Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity.

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species.

Agrobacterium rhizogenes rolB oncogene: An intriguing player for many roles.

The rolB oncogene is one of the so-called rol genes found in the T-DNA region of the Agrobacterium rhizogenes Ri plasmid and involved in the hairy root syndrome, a tumour characterized by adventitious root overgrowth on plant stem. rolB produces in plants a peculiar phenotype that, together with its root-inducing capacity, has been connected to auxin sensitivity. The gene is able to modify the plant genetic programme to induce meristem cells and direct them to differentiate not only roots, but also other cells, tissues or organs. Besides its essential function in hairy root pathogenesis, the rolB role has been progressively extended to cover several physiological aspects in the transgenic plants: from secondary metabolites production and ROS inhibition, to abiotic and biotic stress tolerance and photosynthesis improvement. Some of the observed effects could be determined, at least in part, through microRNAs molecules, suggesting an epigenetic control rolB-mediated. These multifaceted capacities could allow plants to withstand adverse environmental conditions, enhancing fitness. In spite of this expanding knowledge, functional analyses did not detect yet any definitive rolB-derived biochemical product, even if more than one enzymatic activity has been ascribed to it. Moreover, phylogenetic and evolutionary studies evidenced no homology with any plant sequences but, otherwise, it belongs to the Plast family, a group of rolB-homologous bacterial genes. Finally, the finding of sequences similar to rolB in plants not infected by A. rhizogenes suggests a hypothetical plant origin for this gene, implying different possibilities about its evolution.

Effect of far-red light exposure on photosynthesis and photoprotection in tomato plants transgenic for the Agrobacterium rhizogenes rolB gene.

Previous work showed in tomato plants harbouring the Agrobacterium rhizogenes rolB gene overexpression of genes involved in chloroplast function and stress response, significant increase in non-photochemical quenching and chlorophyll a and b content, and reduced chlorophyll a/b ratio. The latter condition being typical of plant shade where far-red is dominant, suggested a role for rolB in improving photosynthesis in such condition. To gain a better insight into these results, the photosynthetic performance of transgenic and control plants was compared by means of variable fluorescence kinetics with a WATER-PAM chlorophyll fluorometer, after 6 days-exposure to white light and to a far-red-enriched light source. Photosynthetic parameters analysed were quantum yield of photosystem II photochemistry Y(II); qL, corresponding to the fraction of open PSII reaction centers in a "lake" model of photosystem II; non-photochemical quenching and Y(NO), describing, respectively, regulated and non-regulated pathways for dissipation of excess energy. Chlorophyll a and b content was also analysed by HPLC. Finally, real-time PCR was performed to quantify the expression level of some of the chloroplast-related genes already shown to be overexpressed in transgenic plants. Quantum yield of photosystem II photochemistry decreased with increasing light intensity, showing no significant differences in both plant genotypes and light regimen. qL, on the other hand, was significantly higher at low PAR intensities, in particular in FR-treated transgenic plants. Fate of remaining light energy, channelled into regulated or non-regulated dissipation pathways, was different in transgenic and control plants, indicating a higher capability for protection from photodamage in rolB plants, particularly after exposure to far-red-enriched light. Chlorophyll a/b ratio was also decreased in transgenic plants under far-red-enriched light with respect to white light. Finally, qPCR showed that the expression of genes encoding small heat shock protein, chlorophyll a/b binding protein and carbonic anhydrase was significantly induced by far-red-enriched condition. Taken together, these data suggest the involvement of rolB in photosynthesis modulation under far-red-rich light in tomato.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants.

Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis.

Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells.

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1ß. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1ß-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.

The rolD oncogene promotes axillary bud and adventitious root meristems in Arabidopsis.

The expression of the Agrobacterium rhizogenes rolD oncogene induces precocious floral transition and strong flowering potential in tobacco and tomato. Here, we describe specific developmental effects induced by expression of rolD in Arabidopsis. We show that floral transition, as histologically monitored, occurred in rolD- plants earlier than in wild type, and this was coupled with a premature and enhanced formation of vegetative and reproductive axillary bud meristems. Furthermore, CYP79F1/SUPERSHOOT/BUSHY (SPS), a gene that negatively controls shoot branching in Arabidopsis and involved in glucosinolate metabolism and in cytokinin and auxin homeostasis, was down-regulated in rolD plants. The multiplication of post-embryonic meristems was also observed in the root system, with enhanced adventitious root formation. This result was confirmed by thin cell layer response in vitro, both under hormone-free and standard rooting conditions. However, the formation of lateral root meristems was not affected by rolD expression. Our results show that rolD accelerates and enhances specific post-embryonic meristems in Arabidopsis.