Maria Luisa Chiusano

Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance

Global warming is a major threat for agriculture and food safety and in many cases the negative effects are already apparent. The current challenge of basic and applied plant science is to decipher the molecular mechanisms of heat stress response (HSR) and thermotolerance in detail and use this information to identify genotypes that will withstand unfavorable environmental conditions. Nowadays X-omics approaches complement the findings of previous targeted studies and highlight the complexity of HSR mechanisms giving information for so far unrecognized genes, proteins and metabolites as potential key players of thermotolerance. Even more, roles of epigenetic mechanisms and the involvement of small RNAs in thermotolerance are currently emerging and thus open new directions of yet unexplored areas of plant HSR. In parallel it is emerging that although the whole plant is vulnerable to heat, specific organs are particularly sensitive to elevated temperatures. This has redirected research from the vegetative to generative tissues. The sexual reproduction phase is considered as the most sensitive to heat and specifically pollen exhibits the highest sensitivity and frequently an elevation of the temperature just a few degrees above the optimum during pollen development can have detrimental effects for crop production. Compared to our knowledge on HSR of vegetative tissues, the information on pollen is still scarce. Nowadays, several techniques for high-throughput X-omics approaches provide major tools to explore the principles of pollen HSR and thermotolerance mechanisms in specific genotypes. The collection of such information will provide an excellent support for improvement of breeding programs to facilitate the development of tolerant cultivars. The review aims at describing the current knowledge of thermotolerance mechanisms and the technical advances which will foster new insights into this process.

Transcriptomic Changes Drive Physiological Responses to Progressive Drought Stress and Rehydration in Tomato

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical, and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation, and chlorophyll fluorescence), abscisic acid (ABA), and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes (DEGs) between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting, and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Identification of novel small ncRNAs in pollen of tomato

BackgroundThe unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions.ResultsUsing the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions.ConclusionsThus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.

NexGenEx-Tom: a gene expression platform to investigate the functionalities of the tomato genome

BackgroundNext Generation Sequencing technologies (NGS) unexpectedly pushed forward the capability of solving genome organization and of widely depicting gene expression. However, although the flourishing of tools to process the NGS data, versatile and user-friendly computational environments for integrative and comparative analyses of the results from the increasing amount of collections are still required.The gene expression of tomato tissues has been widely investigated in the years, thanks to both EST sequencing and different microarray platforms. However, the resulting collections are heterogeneous in terms of experimental approaches, genotypes and conditions, making the data far from representing a gene expression atlas for the species. Therefore, the recent release of NGS transcriptome collections from several tissues and stages from physiological conditions for specific tomato genotypes provides a relevant resource to be appropriately exploited to address key questions on gene expression patterns, such as those related to fruit ripening and development in tomato. The organization of the results from the processed collections in web accessible environments, enriched with tools for their exploration, may represent a precious opportunity for the scientific research in tomato and a reference example for similar efforts.DescriptionHere we present the architecture and the facilities of NexGenEx-, a web based platform that offers processed NGS transcriptome collections and enables immediate analyses of the results. The platform allows gene expression investigations, profiling and comparisons, and exploits different resources.Specifically, we present here the platform partition NexGenEx-Tom, dedicated to the organization of results from tomato NGS based transcriptomes.ConclusionIn the current version, NexGenEx-Tom includes processed and normalized NGS expression data from three collections covering several tissue/stages from different genotypes. Beyond providing a user-friendly interface, the platform was designed with the aim to easily be expanded to include other NGS based transcriptome collections. It can also integrate different genome releases, possibly from different cultivars or genotypes, but even from different species. The platform is proposed as an example effort in tomato, and is described as a profitable approach for the exploitation of these challenging and precious datasets.

Integrated bioinformatics to decipher the ascorbic acid metabolic network in tomato

Ascorbic acid is involved in a plethora of reactions in both plant and animal metabolism. It plays an essential role neutralizing free radicals and acting as enzyme co-factor in several reaction. Since humans are ascorbate auxotrophs, enhancing the nutritional quality of a widely consumed vegetable like tomato is a desirable goal. Although the main reactions of the ascorbate biosynthesis, recycling and translocation pathways have been characterized, the assignment of tomato genes to each enzymatic step of the entire network has never been reported to date. By integrating bioinformatics approaches, omics resources and transcriptome collections today available for tomato, this study provides an overview on the architecture of the ascorbate pathway. In particular, 237 tomato loci were associated with the different enzymatic steps of the network, establishing the first comprehensive reference collection of candidate genes based on the recently released tomato gene annotation. The co-expression analyses performed by using RNA-Seq data supported the functional investigation of main expression patterns for the candidate genes and highlighted a coordinated spatial–temporal regulation of genes of the different pathways across tissues and developmental stages. Taken together these results provide evidence of a complex interplaying mechanism and highlight the pivotal role of functional related genes. The definition of genes contributing to alternative pathways and their expression profiles corroborates previous hypothesis on mechanisms of accumulation of ascorbate in the later stages of fruit ripening. Results and evidences here provided may facilitate the development of novel strategies for biofortification of tomato fruit with Vitamin C and offer an example framework for similar studies concerning other metabolic pathways and species.

pATsi: Paralogs and Singleton Genes from Arabidopsis thaliana:

Arabidopsis thaliana is widely accepted as a model species in plant biology. Its genome, due to its small size and diploidy, was the first to be sequenced among plants, making this species also a reference for plant comparative genomics. Nevertheless, the evolutionary mechanisms that shaped the Arabidopsis genome are still controversial. Indeed, duplications, translocations, inversions, and gene loss events that contributed to the current organization are difficult to be traced. A reliable identification of paralogs and single-copy genes is essential to understand these mechanisms. Therefore, we implemented a dedicated pipeline to identify paralog genes and classify single-copy genes into opportune categories. PATsi, a web-accessible database, was organized to allow the straightforward access to the paralogs organized into networks and to the classification of single-copy genes. This permits to efficiently explore the gene collection of Arabidopsis for evolutionary investigations and comparative genomics.

Survey of Genes Involved in Biosynthesis, Transport, and Signaling of Phytohormones with Focus on Solanum lycopersicum

Phytohormones control the development and growth of plants, as well as their response to biotic and abiotic stress. The seven most well-studied phytohormone classes defined today are as follows: auxins, ethylene, cytokinin, abscisic acid, jasmonic acid, gibberellins, and brassinosteroids. The basic principle of hormone regulation is conserved in all plants, but recent results suggest adaptations of synthesis, transport, or signaling pathways to the architecture and growth environment of different plant species. Thus, we aimed to define the extent to which information from the model plant Arabidopsis thaliana is transferable to other plants such as Solanum lycopersicum. We extracted the co-orthologues of genes coding for major pathway enzymes in A. thaliana from the translated genomes of 12 species from the clade Viridiplantae. Based on predicted domain architecture and localization of the identified proteins from all 13 species, we inspected the conservation of phytohormone pathways. The comparison was complemented by expression analysis of (co-) orthologous genes in S. lycopersicum. Altogether, this information allowed the assignment of putative functional equivalents between A. thaliana and S. lycopersicum but also pointed to some variations between the pathways in eudicots, monocots, mosses, and green algae. These results provide first insights into the conservation of the various phytohormone pathways between the model system A. thaliana and crop plants such as tomato. We conclude that orthologue prediction in combination with analysis of functional domain architecture and intracellular localization and expression studies are sufficient tools to transfer information from model plants to other plant species. Our results support the notion that hormone synthesis, transport, and response for most part of the pathways are conserved, and species-specific variations can be found.

Exploiting a Reference Genome in Terms of Duplications: The Network of Paralogs and Single Copy Genes in Arabidopsis thaliana.

Arabidopsis thaliana became the model organism for plant studies because of its small diploid genome, rapid lifecycle and short adult size. Its genome was the first among plants to be sequenced, becoming the reference in plant genomics. However, the Arabidopsis genome is characterized by an inherently complex organization, since it has undergone ancient whole genome duplications, followed by gene reduction, diploidization events and extended rearrangements, which relocated and split up the retained portions. These events, together with probable chromosome reductions, dramatically increased the genome complexity, limiting its role as a reference. The identification of paralogs and single copy genes within a highly duplicated genome is a prerequisite to understand its organization and evolution and to improve its exploitation in comparative genomics. This is still controversial, even in the widely studied Arabidopsis genome. This is also due to the lack of a reference bioinformatics pipeline that could exhaustively identify paralogs and singleton genes. We describe here a complete computational strategy to detect both duplicated and single copy genes in a genome, discussing all the methodological issues that may strongly affect the results, their quality and their reliability. This approach was used to analyze the organization of Arabidopsis nuclear protein coding genes, and besides classifying computationally defined paralogs into networks and single copy genes into different classes, it unraveled further intriguing aspects concerning the genome annotation and the gene relationships in this reference plant species. Since our results may be useful for comparative genomics and genome functional analyses, we organized a dedicated web interface to make them accessible to the scientific community

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.

Bioinformatics resources for pollen.

Key messageBioinformatics for Pollen.AbstractPollen plays a key role in crop production, and its development is the most delicate phase in reproduction. Different metabolic pathways are involved in pollen development, and changes in the level of some metabolites, as well as responses to stress, are correlated with the reduction in pollen viability, leading consequently to a decrease in the fruit production. However, studies on pollen may be hard because gamete development and fertilization are complex processes that occur during a short window of time. The rise of the so-called -omics sciences provided key strategies to promote molecular research in pollen tissues, starting from model organisms and moving to increasing number of species. An integrated multi-level approach based on investigations from genomics, transcriptomics, proteomics and metabolomics appears now feasible to clarify key molecular processes in pollen development and viability. To this aim, bioinformatics has a fundamental role for data production and analysis, contributing varied and ad hoc methodologies, endowed with different sensitivity and specificity, necessary for extracting added-value information from the large amount of molecular data achievable. Bioinformatics is also essential for data management, organization, distribution and integration in suitable resources. This is necessary to catch the biological features of the pollen tissues and to design effective approaches to identifying structural or functional properties, enabling the modeling of the major involved processes in normal or in stress conditions. In this review, we provide an overview of the available bioinformatics resources for pollen, ranging from raw data collections to complete databases or platforms, when available, which include data and/or results from -omics efforts on the male gametophyte. Perspectives in the fields will also be described.