Antonietta Leone

Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)-mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.

Plant Tolerance to Heat Stress: Current Strategies and New Emergent Insights

Temperatures above the optimal temperature range for plant growth and reproduction cause deleterious cellular damage, which in turn affects plant productivity. To relieve these effects, plants adapt to high temperature by activating a series of physiological and biochemical changes necessary to reestablish a new cellular homeostasis compatible with the increase in temperature. The genetic control of the heat shock (HS) response is quite complex and requires the activation of a network of genes, involved in the perception and transduction of the HS signal, which, in turn, trigger the up-regulation of other target genes. The induced genes code for proteins and enzymes (HS proteins, active oxygen detoxifying enzymes), playing a direct role in the protection of cellular and subcellular organelles or genes encoding enzymes involved in the biosynthesis of protective compatible compounds (sugars, polyols, betaines). Membrane lipid instauration, controlled by desaturase genes, is also a critical component of thermotolerance. This chapter covers the principal aspects of the plant HS response and the role of different class of genes in the acquisition of thermotolerance. The molecular breeding strategies currently available to alter genetically the level of the protective proteins, enzymes and molecules, that may ameliorate plant tolerance and productivity under high temperature stress, will be also discussed.

The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

An Arabidopsis RNA-binding protein contributes to drought and salt stress tolerance. Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Response to low soil water potential in pea genotypes (Pisum sativum L.) with different leaf morphology

Abstract Genetic modifications of canopy structure may result in enhanced yield when water is a limiting factor. Three near-isogenic pea genotypes carrying genes which determine a striking effect on leaf morphology were grown in lysimeters under two extreme water regimes. Pea genotypes of normal and reduced leaf and stipule morphology were cultivated at a plant density of 72 m−2 in drainage lysimeters, where a well watered regime was established by irrigating whenever tensiometric values reached (0.05 MPa). After 70 days from emergence, a second regime was created by withholding water until wilting symptoms were visible; the control plants were irrigated 12 times, whereas the stressed plants only once. Dramatic differences in dry matter accumulation were found between the two irrigation regimes. The physiological response varied accordingly. Under stressed conditions, genotype af, which has the leaflets transformed into tendrils, showed a faster CO2 exchange rate (CER), a lower stomatal resistance, and a lower canopy temperature than the other two genotypes. This allowed a reduced soil water consumption, but a higher pod dry matter accumulation, which doubled in the last 2 weeks. This behaviour was also favoured by a leaf dry matter accumulation, in terms of quantity and duration, significantly higher than that shown by the other two genotypes. Additionally, af plants showed a higher leaf/stem ratio and negligible senescence.

Bio-Nano-Composite Materials Constructed With Single Cells and Carbon Nanotubes: Mechanical, Electrical, and Optical Properties

Here, we report a procedure to obtain novel artificial materials using either fungal or isolated tobacco cells in association with different percentages of carbon nanotubes. The electrical, mechanical, and optical properties of these materials have been determined. The produced bio-nano-composite materials have linear electrical characteristics, high temperature stability up to 180 °C, linear increase of the electrical conductivity with increasing temperature and, in one case, also optical transparency. Using tobacco cells, we obtained a material with low mass density and mechanical properties suitable for structural applications along with high electrical conductivity. We also present theoretical models both for their mechanical and electrical behavior. These findings report a procedure for the next generation bio-nano-composite materials.

Ectopic expression of the Osmyb4 rice gene enhances synthesis of hydroxycinnamic acid derivatives in tobacco and clary sage

In this work, we report the ectopic expression of the Osmyb4 rice gene, encoding the Myb4 transcription factor, in Nicotiana tabacum and Salvia sclarea. Transcriptional analysis of T2 homozygous tobacco plants overexpressing Osmyb4 revealed that Myb4 activated the transcription of several genes of the phenylpropanoid pathway such as PAL, C4H, 4CL1, 4CL2 (encoding phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate: Co A ligase1, 4-coumarate: Co A ligase2). Moreover, the Myb4 increased expression of HQT encoding hydroxycinnamoyl-CoA: quinate transferase, which specifically triggers the accumulation of chlorogenic acid (CGA). In addition, increased acccumulation of rosmarinic acid (RA) was found in transgenic plants of both species. These results open the possibility of using the Osmyb4 gene to increase the production of specific bioactive hydroxycinnamates.

Identification of Limonol Derivatives as Heat Shock Protein 90 (Hsp90) Inhibitors through a Multidisciplinary Approach.

The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high-affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α-limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c-Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol-derivatives as promising scaffolds for the design of novel Hsp90α inhibitors.

Distinct gene networks drive differential response to abrupt or gradual water deficit in potato

Water-limiting conditions affect dramatically plant growth and development and, ultimately, yield of potato plants (Solanum tuberosum L.). Therefore, understanding the mechanisms underlying the response to water deficit is of paramount interest to obtain drought tolerant potato varieties. Herein, potato 10K cDNA array slides were used to profile transcriptomic changes of two potato cell populations under abrupt (shocked cells) or gradual exposure (adapted cells) to polyethylene glycol (PEG)-mediated water stress. Data analysis identified >1000 differentially expressed genes (DEGs) in our experimental conditions. Noteworthy, our microarray study also suggests that distinct gene networks underlie the cellular response to shock or gradual water stress. On the basis of our experimental findings, it is possible to speculate that DEGs identified in shocked cells participate in early protective and sensing mechanisms to environmental insults, while the genes whose expression was modulated in adapted cells are directly involved in the acquisition of a new cellular homeostasis to cope with water stress conditions. To validate microarray data obtained for potato cells, the expression analysis of 21 selected genes of interest was performed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Intriguingly, the expression levels of these transcripts in 4-week old potato plants exposed to long-term water-deficit. qRT-PCR analysis showed that several genes were regulated similarly in potato cells cultures and tissues exposed to drought, thus confirming the efficacy of our simple experimental system to capture important genes involved in osmotic stress response. Highlighting the differences in gene expression between shock-like and adaptive response, our findings could contribute to the discussion on the biological function of distinct gene networks involved in the response to abrupt and gradual adaptation to water deficit.

Erratum to: Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming

Unfortunately, the second author name was wrongly published in the original publication. The correct author name should read as follows.

Biosynthesis of Salvia Specialized Metabolites and Biotechnological Approaches to Increase Their Production

Aromatic Salvia species are particularly valuable for providing several bioactive compounds used as food additives, pigments, cosmetics, perfumes and fine chemicals. Within the Lamiaceae family, the Salvia genus, with more than 900 species, biosynthesizes a plethora of beneficial metabolites including terpenes, steroids and polyphenols. The whole plant can be considered a factory of bioactive compounds, but plant cell and tissue cultures are also an attractive sustainable alternative to cultivation. Salvia cell cultures can readily be initiated from different explants, including leaves, roots, stems, petioles, anthers and seedlings; however high metabolites accumulation in plant tissue and cell culture is a prerequisite for massive production of these bioactive compounds. In this chapter, the occurrence and tissue distribution of specialized metabolites in several Salvia species, especially flavonoids and diterpenoids, will be reviewed along with recent advances in the understanding of biosynthetic pathways as well as regulatory mechanisms leading to their biosynthesis. We will focus on the recent biotechnological approaches aimed at enhancing the final biomass and metabolite accumulation in Salvia cell and tissue cultures. Advances in metabolic engineering strategies will be also summarized, reporting relevant and successful results and potential pitfalls, in order to provide valuable perspectives for developing cell and tissue cultures as a reliable and standardized biomass platform for the extraction of Salvia bioactive metabolites.