Antonella Leone

Beyond transcription: RNA-binding proteins as emerging regulators of plant response to environmental constraints.

RNA-binding proteins (RBPs) govern many aspects of RNA metabolism, including pre-mRNA processing, transport, stability/decay and translation. Although relatively few plant RNA-binding proteins have been characterized genetically and biochemically, more than 200 RBP genes have been predicted in Arabidopsis and rice genomes, suggesting that they might serve specific plant functions. Besides their role in normal cellular functions, RBPs are emerging also as an interesting class of proteins involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions. Here, we review the most recent results and evidence on the functional role of RBPs in plant adaptation to various unfavourable environmental conditions and their contribution to enhance plant tolerance to abiotic stresses, with special emphasis on osmotic and temperature stress.

Lipoxygenase in pea roots subjected to biotic stress

Abstract Cyst nematode Heterodera goettingiana is a major pest for pea crop. Sources of resistance to this disease have been found in germplasm pea, although the mechanism of host resistance is still obscure. Lipoxygenases (LOX) have been proposed to be involved in the defence of host-parasite interactions. Resistant accession displayed at 24–72 h after nematode infection an incompatible interaction with H. goettingiana characterised by the induction of hypersensitive cell death and induction of LOX activity, mRNA and fatty acid hydroperoxides. In contrast, in susceptible pea roots infected with nematodes the induction of LOX activity, mRNA and fatty acid hydroperoxides was less consistent and delayed and no cell death was observed. A single polypeptide, with an apparent molecular mass of 96 kDa, was identified in both resistant and susceptible either uninfected or infected pea roots. Subcellular localisation showed that immunogold labelling was present in the cytoplasm, vacuoles, and plastids of differentiating parenchyma cells of uninfected and infected roots. Labelling increased weakly in differentiating cells of the feeding sites (syncytia) in susceptible pea at 72 h after infection, and was of greater magnitude in resistant tissues. Our results suggest that LOXs might have a role in root-nematode interaction and in resistance mechanisms.

Identification of the Plant Compound Geraniin as a Novel Hsp90 Inhibitor

Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90) binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino)-17-demethoxygeldanamycin (17AGG). Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose−dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down−regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors.

Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.

Carotenoids, Fatty Acid Composition and Heat Stability of Supercritical Carbon Dioxide-Extracted-Oleoresins

The risk of chronic diseases has been shown to be inversely related to tomato intake and the lycopene levels in serum and tissue. Cis-isomers represent approximately 50%–80% of serum lycopene, while dietary lycopene maintains the isomeric ratio present in the plant sources with about 95% of all-trans-lycopene. Supercritical CO2 extraction (S-CO2) has been extensively developed to extract lycopene from tomato and tomato processing wastes, for food or pharmaceutical industries, also by using additional plant sources as co-matrices. We compared two S-CO2-extracted oleoresins (from tomato and tomato/hazelnut matrices), which showed an oil-solid bi-phasic appearance, a higher cis-lycopene content, and enhanced antioxidant ability compared with the traditional solvent extracts. Heat-treating, in the range of 60–100 °C, led to changes in the lycopene isomeric composition and to enhanced antioxidant activity in both types of oleoresins. The greater stability has been related to peculiar lycopene isomer composition and to the lipid environment. The results indicate these oleoresins are a good source of potentially healthful lycopene.

Supercritical CO2-Extracted Tomato Oleoresins Enhance Gap Junction Intercellular Communications and Recover from Mercury Chloride Inhibition in Keratinocytes

A nutritionally relevant phytochemical such as lycopene, found in tomatoes and other fruits, has been proposed to have health-promoting effects by modulating hormonal and immune systems, metabolic pathways, and gap junction intercellular communication (GJIC). This work analyzes lycopene extracts, obtained from tomato and tomato added with grape seeds by using a safe and environmentally friendly extraction process, based on supercritical carbon dioxide technology (S-CO(2)). Analysis of the innovative S-CO(2)-extracted oleoresins showed peculiar chemical composition with high lycopene concentration and the presence of other carotenoids, lipids, and phenol compounds. The oleoresins showed a higher in vitro antioxidant activity compared with pure lycopene and beta-carotene and the remarkable ability to enhance the GJIC and to increase cx43 expression in keratinocytes. The oleoresins, (0.9 microM lycopene), were also able to overcome, completely, the GJIC inhibition induced by 10 nM HgCl(2), mercury(II) chloride, suggesting a possible action mechanism.

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.

Physical Stresses in Plants

The influence of high temperature stress (heat shock or HS) and other environmental stress agents on gene expression of soybean seedlings has been extensively studied. The sequence analysis of HS genes has revealed a high degree of conservation among individual members of several heat shock protein (HSP) families and different classes within a family, but some interesting differences have been noted. These studies have also revealed complex patterns of regulation of expression of the HS genes and accumulation of the HSPs. Based primarily upon the deduced amino acid sequence of the HSPs, immunological cross-reactivity, and intracellular localization, the complex group of low molecular weight (LMW) HSP genes have been organized into multiple classes. In soybean several eDNA and genomic clones encoding 20 to 24 kD LMW HSPs have been isolated which represent new classes of the LMW HSP gene super family based on nucleotide/amino acid sequence and cell fractionation analyses. The mRNAs transcribed from these genes are of lower abundance than those for the 15 to 18 kD Class I and II proteins, and these genes occur as small multigene (i.e. three to four) classes or subfamilies. The mRNAs of three of these classes of LMW HSP genes are translated on ERbound ribosomes and possess hydrophobic leader sequences. The presence of a consensus ER retention sequence on two of these proteins indicates that they probably reside within the ER. The third protein lacks the consensus ER retention signal and presumably is translocated to an as yet unidentified location. The mRNA representing a fourth LMW gene class is translated on unbound cytoplasmic ribosomes, and the predicted protein has aN-terminal sequence with properties similar to that of some proteins which are translocated into mitochondria. Early studies with soybean seedlings indicated that some 22 to 24 kD HSPs are localized in mitochondria. Differential induction by amino analog treatment indicates that genes assigned to the same class based on amino acid similarity and localization can be regulated differently. The possible role of the multiple classes on LMW 15 to 24 kD HSPs in protein protection

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.

Asg1 is a stress-inducible gene which increases stomatal resistance in salt stressed potato

The identification of critical components in plant salt stress adaptation has greatly benefitted, in the last two decades, from fundamental discoveries in Arabidopsis and close model systems. Nevertheless, this approach has also highlighted a non-complete overlap between stress tolerance mechanisms in Arabidopsis and agricultural crops. Within a long-running research program aimed at identifying salt stress genetic determinants in potato by functional screening in Escherichia coli, we isolated Asg1, a stress-related gene with an unknown function. Asg1 is induced by salt stress in both potato and Arabidopsis and by abscisic acid in Arabidopsis. Asg1 is actively transcribed in all plant tissues. Furthermore, Asg1 promoter analysis confirmed its ubiquitous expression, which was remarkable in pollen, a plant tissue that undergoes drastic dehydration/hydration processes. Fusion of Asg1 with green fluorescent protein showed that the encoded protein is localized close to the plasma membrane with a non-continuous pattern of distribution. In addition, Arabidopsis knockout asg1 mutants were insensitive to both NaCl and sugar hyperosmotic environments during seed germination. Transgenic potato plants over-expressing the Asg1 gene revealed a stomatal hypersensitivity to NaCl stress which, however, did not result in a significantly improved tuber yield in stress conditions. Altogether, these data suggest that Asg1 might interfere with components of the stress signaling pathway by promoting stomatal closure and participating in stress adaptation.