Alberto Nonis

Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots

Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate. cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization. Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated co-regulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis/scavenging. Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.

Identification and differential expression dynamics of peach small GTPases encoding genes during fruit development and ripening

The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation.

PRaTo: a web-tool to select optimal primer pairs for qPCR.

An essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it.

Isolation and characterization of terpene synthases potentially involved in flavor development of ripening olive (Olea europaea) fruits.

The flavor and taste of fruits are often determined by terpenes. We identified three cDNAs encoding putative terpene synthases from olive fruits of cv. Frantoio and Grignano. Heterologous expression in a bacterial system demonstrated that one of the terpene synthases, OeGES1, was an active monoterpene synthase that converted geranyl diphosphate to the monoterpene alcohol geraniol. The transcript accumulation pattern of this gene showed a peak during fruit ripening in both genotypes, indicating that the enzyme may be involved in the production of monoterpene flavor compounds in olive fruit. Although the putative terpene synthases OeTPS2 and OeTPS3 clustered with α-farnesene synthases and angiosperm monoterpene synthases, no detectable in vitro activity was found after expression in a bacterial system. Nevertheless, their transcripts sharply accumulated during fruit ripening starting from véraison.

Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

Highlight In cold-stressed apple skins, the ethylene-dependent negative regulation of ROP-GAP rheostat transcripts and apoplastic H2O2 homeostasis suggests an unprecedented putative crosstalk between ROS and ethylene in abiotic stress.

Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.

The miRNA-mediated post-transcriptional regulation of maize response to nitrate

Stress responses depend on the correct regulation of gene expression. The discovery that abiotic as well as biotic stresses can regulate miRNA levels, coupled with the identification and functional analyses of stress-associated genes as miRNA targets, provided clues about the vital role that several miRNAs may play in modulating plant resistance to stresses. Nitrogen availability seriously affects crops productivity and environment and the understanding of the miRNA-guided stress regulatory networks should provide new tools for the genetic improvement of nitrogen use efficiency of crops. A recent study revealed the potential role of a number of nitrate-responsive miRNAs in the maize adaptation to nitrate fluctuations. In particular, results obtained suggested that a nitrate depletion might regulate the expression of genes involved in the starvation adaptive response, by affecting the spatio-temporal expression patterns of specific miRNAs.

Choosing between RT-qPCR and RNA-seq: a back-of-the-envelope estimate towards the definition of the break-even-point

Gene expression studies are widely diffused in life science research. So far, the most popular techniques adopted for transcript quantification are Northern blot, reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative real-time polymerase chain reaction (RTqPCR), microarrays, and RNA sequencing (RNA-seq). These methods can be grouped according to their throughput level. Northern blot is a classic low-throughput technique, whereas RT-PCR and quantitative real-time polymerase chain reaction (qPCR) are medium-throughput techniques thanks to their ability to analyze more than one transcript at a time. High-throughput techniques are devoted to measuring levels of thousands of transcripts per assay (microarrays and RNA-seq). Soon after the introduction of RNA-seq to the market, the running costs were extremely high, so the technique was affordable only to high-budget groups. In recent years, although equipment investments remained relevant, running costs markedly decreased. This scenario fostered the birth of companies offering RNA-seq as a service, so RNA-seq has become affordable. On one hand, RNA-seq is now so cheap that it has to be considered even when one is interested in the expression levels of only a fraction of the transcriptome. On the other hand, RT-qPCR is regularly used to measure numerous transcripts at a time. The state of the art of the techniques and platforms mentioned in this article has been reviewed recently by Devonshire et al. [1]. In this article we try to understand when RNA-seq may be economically convenient compared with RT-qPCR (even when one is interested in the expression levels of just a few genes instead of the whole transcriptome). Therefore, we determine the break-even point between the two techniques by calculating the number of genes/samples for which it is cheaper to use one or the another technique.

Tissue-Specific and Developmental Expression Pattern of Abscisic Acid Biosynthetic Genes in Peach Fruit: Possible Role of the Hormone in the Coordinated Growth of Seed and Mesocarp

Peach fruit tissues start differentiating into mesocarp, endocarp, and seed from the first phases of development. A seed is necessary for fruit set, and it may strongly regulate the growth of the whole fruit through metabolic or hormonal signals. Although the importance of sugar and hormone signaling in growing fruit, such as peach, has been analyzed extensively, no conclusions as to the cross-talk between these signal molecules and their role in seed and flesh development have previously been made. The present study provides insight into the regulatory steps of the carotenoid/ABA biosynthetic pathway to establish possible relationships between growth and accumulation of pigments, sugar, and hormone in the different tissues of peach fruit (Prunus persica L. Batsch, cv Redhaven). In the flesh, the transcriptional pattern of most of the genes involved in the ABA synthesis exhibits a good association with both color changes and hormone accumulation. In contrast, in the seed, along with other nongreen tissues, this association is not conceivable. The behavior of zeaxanthin epoxidase genes (Ppzep) well represents the presence of distinct regulatory mechanisms at various steps of the pathway and in a tissue-specific manner. Moreover, the key role of 9-cis-epoxycarotenoid dioxygenase (NCED) enzyme in regulating ABA synthesis appears to be substantiated by observed Ppnced expression profiles, both in the flesh and in the seed. Based on the results obtained in this study, a crucial connection between ABA biosynthesis, sugar content, and sucrose cleavage enzymes (sucrose synthase), at different stages of fruit development, is proposed.