high-throughput-robust-highly-time-flexible-method-for-surface

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Fondazione Edmund Mach

In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.

Evolution of MIR168 paralogs in Brassicaceae.

Plastid genomes, also known as plastomes, are shaped by the selective forces acting on the fundamental cellular functions they code for and thus they are expected to preserve signatures of the adaptive path undertaken by different plant species during evolution. To identify molecular signatures of positive selection associated to adaptation to contrasting ecological niches, we sequenced with Solexa technology the plastomes of two congeneric Brassicaceae species with different habitat preference, Cardamine resedifolia and Cardamine impatiens.

Plastome organization and evolution of chloroplast genes in Cardamine species adapted to contrasting habitats.

Species evolution depends on numerous and distinct forces, including demography and natural selection. For example, local adaptation and population structure affect the evolutionary history of species living along environmental clines. This is particularly relevant in plants, which are often characterized by limited dispersal ability and the need to respond to abiotic and biotic stress factors specific to the local environment. Here we study the demographic history and the possible existence of local adaptation in two related species of Brassicaceae, Cardamine impatiens and Cardamine resedifolia, which occupy separate habitats along the elevation gradient. Previous genome-wide analyses revealed the occurrence of distinct selective pressures in the two species, with genes involved in cold response evolving particularly fast in C. resedifolia. In this study we surveyed patterns of molecular evolution and genetic variability in a set of 19 genes, including neutral and candidate genes involved in cold response, across 10 populations each of C. resedifolia and C. impatiens from the Italian Alps (Trentino). We inferred the population structure and demographic history of the two species, and tested the occurrence of signatures of local adaptation in these genes. The results indicate that, despite a slightly higher population differentiation in C. resedifolia than in C. impatiens, both species are only weakly structured and that populations sampled at high altitude experience less gene flow than low-altitude ones. None of the genes showed signatures of positive selection, suggesting that they do not seem to play relevant roles in the current evolutionary processes of adaptation to alpine environments of these species. 

Demographic History, Population Structure, and Local Adaptation in Alpine Populations of Cardamine impatiens and Cardamine resedifolia.

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a "triploid bridge", between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in , a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for  and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the "triploid bridge" for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of  evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the  genus and other Arundinoideae.

Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the "Triploid Bridge" in  (Poaceae).

Phytochelatin synthases (PCSs) play essential roles in detoxification of a broad range of heavy metals in plants and other organisms. Until now, however, no PCS gene from liverworts, the earliest branch of land plants and possibly the first one to acquire a PCS with a C-terminal domain, has been characterized. In this study, we isolated and functionally characterized the first PCS gene from a liverwort, Marchantia polymorpha (MpPCS). MpPCS is constitutively expressed in all organs examined, with stronger expression in thallus midrib. The gene expression is repressed by Cd2+ and Zn2+. The ability of MpPCS to increase heavy metal resistance in yeast and to complement cad1-3 (the null mutant of the Arabidopsis ortholog AtPCS1) proves its function as the only PCS from M. polymorpha. Site-directed mutagenesis of the most conserved cysteines of the C-terminus of the enzyme further uncovered that two twin-cysteine motifs repress, to different extents, enzyme activation by heavy metal exposure. These results highlight an ancestral function of the PCS elusive C-terminus as a regulatory domain inhibiting enzyme overactivation by essential and non-essential heavy metals. The latter finding may be relevant for obtaining crops with decreased root to shoot mobility of cadmium, thus preventing its accumulation in the food chain.

Article	Year
Evolution of MIR168 paralogs in Brassicaceae. BMC evolutionary biology\| PubMed ID: 19309501	2009
Plastome organization and evolution of chloroplast genes in Cardamine species adapted to contrasting habitats. BMC genomics\| PubMed ID: 25887666	2015
Demographic History, Population Structure, and Local Adaptation in Alpine Populations of Cardamine impatiens and Cardamine resedifolia. PloS one\| PubMed ID: 25933225	2015
In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases. Molecular biology and evolution\| PubMed ID: 28637270	2017
Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the "Triploid Bridge" in (Poaceae). International journal of molecular sciences\| PubMed ID: 32722033	2020
Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation. Journal of experimental botany\| PubMed ID: 32936292	2020

Enrico Barbaro

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