P Poltronieri, Italian National Research Council, Italy, N Burbulis, Aleksandr Stulginskis
University, Lithuania and C Fogher, Catholic University, Italy
Woodhead Publishing Series in Biomedicine No. 53
From plant genomics to plant biotechnology
ISBN 1 907568 29 8
ISBN-13: 978 1 907568 29 9
November 2013
280 pages 234 x 156mm hardback
Approx. £99.00 / US$165.00 / €125.00
About the authors
Dr Palmiro Poltronieri is researcher at the Agrofood Department of the Italian National Research
Council. He is cofounder of Biotecgen SME - a service company involved in European projects (FP
VI STREP Novel roles for non-coding RNAs -RIBOREG- and the FP VII ABSTRESS, starting in
2012), developing molecular tools such as Ribochip DNA arrays, and protein chip tools. He is
Associate Editor to BMC Research Notes. He holds a Ph.D. in Molecular and Cellular Biology
from Verona University, 1995, and from 1996 to 1997 was 'Japanese Society for Promotion of
Science' post-doctoral fellow at Tsukuba University. Since 1999 as a researcher for the NRC he has
been studying plant protease inhibitors, and their applications. Current interest is on the water stress
response in roots of tolerant and sensitive chickpea varieties, activating the jasmonic acid synthesis
pathway at different timing.
Dr Natalija Burbulis is currently head of the agrobiotechnology laboratory and professor at the
Crop Science and Animal Husbandry Department of the Aleksandr Stulginskis University
(Lithuania). She holds a Ph.D. in Agricultural science obtained from the Lithuanian University of
Agriculture, and for 10 years performed research in plant biotechnology, physiology and
biochemistry. Current studies are in vitro selection of oilseed crops (rapeseed and linseed) genotypes with important agronomic traits, including disease resistance, cold tolerance and oil
quality improvements.
Professor Corrado Fogher, Ph.D., is Associate Professor of Genetics and Responsible for the
transgenic plants sector of the Observatory on Transgenic Organisms in Agriculture at the Faculty
of Agricultural Science of the Catholic University, Piacenza, Italy. He was NATO Fellow (1982-83)
at the Department of Biochemistry, University of Missouri, Columbia, Researcher (1984-85) at the
Department of Cellular Physiology and Molecular Genetics of the Pasteur Institute, Paris, and
Visiting scientist (summer of 1989, 1990, 1991, 1992, 1995) at The Scripps Research Institute, La
Jolla, California. He is Author or co-author of more than 70 peer-reviewed papers. He is Research
Director of thee SMEs, Plantechno, Incura and SunChem.
From Plant Genomics to Plant Biotechnology
Introduction
<TXT>This book aims to provide an overview of research advancements in plant genomics,
functional genomics and plant phenotyping, exploring the next generation technologies (Chapter 1),
small RNAs and RNA silencing (Chapters2and7), epigenetics (Chapter 3), metabolomics
(Chapters4 and 5), transcriptomics and functional genomics in conifers (Chapter 5), in tomato
(Chapter 6), with a special focus on the interactions between hormones and light response genes, in
grape (Chapters 7 and8) and in peach (Chapter 9), doubled haploid technology in breeding of
Brassica napus (Chapter 10), biotechnological approaches in cereal crops (Chapter 11) and
biotechnological approaches for production of bioactives such as resveratrol in biofermentors and in
modified tomato plants (Chapter 12).
Plant functional genomics is presented under different approaches, in crop plants (Chapter 1) and in
conifers (Chapter 5), with summaries of studies in genomics and transcriptomics in berries and fruit
trees (Chapters 7 and 8). These achieved scientific advancements have the potential to improve
specialty crops (fruits, vegetables) and other plants for food and non-food applications.
In the coming years, these technologies will influence scientific advancements with applicable uses,
especially in such fields as agronomy, stress-resistant varieties, improvement of plant fitness,
improving crop yields, and other non-food applications. Skilled human resources are an essential
building block for competitiveness. Supporting training and the acquisition of expertise, for young
scientists in particular, will help to widen their skill base and to develop links within and between
the academic and industrial research environments.
This book is aimed not only at plant scientists but also at academy staff and students, thanks to the
involvement of severalauthors with international genome sequencing projects and functional
genomics (the Solanaceae (tomato and potato), Rosaceae (strawberry, peach),grape, and conifers
genomics groups). In the topics covered, differences in genome structure, organization, small
RNAs, and types of fruit are presented and discussed from the point of view of different species and
groups, benefiting both plant students and specialists focused on individual plants.
In several chapters, ongoing and former international projects are presented, together with new
approaches and technologies, often led by private companies, to produce plant trees and tree
transgenics. Furthermore, the book aims to focus the attention of public authorities and the scientific
community on the problematics and the monitoring of trials with new transgenic plants, providing
some links and websites on monitoring activities of specific COST actions and international
research projects.
<CN>1
<CT>From plant genomics to -omics technologies
<AU>Palmiro Poltronieri, Institute of Sciences of Food Productions, ISPA-CNR, Via
Monteroni, 73100 Lecce, Italy
At the beginning of the last decade a revolution in high-throughput DNA sequencing, based on a
high number of capillaries, high automation, robotic handling and microplate preparation of
cDNAs, allowed collections of complete transcribed sequences (full-length cDNA libraries)
(Carninci et al. 2003) to be produced for the human and mouse complete genomes. This approach
was immediately applied to model plants such as Arabidopsis. The general opinion was that DNA
coding sequences were present in genomes at a relatively small number (approx. 25 000–30 000
genes), surrounded by junk non-coding sequences. Since 2003, large-scale studies have aimed at the
identification of RNA transcripts non-coding for proteins (ncRNAs), as a massive output of
transcription (Frith et al. 2006). RNomics took the stage from the hypothesis that DNA is thestatic
element of genetic information, thehardware, while RNA is the active information, the software.
The ability of RNAs to orchestrate chromatin states, DNA transcription, differential splicing, RNA
translation, post-transcriptional modification and protein stability determines a hidden layer of
complexity of genetic information. In thisway, not only did protein product numbers increase
through the use of different transcription starts and different splicing sites, but also genes producing
regulatory RNAs (long and small RNAs) were taken into account.
<TXTIND>ENOD40 (Campalans et al. 2004), one of the first plant riboregulators, is produced in
legume roots during nodulation, inducing the reprogramming of legume root cells. It functions as a
structured RNA, through its binding to an RNA binding protein, but is also transcribed into a small
peptide, thus belonging to a novel category of plant dual RNAs (Bardou et al. 2011).
<TXTIND>In the years from 2004 to 2007, the project ‘Riboreg’, in the frame of the European
Commission VIth Framework Programme (FP6), brought together scientists such as Martin Crespi,
Hervé Vaucheret, Joszef Burgyan, Javier Paz-Ares, Sakari Kauppinen and Jean-Marc Deragon,
focused on RNAs in plants (Poltronieri and Santino 2012). A cooperative activity supported the
production of the first Arabidopsis DNA microarray targeting ncRNAs and RNA binding proteins,
to study transcripts in different tissues and in legume plants subjected to environmental stresses
(Laporte et al. 2007).
DNA microarray platforms are today available for many plants, from Affymetrix GeneChip
technology to in-situ synthesised microarrays (CombiMatrix, NimbleGen, Oxford Gene
Technology, Agilent). Nowadays, transcriptomic studies are possible even for plants without
sequenced genomes, through the development of EST libraries, cDNA collections, and highthroughput transcript profiling and next-generation sequencing (NGS). The new sequencing
technologies (454/Roche GS FLX, SOLiD, Illumina GAIIX, new NGS platforms) have set up the
basis for genome-wide comprehensive transcriptomics and analysis of RNAs (Metzker 2008;
Oshlack et al. 2012). One recent effort focused on the transcriptome of Catharanthus roseus
(SmartCell EU project, http://www-smart-cell.org).
1.1 SuperSAGE
Serial Analysis of Gene Expression (SAGE) was a method exploited during the pre-genomic era to
individuate each transcript based on sequencing short tags of 16 to 18 bases in length. Nowadays
SuperSAGE, based on longer reads, such as tags of 26 bases in length, allows powerful serial
analysis of gene expression (Matsumura et al., 2012) (Figure 1.1).
Several protocols exploit restriction enzymes releasing 26-mer nucleotides and the application of
tags to recognise the 5’ and 3’ ends of sequenced nucleic acids, allowing whole transcriptome
studies to be performed. High-throughput SuperSAGE or DeepSuperSAGE is based on massive
sequence analysis on the new, high-throughput NGS platforms. HT-SuperSAGE is suitable for use
with the Illumina Genome Analyzer and the SOLiD sequencer (Matsumura et al., 2010). This
approach allows deep transcriptome analysis and multiplexing, while reducing time, cost and effort
of the analysis.
SuperSAGE-Arrays were used for high-throughput transcription profiling studies, in legume
genomes without completed DNA sequencing, to find differentially expressed genes in stresstolerant and stress-susceptible genotypes. The DeepSuperSAGE protocol applied to chickpea
(Molina et al., 2008,2011) allowed early global transcriptome changes in drought and salt-stressed
chickpea roots and nodules to be detected and metabolic pathways relevant to these stress responses
to be individuated. Different varieties responded differently to abiotic stresses, showing significant
intra-specific genetic variability. These studies identified new up-regulated and down-regulated
genes and isoforms with tissue-specific expression, and assigned gene function through homology
with genes already present in databases.
The SuperSAGE data were used to design Taqman primers splice variant-specific and isoformspecific for chickpea genes in Real-Time PCR studies in root tissues of two not yet studied varieties
in drought stress conditions, validating these data with high performance liquid chromatography
(HLPC) and Mass analysis of the intermediates produced and active hormone forms (De Domenico
et al., 2012).
Recent sequencing technologies have revitalised sequencing approaches in genomics and have
produced opportunities for various emerging analytical applications. A new European FP7 project,
AB-Stress, based on DeepSuperSAGE, epigenetics and DNA methylation changes, aims to
elucidate the stress-induced small RNome in legumes (pea and Medicago) plants during biotic and
abiotic stresses (Poltronieri and Santino 2012).
<A>1.2 CAGE –cap analysis of gene expression
<TXT>DNA Next-Generation Sequencing technologies have also been applied to the identification
of the 5′ end of cDNAs and to the differentiation of transcription start sites of expressed genes.
CAGE is a 5′ sequence tag technology applied to globally determine transcription start sites in the
transcribed genome and to measure the expression levels: the production of tags is combined with
Next-Gen sequencing for high-throughput processivity (Takahashi et al. 2012). In principle, a
CAGE protocol resembles a SuperSAGE protocol, except for the selective capturing of 5′ capped
mRNAs, a method previously exploited by Carninci in the preparation of full-length cDNA
libraries. Recently, CAGE has been adapted to the HeliScope single molecule sequencer. Despite
significant simplifications in the CAGE protocol, it is still a labour intensive protocol (Itoh et al.
2012).
<A>1.3 -Omics and new advancement in plant functional genomics
<TXT>Systems biology enables determination of how the interconnected networks of genes and
gene products work together in steering biological processes, for instance, to produce fruit and
grain, or to determine the performance of the plant under different specific environmental
conditions (Mochida and Shinozaki 2011). Systems biology will allow scientists to reveal how
natural genetic variation creates biodiversity and, together with innovative genomic technologies,
will support researchers in the discovery of methods for breeding plants.
There is a need for resources and analytical tools for functional genomics, through different
approaches, application of ‘omics’ (transcriptomics, proteomics, metabolomics) technologies, plant
phenotyping, Quantitative Trait Loci (QTL) analysis and identification of genes by expression QTL
(eQTL) (Figure 1.2), in order to understand the molecular systems that regulate various plant
functions.
Great numbers of plant genomes have been released in recent years, exploited for understanding of
vascular plants (Bancks et al. 2011) or highly important tree species (Grattapaglia et al. 2012,
Slavov et al. 2012), phylogenetic studies or advancements in phenotype analysis (Chia et al. 2012;
The Tomato Genome Consortium 2012). A recent overview ofplant genomes and their exploitation
discusses this wealth of data on plant genomes (Ranjan et al. 2012). Traditional studies developing
plant resources, such as conventional breeding and marker-assisted selection, need to be supported
by the genomics and -omics information, thanks to the new high-throughput platforms. These
efforts can produce improvements in food crops and non-food plants, obtaining an increase in the
production of plants with desired traits.
TILLING (Targeting Induced Local Lesions In Genomes) and collections of plant mutants, reverse
and forward genetics (tissue-specific expression and gene silencing) have been extended from
model plants (Arabidopsis, Medicago) to important food crops.
In tomato, the development of genetic and genomic resources has led to the development of
functional genomic resources of tomato as a model cultivar with great importance for human
nutrition (Ranjan et al. 2012; Matsukura et al. 2008). Tomato populations treated with 1.0% ethyl
methanesulfonate (EMS) showed a frequency (one mutation per 737 kb) suitable for producing an
allelic series of mutations in the target genes (Okabe et al. 2011; Minoia et al. 2010). Micro-Tom
TILLING platforms were used for efficient mutant isolation, as a tool to study fruit biology and for
obtaining novel genetic material to be used to improve agronomic traits. A tomato in silico
database, TOMATOMA, is a relational system interfacing modules between mutant line names and
phenotypic categories (Saito et al. 2011).
Small RNAs include microRNAs, siRNAs and tasi-RNAs (Eamens et al. 2011; Poltronieri and
Santino 2012).MicroRNAs target mRNAs by forming duplexes on the complementary seed
sequences (7 bases in length) in mRNA transcripts. miRNAs negatively affect their targets through
a variety of transcriptional and post-transcriptional mechanisms, such as mRNA degradation or
blocking transcription.
RNA silencing and RNA interference allow specific knockdown of individual gene targets. At low
concentration, microRNAs are able to affect the expression of several genes and of hundreds of
mRNAs of one gene target. Because miRNAs target several different mRNA species, often in a
tissue-specific manner, the delivery of RNAs complementary to miRNAs, as miRNA blockers, may
affect and control cell growth more strongly than antisense RNA and RNA analogs. Hence, the
ability of individual miRNAs to target multiple genes and pathways is potentially a major
advantage. Several methods have been developed to inhibit a specific microRNA, such as target
mimicry (Rubio-Somoza and Manavella 2011) or the siRNA sponges, in which long RNA strands
containing hundreds of thousands to millions of nucleotides are designed to be cleaved by
cells’RNA processing machinery into siRNAs inside the cells, to produce a high copy number of
expressed antagomiRs (Lee et al. 2012).
Diverse and complementary technologies to study plant adaptation in response to biotic and abiotic
stress will benefit from top-down and bottom-up genomics approaches to identify potential gene
candidates for innovative molecular breeding strategies. Gene overexpression and gene knock-out
in plant tissue cultures (Ariel et al. 2010) and RNA interference will take the stage in coming years
(Rubio-Somoza and Manavella 2011).
The exploitation of RNA silencing and antisense technologies for controlling gene expression
hasbeen translated into new plant phenotypes and tree populations with novel traits. Several
international collaborations are at an advanced stage, forexample, European COST activities
FA0804: http://molfarm.ueb.cas.cz/‘Molecular farming: plants as a production platform for high
value proteins’,FA1006 http://www.plantengine.eu/‘Plant Metabolic Engineering for High Value
Products’, and the EU collaborative project ‘Green factories for the next generation of
pharmaceuticals’,SmartCell. Other projects aim to develop novel tree genetic strategies, such as the
NovelTree project http://cordis.europa.eu/projects/rcn/88733_en.html,and to improve major forest
genetics and forestry research infrastructures (Trees4Future,http://www.trees4future.eu/). There are
two coordinated approaches to the topic of plant biotechnology:the first, COST FP0905, for
monitoring transgenic trees in vitro and in field trials, ‘Biosafety of forest transgenic trees:
improving the scientific basis for safe tree development and implementation of EU policy
directives’,http://www.cost-action-fp0905.eu/(Walter
et
al.
2010);and
the
second
FA0806,‘Plant virus control employing RNA-based vaccines’,http://costfa0806.aua.gr/.
COST
The exploitation of RNA silencing and antisense technologies for controlling gene expression has
already translated into new plant phenotypes and tree varieties adapted to cold climates (such as the
SENESCO Inc. proprietary technology to produce transgenic plants and trees).
Recently Carol Auer summarised the state-of-the-art of plant biotechnologies with a special focus
on new approaches based on small RNAs, RNA interference and production of RNA-mediated
traits in plants (Auer 2011). The potential of RNA-regulated traits in non-food plants and biofuelproducing plants is enormous. Accordingly, new methods for risk analysis are required to perform
analyses of off-target effects and persistence of RNAs in the environment.
Complexity Science, Informing Science, -Omics Engineering could effectively support and
integrate methodologies of different academic fields. To this end, cloud computing and the sharing
of data networks are possible today using new tools and software, designed to work in Linux based
environments, while new systems will become open for use on Microsoft, such as the
Windows2Galaxy project. The continued adoption of Galaxy by the life sciences community
depends on the enhancement of features and development of new functionality. A number of new
features were recently highlighted by members of the Galaxy development team (Li et al. 2012).
Some recent scientific developments have shown an impact on food and non-food crops:
breakthroughs in understanding how plant cells recognise different hormones and which signalling
pathways are activated by hormones (Razem and Baron 2006; Fuji et al. 2009; Yin et al. 2009) and
the links between epigenetics and abiotic stress memory (Urano et al. 2008); the role of plant
sRNAs and epigenetics in the regulation of development and stress response (Chuck and O’Connor
2010; Matsui et al. 2008; Mercian et al. 2007); and understanding how interactions between genome
elements (DNA, RNA) and the environment make a plant body. Understanding how non-coding
RNAs work will reveal novel mechanisms involved in growth control and differentiation.
<TXTIND>Skilled human resources are an essential building block for competitiveness. Supporting
young scientists and their training in new technologies will help to widen their skill base and to
develop links within and between the academic and industrial research environments. In the
forthcoming years these advancements will support the production of plant varieties better suited to
resist biotic and abiotic stresses, for food and non-food applications.
1.4 Bibliography
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Figure 1.1 SuperSAGE protocol scheme: cDNA cleavage, formation of DNA tags and ligation into
ditags. Source: GenXpro, Germany
Systems Biology
Figure 2. Multidisciplinary approaches in plant science.
Index
Chapter 2 Plant microRNAs
Moreno Colaiacovo and Primetta Faccioli,Genomics Research Centre, Agricultural Research
Council, Fiorenzuola d’Arda, Italy
Chapter 3
Epigenetic control by plant Polycomb proteins: new perspectives and emerging roles in stress
response
Filomena De Lucia,Institut Pasteur, France, and Valérie Gaudin, Institut Jean-Pierre Bourgin,
France
Chapter 4 Metabolite profiling for plant research
Nalini Desai and Danny Alexander,Metabolon, Inc.,USA
Chapter 5
The uniqueness of conifers
Carmen Diaz-Sala, Department of Plant Biology, University of Alcalá, Spain,José Antonio
Cabezas, National Research Institute for Agricultural and Food Technology (INIA), Spain,
Brígida Fernández de Simón, National Research Institute for Agricultural and Food Technology
(INIA), Spain, Dolores Abarca, Department of Plant Biology, University of Alcalá, Spain, M.
Ángeles Guevara, National Research Institute for Agricultural and Food Technology (INIA),
Spain, Mixed Unit of Forest Genomics and Ecophysiology, INIA/UPM, Spain, Marina de
Miguel, National Research Institute for Agricultural and Food Technology (INIA), Spain,
Estrella Cadahía, National Research Institute for Agricultural and Food Technology (INIA),
Spain, Ismael Aranda, National Research Institute for Agricultural and Food Technology (INIA),
Spain, and María-Teresa Cervera, National Research Institute for Agricultural and Food
Technology (INIA), Spain,Mixed Unit of Forest Genomics and Ecophysiology, INIA/UPM, Spain
Chapter 6
Cr yptochrome genes modulate global transcriptome of tomato
Loredana Lopez andGaetano Perrotta,ENEA, Trisaia Research Centre, Italy
Chapter 7
Genomics of grapevine: from genomics research on model plants to crops and from science to
grapevine breeding
Fatemeh Maghuly, BOKU VIBT, Austria,Giorgio Gambino, Plant Virology Institute, National
Research Council (IVV-CNR), Italy,Tamas Deak, Corvinus University of Budapest, Hungary, and
Margit Laimer,BOKU VIBT, Austria
Chapter 8
Grapevine genomics and phenotypic diversity of bud sports, varieties and wild relatives
Gabriele Di Gasperoand Raffaele Testolin, Dipartimento di Scienze Agrarie e Ambientali,
University of Udine, Italy and Institute of Applied Genomics / Istituto di Genomica Applicata,
Parco Scientifico e Tecnologico Luigi Danieli, Italy
Chapter 9
Peach ripening transcriptomics unveils new and unexpected targets for the improvement of
drupe quality
Nicola Busatto, Abdur Md Rahim andLivio Trainotti,University of Padova, Italy
Chapter 10
Application of doubled haploid technology in breeding of Brassica napus
Natalija Burbulis,Aleksandras Stulginskis University, Lithuania and Laima S. Kott, University of
Guelph, Canada
Chapter 11
Plant biodiversity and biotechnology: A focus on cereals
Naglaa A. Ashry,Field Crops Research Institute, ARC, Egypt
Chapter 12
Natural resveratrol bioproduction
Angelo Santino, Marco Taurino, Ilaria Ingrosso and Giovanna Giovinazzo,Institute of Sciences of
Food Productions, CNR-ISPA, Italy