activate several signal transduction pathways, in relation to their morphological, physiological and functional properties predisposing an efficient cell communication, signalling and stimuli sensing. The quick and well localized pattern of cell death induced by O3 in sensitive poplar leaves was accompanied by some hallmarks of programmed cell death: nuclear shrinkage, chromatin condensation and cell wall collapse.