Development of Genomic Resources in \u3cem\u3eVitis Riparia\u3c/em\u3e for Discoveries on Pre- And Post-Transcriptional Molecular Regulators of Early Induction into Endodormancy

Grapevine is one of the most important fruit crops in the world, responsible for billions in global sales annually. The largest threat to grapevine and other crop production is global climate change resulting human activities. This brings unpredictable and drastic changes in ambient air temperatures to many climates in which grapes are grown. Lower temperatures and inclement weather are already responsible for millions in lost revenue due to tissue damage of established plants. Thus, protecting grapevine crops from weather-related damage is the biggest concern to growers aside from pathogen- and diseaserelated crop damage. The primary mechanism for winter survival in woody perennial plants is bud endodormancy, a state of hibernation that is activated in response to decreasing temperatures and photoperiod. The current understanding of this process is limited, but it is believed that induction into endodormancy is controlled by a combination of hormones and transcriptional regulators internal to the cell. Grapevines have variable resistance to cold depending on species. Of the approximately 80 identified grapevine species, North American and Asian grapevines have more enhanced winter survival. Vitis riparia, the riverbank grapevine, is one of the most resistant of the genus and has been identified to enter endodormancy at longer day lengths. Investigating why V. riparia responds differently may reveal key genes and molecular mechanisms needed for photoperiod induced endodormancy induction. To investigate this speciesspecific response, we first sought to establish a genome assembly for this nonmodel species. Sequencing and assembly of DNA from V. riparia resulted in 69,616 scaffolds at an N50 of 518,740. Reference, mapping, and nonhomologous estimates of misassembly suggest that this draft assembly is of a high quality. cDNA sequence prediction from multiple RNA-seq studies resulted in 40,019 genes. Variations in gene families demonstrated that there were genetic differences between V. riparia and V. vinifera which could explain the difference in response to photoperiod and winter survival. One of the best indicators in plants of the physiological response to external regulators is changes in gene expression. We measured changes in expression during endodormancy transition in two F2 genotypes at multiple time periods of exposure to short day (SD, 13h) and long day (LD, 15h) photoperiods. Expression of genes associated with cell cycle control and phenylpropanoid biosynthesis were downregulated in response to SD treatment. The F2-110 genotype which more closely resembled V. riparia had greater natural expression of auxin signaling genes than the F2-040. This was further confirmed by coexpression networks that were highly correlated with short day induced endodormancy transition and F2-110 genotypes. Regulation of endodormancy induction is a primary concern for this study. We performed small-RNA seq to find miRNA that were differentially regulated during dormancy transition. A machine learning based prediction of miRNA identified 216 regulatory sequences in the non-model V. riparia genome. We found that miRNA families 166 and 167 were predominantly upregulated during dormancy transition. This coincided with downregulation of cell cycle control genes and suppression of cyclins and expansins by the MYB3R1 transcription factor. Motif enrichment of gene co-expression clusters identified PLETHORA 1 as a major regulator of the stem cell state during dormant conditions. These results suggest that auxin is a major regulator of endodormancy through control of cell differentiation in the bud apical meristem. Auxin signaling may therefore also be a contributor to the enhanced dormancy response in V. riparia due to an increased sensitivity to auxin in the buds. Further research is needed to determine auxin’s role in regulation of the process of endodormancy and what effect it has in crop winter survival

MicroRNAs and target genes involved in E. globulus xylogenesis: in silico prediction and experimental validation

Mestrado em Biologia Funcional - Instituto Superior de AgronomiaPortugal is one of the largest producers of pulp and paper derived from Eucalyptus globulus, which makes this a valuable species for the country. Wood is a complex and variable material, and its posttranscriptional regulation knowledge is only beginning. MicroRNAs (miRNA) are small size (21-24nt), endogenous non-coding RNAs, involved in post-transcriptional regulation. MiRBase v20 database encloses thousands of entries, however none from Eucalyptus. In this study we aim to validate E. globulus miRNAs candidates; to predict in silico and validate experimentally the miRNAs targets; and analyze the gene expression of validated targets. Four miRCa-02, miRCa-04, miRCa-08 and miRCa-09 candidates were validated by Northern blot and there in silico prediction revealed 42 target genes. Fourteen predicted target genes were tested through the RLM 5’-RACE methodology, but only three predicted targets were validated (Eucgr.E01509, Eucgr.C01382 and Eucgr.J02113 predicted target genes for miR171, miRCa-04 and miRCa-08, respectively). Expression of these three target genes analyzed by RT-qPCR suggests that the distinct expression levels found may be related with to wood formation in Eucalyptus globulus. For the first time, four Eucalytus miRNAs and their target genes were disclosed and validated by bioinformatic and molecular tools

Additional insights into the adaptation of cotton plants under abiotic stresses by in silico analysis of conserved miRNAs in cotton expressed sequence tag database (dbEST)

Abiotic stress is the primary cause of crop losses worldwide. In addition to protein coding genes, microRNAs (miRNAs) have emerged as important players in plant stress responses. Though miRNAs are key in regulating many aspects of plant developmental plasticity under abiotic stresses, very few information are available in cotton. Hence, this study was conducted to identify the phylogenetically conserved miRNAs in cotton, using computational approaches. In this paper, we reported a set of miRNAs such as miR159, miR165, miR170, miR319, miR529, miR828, miR869, miR1030, miR1884, and miR2118 that are likely to be involved in abiotic stress response. Although, few of them have been described in literature for their specific role in fiber development, literature survey have shown that they may also be involved in abiotic stress response. Interestingly, miRNAs reported in this study were found to have several targets that are involved in abiotic stress resistance. Considering all together, it was concluded that these newly identified conserved microRNAs in cotton have great potential in future efforts to improve abiotic stress tolerance in cotton.Key words: miRNA, cotton, abiotic stress resistance, in silico analysis

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it.Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background. Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. Principal Findings. We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitisspecific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). Conclusions. Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape

MicroRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.)

none2siBACKGROUND: Rice (Oryza sativa L.), one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. RESULTS: In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase) gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein), OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase) and OsDBH (DEAD-Box Helicase) genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5'RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control) in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in miRNA expression were limited. CONCLUSIONS: Osa-MIR414, osa-MIR164e and osa-MIR408 were experimentally validated for the first time in plants as targeting the OsABP, OsDBH and OsDSHCT genes. Our data showed that that the genes were up-regulated and the miRNAs were down-regulated in relation to salt stress. The negative correlation between the miRNAs and their targets was proven.Macovei, Anca; Tuteja, NarendraMacovei, Anca; Tuteja, Narendr

Identification and Characterization of Mitogen-Activated Protein Kinase (MAPK) Genes in Sunflower (Helianthus annuus L.)

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that regulate biotic and abiotic stresses in plants through signaling cascades comprised of three major subfamilies: MAP Kinase (MPK), MAPK Kinase (MKK), and MAPKK Kinase (MKKK). The main objectives of this research were to conduct genome-wide identification of MAPK genes in Helianthus annuus and examine functional divergence of these genes in relation to those in nine other plant species (Amborella trichopoda, Aquilegia coerulea, Arabidopsis thaliana, Daucus carota, Glycine max, Oryza sativa, Solanum lycopersicum, Sphagnum fallax, and Vitis vinifera), representing diverse taxonomic groups of the Plant Kingdom. A Hidden Markov Model (HMM) profile of the MAPK genes utilized reference sequences from A. thaliana and G. max, yielding a total of 96 MPKs and 37 MKKs in the genomes of A. trichopoda, A. coerulea, C. reinhardtii, D. carota, H. annuus, S. lycopersicum, and S. fallax. Among them, 28 MPKs and eight MKKs were confirmed in H. annuus. Phylogenetic analyses revealed four clades within each subfamily. Transcriptomic analyses showed that at least 19 HaMPK and seven HaMKK genes were induced in response to salicylic acid (SA), sodium chloride (NaCl), and polyethylene glycol (Peg) in leaves and roots. Of the seven published sunflower microRNAs, five microRNA families are involved in targeting eight MPKs. Additionally, we discussed the need for using MAP Kinase nomenclature guidelines across plant species. Our identification and characterization of MAP Kinase genes would have implications in sunflower crop improvement, and in advancing our knowledge of the diversity and evolution of MAPK genes in the Plant Kingdom

Molecular Tools for Adapting Viticulture to Climate Change

Adaptation of viticulture to climate change includes exploration of new geographical areas, new training systems, new management practices, or new varieties, both for rootstocks and scions. Molecular tools can be defined as molecular approaches used to study DNAs, RNAs, and proteins in all living organisms. We present here the current knowledge about molecular tools and their potential usefulness in three aspects of grapevine adaptation to the ongoing climate change. (i) Molecular tools for understanding grapevine response to environmental stresses. A fine description of the regulation of gene expression is a powerful tool to understand the physiological mechanisms set up by the grapevine to respond to abiotic stress such as high temperatures or drought. The current knowledge on gene expression is continuously evolving with increasing evidence of the role of alternative splicing, small RNAs, long non-coding RNAs, DNA methylation, or chromatin activity. (ii) Genetics and genomics of grapevine stress tolerance. The description of the grapevine genome is more and more precise. The genetic variations among genotypes are now revealed with new technologies with the sequencing of very long DNA molecules. High throughput technologies for DNA sequencing also allow now the genetic characterization at the same time of hundreds of genotypes for thousands of points in the genome, which provides unprecedented datasets for genotype-phenotype associations studies. We review the current knowledge on the genetic determinism of traits for the adaptation to climate change. We focus on quantitative trait loci and molecular markers available for developmental stages, tolerance to water stress/water use efficiency, sugar content, acidity, and secondary metabolism of the berries. (iii) Controlling the genome and its expression to allow breeding of better-adapted genotypes. High-density DNA genotyping can be used to select genotypes with specific interesting alleles but genomic selection is also a powerful method able to take into account the genetic information along the whole genome to predict a phenotype. Modern technologies are also able to generate mutations that are possibly interesting for generating new phenotypes but the most promising one is the direct editing of the genome at a precise location

Berry transcriptome comparison of ten Italian grapevine varieties

Lo sviluppo della bacca di vite pu\uf2 essere descritto come una successione di cambiamenti fisiologici e biochimici che riflettono la modulazione trascrizionale di molti geni. Nello scorso decennio molti studi trascrittomici sono stati eseguiti per descrivere in modo pi\uf9 approfondito questo processo di sviluppo dinamico e complesso. Tuttavia, la maggior parte di questi studi trascrittomici si sono focalizzati solo su un\u2019unica variet\ue0 per volta e quindi vi \ue8 ancora una mancanza di risorse per poter effettuare comparazioni sullo sviluppo della bacca in differenti variet\ue0 di vite. Questa tesi riguarda la prima comparazione del trascrittoma della bacca di vite effettuato attraverso RNA sequencing di 120 campioni di RNA, corrispondenti alle bacche di dieci variet\ue0 raccolte a quattro stadi fenologici, due precedenti e due successivi all\u2019invaiatura, in triplicato biologico. Quest\u2019analisi RNA-seq ha mostrato un\u2019evidente e profonda transizione del trascrittoma dalla fase verde alla maturazione che avviene all\u2019invaiatura indipendentemente da colore della buccia e variet\ue0, che coinvolge la soppressione di diversi processi metabolici relativi alla crescita vegetativa, e l\u2019induzione di solo poche vie, come processi di metabolismo secondario e di risposta a stimoli biotici. Questo importante riprogramma del trascrittoma durante la maturazione \ue8 stato evidenziato da diversi approcci: correlazione con distanza di Pearson, analisi a componenti principali (PCA), O2PLS-DA, ricerca di biomarcatori, analisi clustering e network di correlazione. La creazione della prima via trascrittomica di sviluppo della bacca di vite, corrispondente a geni aventi un profilo di espressione simile durante tutto lo sviluppo indipendentemente dalla variet\ue0, ha permesso di identificare geni coinvolti nei maggiori processi biologici che avvengono durante la maturazione del frutto. Infine, l\u2019espressione dei geni appartenenti alla via biosintetica dei fenilpropanoidi/flavonoidi si sono mostrati insufficienti da soli nello spiegare le differenze trascrittomiche tra variet\ue0 rosse e bianche; tuttavia si presuppone che questi \u2013 probabilmente per effetto dell\u2019accumulo di antociani nella buccia della bacca dall\u2019inizio della maturazione \u2013 influenzino comunque il programma della fase di maturazione, determinando il coinvolgimento e reclutamento di geni appartenenti ad altri processi biologici.Grape berry development can be described as a succession of physiological and biochemical changes reflecting the transcriptional modulation of many genes. In the last decade, many transcriptomic studies have been carried out to deeper describe this dynamic and complex development. Nonetheless, most of those transcriptomic studies focused on one single variety at a time and then there is still a lack of resources for comparing berry development in different grape varieties. This thesis describes the first berry transcriptome comparison carried out by RNA sequencing of 120 RNA samples, corresponding to 10-variety berries collected at four phenological growth stages, two pre- and two post-v\ue9raison, in biological triplication. This RNA-Seq analysis showed an evident deep green-to-maturation transcriptome shift occurring at v\ue9raison independently on skin colour and variety, which involves the suppression of diverse metabolic processes related to vegetative growth, and the induction of only a few pathways, such as secondary metabolic processes and responses to biotic stimuli. This fundamental transcriptome reprogramming during ripening was highlighted by distinct approaches: Pearson\u2019s correlation distance, PCA, O2PLS-DA, biomarker discovery, clustering analysis and correlation network method. The establishment of the first grape berry development transcriptomic route, corresponding to the genes having similar patterns of expression during whole development independently on the variety, allowed identifying genes involved in the main biological processes occurring during berry development. Finally, the expression of phenylpropanoid/flavonoid biosynthetic pathway-related genes was found to be insufficient by itself to explain the differences between red- and white-grape transcriptomes, however it was supposed to influence \u2013 supposedly by the effect of anthocyanins accumulation in berry skin since the onset of ripening \u2013 maturation-phase transcriptional program, determining the recruitment of genes belonging to other biological processes