From Plant Genomics to omics technologies

Palmiro Poltronieri
Institute of Sciences of Food Productions, ISPA-CNR, Via Monteroni, 73100 Lecce, Italy,

Abstract
The new technologies for DNA and RNA sequencing have made possible to identify and analyse
individual transcripts and differentially spliced isoforms at a relatively low cost. Several plant
genomes are today known as also what is the genome output, a multitude of transcripts. The
ongoing studies try to understand what is their function and to locate their products into networks
and pathways. One useful tool applied to this means is RNA silencing. Antisense RNA belongs to
gene knock-out technologies that are used either in cell and tissue cultures as well as in whole
plants. Novel applications and the production of transgenic plants and trees require a surveillance
on the positive and negative effects on the environment and in general on life biodiversity that may
result from their introduction.

Keywords: RNA; Expressed Sequence Tags (ESTs); Serial Analysis of Gene Expression (SAGE);
cDNAs; Cap Analysis of Gene Expression (CAGE); transcriptomics; Next Generation Sequencing
(NGS); proteomics; metabolomics; Systems Biology; Targeting Induced Local Lesions In Genomes
(TILLING); Quantitative Trait Locus (QTL); functional genomics; RNA silencing.

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 to produce collections of complete transcribed sequences (full length cDNA
libraries) (Carninci et al. 2003) 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. 25000-30000

genes), surrounded by junk non-coding sequences. Since 2003, large scale studies 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 a static
element of genetic information, a hardware, 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 determine a hidden layer of
complexity of genetic information. In such way, not only protein product numbers increased
through the use of different transcription starts and different splicing sites, but also genes producing
regulatory RNAs (long and small RNAs) were taken in account.
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 either 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).
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, Herv
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 (i.e. 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 through
High-throughput 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).

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 to perform whole
transcriptome studies. High-throughput SuperSAGE or DeepSuperSAGE, is based on the massive
sequence analysis on the new, high-throughput, NGS platforms. HT-SuperSAGE is suitable to be
used 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
for 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 and 2011) allowed to detect early global transcriptome changes in drought and
salt-stressed chickpea roots and nodules and to individuate metabolic pathways relevant to these
stress responses. Different varieties responded differently to abiotic stresses, assessing a 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 isoform-

specific 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 HLPC and Mass analysis of the
intermediates produced and active hormone forms (De Domenico et al., 2012).
Recent sequencing technologies have revitalized 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).

CAGE - Cap analysis of gene expression

Next-Generation Sequencing technologies have been applied also to the identification of 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 star 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 up to now a labour intensive protocol (Itoh et
al. 2012).

-OMICS and new advancement in plant functional genomics

Systems biology enables to determine 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.
In these years, plant genomes have been released in a great number, exploited either for
understanding of vascular plants (Bancks et al. 2011), 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 on plant 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 lead 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
block of 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 of 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 an (Ariel et al. 2010) and RNA interference will take the stage in the next
years (Rubio-Somoza and Manavella 2011).
The exploitation of RNA silencing and antisense technologies for controlling gene expression have
been translated in new plant phenotypes and tree populations with novel traits. Several international
collaborations are at an advanced stage, as example, European COST activities FA0804:

http://molfarm.ueb.cas.cz/ Molecular farming: plants as a production platform for high value

proteins; and 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/). Plant
biotechnology is the topic of two coordinated approaches, the first for monitoring transgenic trees in
vitro and in field trials, i.e. the COST FP0905: 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 COST FA0806 Plant virus control
employing RNA-based vaccines http://costfa0806.aua.gr/.
The exploitation of RNA silencing and antisense technologies for controlling gene expression have
already translated in new plant phenotypes and tree varieties adapted to cold climates (such as the
SENESCO 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 biofuel
producing 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 today possible 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 are recognising 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 (Mercian et al.
2007; Chuck and OConnor 2010; Matsui et al. 2008); understanding how interactions between
genome elements (DNA, RNA) and the environment make a plant body. The understanding how
non-coding RNAs work will reveal novel mechanisms involved in growth control and
differentiation.
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.

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Figure 1.1. SuperSAGE protocol scheme: cDNA cleavage, formation of DNA tags and ligation into
ditags (source: GenXpro, Germany).

Figure 1.2. Approaches to applications of Systems Biology and Omics technologies in plant crops
improvement.

Systems Biology