1

THE ‘-OMICS’ TECHNOLOGIES AND CROP IMPROVEMENT

R.C. Setia and Neelam Setia
Department of Botany
Punjab Agricultural University, Ludhiana-141004, India
email: rc_setia@yahoo.co.in

ABSTRACT

In view of ever-growing population and decreasing natural resources, there is need to enhance
food production that can possibly be achieved by improving upon qualitative and quantitative
traits of crop plants by adopting new analytical tools and technologies. During the last decade
or so rapid progress has been made in plant biology, especially with the introduction of high
throughput ‘omics’ technologies. The three main omics technologies – genomics, proteomics
and metabolomics involve quantification and characterization of genome, proteome and
metabolome, respectively, with extremely rapid, miniaturized and automated methods. These
technologies are aimed at unraveling the overall expression of genes, proteins and metabolites
in a functionally relevant context, and provide insights into the molecular basis of various
fundamental processes involved in growth and development of plants and their environment.
Advances in plant genomics research have opened up new perspectives and opportunities
for improving crop plants and their productivity. The development of sequencing techniques
and availability of genomes’ information on model organisms, Arabidopsis thaliana, rice,
etc., have greatly influenced the disciplines of plant and crop sciences. Gene discovery and
gene expression profiling technologies are creating an unprecedented opportunity for plant
breeders who can now apply molecular markers to assess and enhance diversity in their
germplasm collections, to introgress valuable traits from new sources and identify genes
that control key traits. The genomics technologies have been found useful in deciphering
the multigenicity of biotic and abiotic plant stress responses through genome sequences,
stress specific cell and tissue transcript collections, transcript, protein and metabolite profiles
and their dynamic changes, protein interactions and mutant screens. As a consequence of
the use of high throughput omics methods a vast amount of raw data is generated which is
stored, processed and analyzed with the help of bioinformatics tools. This review provides
an introduction to the three core omics technologies, relevant methodologies and applications
with emphasis on crop improvement strategies.

Keywords: Omics, genomics, transcriptomics, proteomics, metabolomics, bioinformatics, crop

improvement

Crop Improvement: Strategies and Applications z Editors: R.C. Setia, Harsh Nayyar and Neelam Setia
© 2008 I.K. International Publishing House Pvt. Ltd., New Delhi, pp 1-18
2 Crop Improvement: Strategies and Applications

INTRODUCTION
The twentieth century has seen tremendous increase in food production with the introduction of
high yielding crop varieties, especially since the first “green revolution” that helped in keeping pace
with the population growth. The increase in productivity has been powered by changes in the genetic
potential of crops and relevant management practices. The conventional plant breeding strategies
based on phenotypic selection and principles of statistical qualitative genetics have led to continuous
increase in seed yield (mainly due to increased harvest index) and improved yield stability
(Wollenweber et al., 2005, Hammer and Jordan, 2007). Though long term selection experiments
have indicated sufficient potential for genetic improvement of qualitative traits over many generations
(Dudley and Lambert, 1992), a decline in crop yield increase has become evident (Conway and
Toenniessen, 1999; Mann, 1999) that may result in a gap between demand and supply due to high
population growth rate (Wollenweber et al., 2005).
The global population projections are grim reminder of the imperative need to increase food
production and double it by the year 2040 from the present level. The future food security for ever-
growing population will depend on acceleration of yield gains per unit land and per unit of input for
the major food crops at rates well above the historical trend of past 50 years. However, natural
resources are decreasing rapidly for agriculture as a result of economic development, which is
diverting these resources for non-agricultural uses. The leading resource and environmental constraints
faced by the world’s farmers today include soil loss and degradation; water logging, drought and
salinity; the co-evolution of pests, pathogens and host; and impact of climate change (Tilman et al.,
2001). In the arid and semiarid areas of world, water scarcity is becoming an increasingly serious
constraint on growth of agriculture production (Raskin et al., 1998;Gleick, 2000). International Water
Management Institute has projected that by the year 2025, most regions of the world will experience
either absolute or severe water scarcity (Ruttan, 2005). Thus, drought has become the single most
limiting constraint to crop production worldwide, and the need for a ‘blue revolution’ in which
water use efficiency (WUE) of crop plants is improved, has been highlighted (Zhang and Yang,
2004).
The growing worldwide demand for enhancing yields of major crops is placing pressure on
breeding programmes to provide elite cultivars that can adopt to range of environments without
compromising agronomic performance, grain quality or disease resistance. Plant scientists have been
making advances in understanding the biochemical and molecular processes that underlie important
metabolic, physiological and developmental traits that affect the ability of plants to cope with
unfavourable environmental conditions (abiotic and biotic stresses) for several decades. However, it
was often difficult to exploit information for plant breeding, because level of understanding was not
deep enough, and necessary techniques were not available. For closing the ‘yield gap’ and increasing
yield for securing food security and food quality, identification of bottlenecks of plant development
under prevailing environmental conditions and their elimination is of pivotal interest. Molecular
transformation is commonly offered as a hope to overcome the apparent stagnation in crop yield
potential.
Many crop traits are quantitative, complex and controlled by multiple interacting genes. Advances
in molecular biology are now providing the tools to study the genetical make-up of plants, which
allows us to unravel the inheritance of all traits whether they are controlled by single (major) genes,
or many genes (of smaller effect) acting together, known as the quantitative trait loci (QTL). The
molecular marker technologies available since the 1980’s, enabled the variation in traits to be dissected
 The ‘-OMICS’ Technologies and Crop Improvement 3

into the effects of QTL. With the progress of QTL mapping, new breeding approaches such as
marker-assisted selection and breeding by design (Miflin, 2000; Peleman and vander Voort, 2003)
have emerged. But existing QTL-analysis methods do not have precision required to handle plant’s
intrinsic complexities such as polygenic control, epistasis and genotype-environment interactions.
Support from other disciplines like functional genomics, system biology and crop physiology can
jointly improve the genetic analysis and breeding efficiency. Genetic improvements in crop plants
beyond current capabilities are needed to meet the growing world demand not only for food, but
also for greater diversity of food, high quality of food and safer food produced in less land, while
at the same time conserving the soil, water and genetic resources.
During recent past, plant biologists witnessed the most explosive growth of information in the
history of science. Not only is this avalanche of information providing new insights into how plant
works, but it is also creating entirely new scientific disciplines. The newly developed various ‘omics’
technologies have brought revolution in plant science research. Consequently, the availability of
complete genome sequences for reference plant species Arabidopsis thaliana and more recently for
rice, poplar and other organisms hold great potential for research aimed at crop improvement and
crop protection (Borovitz and Chory, 2004). Scientists, through a variety of functional genomic
approaches, are characterizing the genes that control key processes. Thus, in this era of new emerging
technologies, plant scientists/breeders worldwide are recognizing the power that ‘-omics’ can bring
to their efforts for crop improvement.
The emergence of the novel ‘omics’ technologies, such as genomics, proteomics and
metabolomics, is now permitting researchers to identify the genetic underpinnings of crop
improvement, namely the genes, that contribute to the improved productivity and quality of modern
crop varieties. These omics technologies enable a direct and unbiased monitoring of the factors
affecting crop growth and yield formation, and provide the data that can be directly utilized to
investigate the complex interplay between the plant, its metabolism, and also the stress represented
by the environment or the biological threats by insects, fungi or other pathogens. These technologies
also help in thorough investigation of the biology behind agronomic traits at the physiological,
biochemical and molecular levels, and permit the elucidation of molecular circuitry of the crop
plants, ultimately paving way for improved crop production (www.genedata.com). Indeed, the various-
‘omics’ have become a staple of Plant Physiology (Raikhel, 2005).
The term ‘omics’ refers to the comprehensive analysis of the biological system. Informally, the
neologism omics has come to refer to a comprehensive study involving the acquisition of vast data
sets. An omics approach can be considered to be large-scale data rich biology consisting of a heavy
data mining or bioinformatics component. The modern concept of omics was initiated by Human
Genome Project, which was launched in 1986 (Wheelock and Miyagawa, 2006). Omics has been
driven more by emerging experimental technologies than by the novel hypothesis (Yin and Struik,
2007).
Genomics, proteomics and metabolomics are the three core omics technologies, which respectively
deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
These technologies involve large data sets and high throughout methods (fast methods for gathering
data). In the 1980’s, genomics arose as term describing the mapping and sequencing of genomes as
well as the analysis of the information content present in genetic sequences. Subsequently, when the
complete genome sequences were considered as a basis for systematic functional analysis, the
genomics was divided into two disciplines—structural genomics and functional genomics. While the
4 Crop Improvement: Strategies and Applications

structural genomics corresponds to the initial phase of genome analysis resulting ultimately in the
revelation of the complete DNA sequence of an organism, the functional genomics makes the use
of genome sequence to assess, on large scale, the functions of genes (Leister, 2005). Proteomics and
metabolomics are the main tools of functional genomics. Transcriptomics, which deals with the
study of transcriptome (a set of gene transcripts or messenger RNAs in a cell), is also considered to
be a component of functional genomics. All these are generally referred to as post-genomic
technologies that provide primary omics methods of characterizing sets of molecules produced by
genomes. However, do these new fields fit under the larger umbrella of genomics, or are they
distinct? It is a matter of choice of the person who you ask, and in what context (Campbell and
Heyer, 2006). Most of the people encompass all the three disciplines and technologies under
‘genomics’ and use this term in a broader sense. For example, `genomics’ includes everything from
gene sequencing, annotation of function genes, and genome architecture to studying patterns of
gene expression at transcriptome level (transcriptomics), proteome level (proteomics) and metabolite
flux (metabolomics). Due to the magnitude and complexity of omic data, these disciplines are
underpinned by information technology support through bioinformatics. In the following text, we
provide a comprehensive introduction to the basic aspects of genomics, proteomics and metabolomics,
and an overview of the associated techniques as well as (potential) applications of these technologies
in crop improvement strategies.

GENOMICS
The study of the way genes and genetic information are organized within the genome, the methods
of collecting and analyzing this information and how this organization determines their biological
functionality is referred to as genomics (Campos-de Quiroz, 2002). The genetic material in plants
and animals is deoxyribosenucleic acid (DNA), which is present in the nucleus of every cell. DNA
is the double stranded molecule made up of four different basic building blocks called nucleotide
bases—adenine (A), cytosine (C), guanine (G) and thymine (T). The functional unit of DNA is
called gene. In a gene, the sequence of ACGT on a strand of DNA specifies the sequence of amino
acids that make up a protein. In order for a protein to be synthesized, the DNA in a gene is first
transcribed to messenger RNA (mRNA), also called the gene transcript, which is similar to DNA
but is single stranded. The mRNA is then translated into a sequence of amino acids through ribosomes
found in the cytoplasm of the cell. The proteins and products are fundamentally responsible for
all cellular behaviour. The protein function is altered by changes in the sequence of amino acids
(http://linux.ittoolbox.com). Elucidating the pattern of arrangement of nucleotide bases in the entire
genome is called genomic sequencing.
Plant genomes are best described in terms of genome size, gene content, extent of repetive
sequences and polyploidy/duplication events (Campos-de Quiroz, 2002). Genomics investigates how
variation in genes affects protein structure and function throughout the life of a cell. When a cell
senses changes in its environment, it responds by accessing different components of genome.
Normally, this access comprises the expression of genes encoding instructions for the production of
new cellular proteins via production of mRNA molecules or the gene transcripts by a process called
transcription. The set of all the transcripts produced in one or a population of cells is called
transcriptome. Transcriptomics examines the systematic quantification of the levels of all or a large
portion of the transcripts expressed within a given cell population under particular environmental
condition, often using high-throughput techniques (http.//www.en.wikipedia.org). Since sequences
 The ‘-OMICS’ Technologies and Crop Improvement 5

of mRNAs (transcripts) mirror the DNA sequence of the genes from which they are transcribed, it
is possible to determine when and where a gene is turned on or off in various types of cells and
tissues by analyzing the transcriptome. It is often possible to count the number of transcripts to
determine the amount of gene activity, also called expression level, in a certain cell or tissue type.
The higher the number of transcripts, generally the more important that transcript is to the functioning
of cell or tissue (http://www.genome.gov/transcriptome). The identification and characterization of
different candidate genes influencing economically important traits, and those involved in the response
to a particular stress enhance the possibility of promoting crop improvement through direct genetic
modifications (Dunwell et al., 2001).

Genomics Techniques
Genome is the total genetic content of the cells. Genome sequencing refers to the techniques used
to determine the order of the four nucleotides (adenine (A), guanine (G), cytosine (C) and thymine
(T) that make up the genetic code in a DNA sample. While there are several methods for sequencing
DNA, the most popular and commonly used DNA sequencing methods are the dideoxy or chain
termination method, which was developed by Sanger et al. in 1977, and the shotgun sequencing
method.
Dideoxy or Chain Termination Method: Also known as Sanger method, it has undergone
several modifications (since its invention) including automation, and now-a-days sequencers are
used in large scale DNA sequencing. This method employs DNA synthesis in the presence of
dideoxynucleotides in addition to the normal nucleotides found in DNA. Dideoxynucleotides (ddNTPs)
are essentially the same as nucleotides (NTPs), except that they contain a hydrogen group on 3’
carbon instead of hydroxyl group (OH).These modified nucleotides, when incorporated into the
newly synthesized DNA, prevent the addition of further nucleotides due to the absence of 3′-OH
group. This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and
the next incoming nucleotide. Thus, the DNA chain is terminated (http://statwww.berkeley.edu).
The initial stage of sequencing is denaturation of double stranded DNA molecules into single strands
using heat. The single stranded DNA molecule serves as a template for synthesizing a series of
complementary strands that terminate at specific nucleotides. Next, a short primer (a short strand of
DNA) is annealed to the template, and the primer bound DNA is distributed into four reaction
tubes. Either the primer or one of the nucleotides should be radioactively labelled, so that the final
product can be detected on the gel. Then this primer is elongated by adding the DNA polymerase
and the four deoxyribonucleotide phosphates (dATP, dCATP, dGTP and dTTP). In addition, each
tube contains a small amount of one of the four base-specific analogs, the dideoxylnucleotide
phosphate (ddNTP). As DNA synthesis proceeds, the DNA polymerase randomly incorporates the
ddNTP (instead of deoxynucleotide) into the growing DNA strand, thus, terminating DNA synthesis
due to the reasons mentioned above. As the reaction proceeds, the tubes accumulate a whole
succession of DNA molecules that differ in length by one nucleotide at their 3′-ends. Once completed,
the DNA fragments from each of the four reaction tubes are run in separate lanes on a polyacrylamide
gel in order to separate the different sized bands from one another. After the contents have been run
across the gel, the gel is then exposed to either UV light or X-ray, depending on the method used
for labelling the DNA. The nucleotide sequence of DNA can be read directly from the bottom to
top, corresponding to the 5′→ 3′ sequence of DNA complementary to the template (Klug and
Cummings, 2003; Ban, 2006).
6 Crop Improvement: Strategies and Applications

Automated sequencing has been developed for large scale DNA sequencing in a shorter period
of time. The new techniques are, however, based on the same principles of Sanger’s method. In the
automated procedure, the four dideoxynucleotide analogs (ddNTP – ddATP, ddCTP, ddGTP and
ddTTP) are labeled with different fluorescent dyes, so that at the end of the reaction, the chains
terminating in A are labelled with one colour, those ending with C with another colour, and so
forth. The reaction is performed in a single tube and loaded into one lane on the gel, in contrast to
four lanes in the original Sanger’s method. The fluorescent detector or the laser in the sequencing
machine reads the gel to determine the identity of each band according to the wavelengths at which
is it fluoresces. The results are depicted in the form of chromatogram, which analyzed using
appropriate software for nucleotide sequencing (Russel, 2002). Now-a-days, the detection and reading
of sequence is also completed by an automatic capillary array DNA sequencer. Each sample is
loaded into capillary through which the fragments are separated according to their size. A laser is
used to detect the fluorescence specific for each nucleotide, as the DNA migrates to the bottom of
the capillary. Sequencing software is then applied to analyze the output from the DNA sequencer
and provide a chromatogram of the DNA sequence—the end product (Ban, 2006).
Shotgun Sequencing Method: It is another widely used method that is particularly suited to
high- throughput assembly-line style methodology for determining the entire genome of organisms
to be sequenced relatively rapidly. The process of shotgun sequencing starts by shredding
(‘shotgunning’) the DNA molecule into millions of random fragments. The fragments are then inserted
into cloning reactors in order to amplify the DNA to levels needed by the sequencing reaction. The
commonly used cloning vectors are plasmids (circular pieces of DNA), which are then grown in
Escherichia coli bacterium. The plasmid DNA sequence is engineered to enable sequencing reaction
to proceed into the inserted fragments. The ends of each of the original fragments can thus be read
by automated sequencing machines. The sequencing of these fragments is then ordered based on
overlaps in the genetic code followed by piecing together the original genome using specialized
software programs called assemblers(http://en.wikipedia.org/wiki/DNA_sequencing).
Polymerase Chain Reaction: The invention of polymerase chain reaction (PCR) by Mullis et
al. (1986) made a significant breakthrough in the field of molecular biology. By using this technique,
it is possible to generate many copies of a specific DNA sequence through a series of reactions in
a test tube (in vitro), and rapidly amplify target DNA sequences initially present in infinitesimally
small quantities in a population of other DNA molecules. This is essential in order to have enough
starting DNA template for further experiments, such as sequencing. In PCR method, the target
DNA is denatured into single strands. Each strand is then annealed to a short, complementary
primer. The primers are synthetic oligonucleotides that are complementary to sequences flanking
the region to be amplified. DNA polymerase and nucleotides extend the primers in 3′ direction
using the single stranded DNA as template, resulting in double stranded DNA molecules with the
primers incorporated into the newly synthesized strand. In a second PCR cycle, the products of the
first cycle are denatured into single strands, primers are annealed, and DNA polymerase then
synthesizes new strands. Repeated cycles can amplify the original DNA sequence by more than a
million fold (cf. Klug and Cummings, 2003).
Genome Annotation: Once the genome sequence is available, it should be annotated, which
means finding the potential genes and assigning functions to them. Most of this is done in silico,
i.e., with the aid of computer programs. Function assignment relies extensively on sequence similarity.
This means that a function is assigned to a gene in a newly sequenced genome based on its similarity
 The ‘-OMICS’ Technologies and Crop Improvement 7

to the already available gene sequences in sequence databases, such as GenBank. The most widely
used similarity detection tool is the BLAST programme (Alstchul et al., 1997; Setubal and da Silva,
2004). The classification of genes is done according to their assigned function. Generally, each
genome project develops its own classification scheme, but most are based on the one originally
developed for Escherichia coli bacterium (Riley, 1993).
There are various other methods available for genomic studies, and a number of them have
been dealt with in some of the accompanying review articles in this volume.
Several genomes have been sequenced to a high quality in plants, including Arabidopsis thaliana
and rice; draft genomes are available for poplar, lotus, and sequencing efforts are in progress for
others including tomato, maize, Medicago truncatula, sorghum, etc. (Rhee et al., 2006). DNA
sequencing provides information about the number, nature, and organization of genes in a genome
and elucidates the mutational events that alter both genes and gene products, confirming that genes
and proteins are colinear molecules.

Transcriptomics
As mentioned above, transcriptomics deals with the analysis of gene expression patterns across a
wide array of cellular responses, phenotypes and conditions. The identification of candidate genes
influencing any important trait can be approached through an analysis of gene transcripts or mRNAs.
It involves detecting the expression level of one or more specific RNAs out of thousands of other
RNAs, and producing a snapshot image of genes being translated at any given moment. There are
several systems available to analyze parallel expression of many genes, such as DNA microarray
technology (Schena et al., 1995), or tag-based technologies, such as serial analysis of gene expression
(SAGE) (Velculescu et al., 1995), which allow for exact measurement of any transcript, known or
unknown (http://en.wikipedia.org/wiki/gene expression). Several other methods are also available
for detection and quantification of mRNAs (see Meyers et al., 2004).
DNA microarray: The DNA microarray (also known as DNA chip or microarray) is a high
throughput, most widely used technique for gene expression studies and represents a key element in
today’s functional genomics research (Aharoni and Vorst, 2001). In brief, this technique involves
firstly the extraction of specific cell(s) from two or more biological samples by using laser capture
microdissection (LCM) technique, and then extracting the total RNA from the cells captured. This
is followed by multiplication of the copies of each RNA expressed. RNAs thus produced (referred
to as targets) can then be radioactively labelled or labelled with fluorescent dyes and hybridized
onto DNA chip under computer control. These chips (nylon membranes with glass or silicon surfaces
containing thousands of DNA probes) are designed to visualize which genes are being transcribed
to RNA within the cell when the sample was taken. Scanners are used to read the signals and
fluorescence measurements are made for each dye at each spot on chip. Specialized software and
data management tools are then used for data extraction and analysis (http://
www.parisdevelopment.com). The microarray technology has been utilized to examine a range of
plant processes including circadian clock, plant defence, environmental stress responses, fruit ripening,
phytochrome A signalling, seed development and nitrate assimilation (Aharoni and Vorst 2001).
Serial Analysis of Gene Expression (SAGE): The major difference between DNA microarray
hybridization and serial analysis of gene expression (SAGE) techniques is that the latter does not
require prior knowledge of the sequences to be analyzed, as SAGE is a sequencing-based gene
expression profiling technique (Velculescu et al.,2000). For organisms with poorly characterized
8 Crop Improvement: Strategies and Applications

genomic and expressed sequences, SAGE can be used to obtain complete transcriptional profiles of
expressed genes, albeit unknown genes. A recent adaptation of SAGE, called Long SAGE, allows
the derived transcriptome to be used in annotating expressed genes in the genome (Saha et al.,2002).
In this sense, SAGE is a truly global and unbiased gene expression technique. The technique of
SAGE uses multiple enzymatic, PCR amplification, purification, and cloning steps. The SAGE
protocol starts with the purification of mRNA bound to solid phase oligo(dT) magnetic beads. The
cDNA is synthesized directly on the oligo(dT) bead and then digested with the anchoring enzyme
NlaIII (AE) to reveal the 3'-most restriction site anchored to the oligo(dT) bead. Most SAGE
experiments have used the 4-bp recognition site anchoring enzyme NlaIII, predicted to occur every
256 bp and thus present on most mRNA species. However, creating a second SAGE library with a
different anchoring enzyme may be useful for detecting transcripts without a NlaIII site and also for
reconfirming transcript identity in those with both anchoring restriction sites. This may significantly
lessen the work associated with data analysis, but the marginal utility of such an approach remains
to be demonstrated. Next, the sample is equally divided into two separate tubes and ligated to two
different linkers, A or B. Both linkers contain the recognition site for BsmFI, a type IIS restriction
enzyme that cuts 10-bp 3' from the anchoring enzyme recognition site. BsmFI generates a unique
oligonucleotide known as the SAGE tag, hence called the tagging enzyme (TE). The SAGE tags
released from the oligo(dT) beads are then separated, blunted, and ligated to each other to give rise
to ditags. The ditags are PCR amplified, released from the linkers, gel purified, serially ligated,
cloned, and sequenced using an automated sequencer (Patino et al., 2002).

Applications
The plant genomics provides a platform for analyzing and understanding the genetic and molecular
basis of all biological processes in plants that are relevant to the species. The understanding is
fundamental to allow efficient exploitation of plants as biological resources in the development of
new cultivars with improved quality and reduced economic and environmental costs (Vassilev
et al., 2005). The genomics allows the scientists to analyze thousands of genes in parallel and to
understand the complex crop traits, such as yield and yield stability (Struik et al., 2007). It also
reduces the gap between phenotype and genotype and helps to comprehend not only the isolated
effect of a gene, but also the way its genetic content and genetic networks it interacts with can
modulate its activity (Campos-de Quiroz, 2002). Genomics has provided objectivity in plant breeding
as never before. It helps in assaying genetic make up of the individual plants rapidly, so as to select
desirable genotypes in breeding populations, and to design the superior genotypes for ‘breeding by
design’ approach. With genomic approaches, the marker-assisted breeding or marker-assisted selection
will gradually evolve into ‘genomics-assisted breeding’ for crop improvement (Varshney et al.,
2005). Genomics research has also successfully unraveled various metabolic pathways and provided
molecular markers for agronomic traits. The identification of genes that control economically important
traits provide the basis for new progress in genetic improvement of crop species, complementing
traditional methods based on assisted crosses (Dunwell et al., 2001). The knowledge of genomics is
valid for development of new plant diagnostic tools. It will help to find new solutions for improved
germplasm in crop plants and for chemical protection of crops. Thus, a genome programme can be
envisioned as a highly important tool for crop improvement.
 The ‘-OMICS’ Technologies and Crop Improvement 9

PROTEOMICS
While the genome contains the coded information that allows an organism to live and reproduce,
the essential functions of living cells are accomplished by gene products, mainly proteins. Although
ribonucleic acids are also essential, the proteins provide scaffold, regulatory and catalytic functions
that drive metabolism (Whitelegge, 2002). Proteins are fairly large molecules made up of strings of
amino acids linked like a chain. While there are only 20 amino acids, they combine in different
ways to form tens of thousands of proteins, each with a unique, genetically defined sequence that
determines the proteins specific shape and function. A full complement of proteins expressed by
genome of a cell, a tissue or an organism at a specific time point constitutes its proteome (Renaut
et al., 2006). Proteomics is a leading technology that covers the systematic analysis of proteins
expressed by a genome, from identification of their primary amino acid sequence to the determination
of their relative amounts, their state of post-translocation modification and association with other
proteins or molecules of different types to characterization of protein activities and structures (Barbier-
Brygoo and Joyard, 2004; Rhee et al., 2006). While the genome of an organism is a relatively fixed
entity, the proteome is dynamic or changes constantly (similar to transcriptom). Therefore, there are
potentially many proteomes present in an organism, and the picture of one that is presented at any
single point in time will depend on many factors including developmental stage of the plant, response
to abiotic and biotic stress, the organ or tissue being examined or even the cellular compartments
being studied (Renaut et al., 2006).
In many ways proteomics runs parallel to genomics. Genomics starts with the gene and makes
inferences about its products (proteins), whereas proteomics begins with the functionally modified
proteins and works back to the gene responsible for its production. The path from gene to protein
is better described as gene to mRNA to polypeptide to protein, with the last step encompassing a
myriad of alternative steps involving biochemistry and cell biology (Roberts, 2002). In fact, proteomics
is a tool for functional genomics in plants. Proteomics is also becoming a powerful tool to analyze
biochemical pathways and the complex responses of plants to environmental stimuli. This has enabled
researchers to associate changes in protein expression profiles in different physiological moments
and to define the functions of expressed proteins. Further, proteins serve as important components
of major signaling and biochemical pathways. Therefore, studies at protein level are essential to
reveal molecular mechanisms underlying plant growth, development, and interactions with
environment (Chen and Harmon, 2006). The compatibility of proteomics is especially useful for
crops, as it may give clues not only about nutritional value, but also about yield and how these
factors are affected by adverse conditions (Solekdeh and Komatsu, 2007).

Proteomics Techniques
There are numerous emerging techniques available for proteome analysis, each attempting to provide
improved separation, resolution and automation depending on different experimental purposes and
the chemical and physical properties of the target proteins. In general, two analytical techniques are
employed in current proteomic research: two-dimensional polyacrylamide gel electrophoresis (2D-
PAGE or 2-DE) for the separation and visualization of crude extracts (Klose, 1975), and mass
spectrometry (MS) for the identification and characterization of the separated proteins (Fenn et
al.,1989; Karas et al., 1989). 2-DE is based on isoelectric focussing (IEF), by which the proteins
are separated according to their pI in pH gradient polyacrylamide gels (first dimension) and SDS
(sodium dodecyl sulphate)-PAGE, by which the proteins are separated according to their molecular
10 Crop Improvement: Strategies and Applications

weights (second dimension) (Klose, 1975,1999; Klose and Kobalz, 1995). Visualization of separated
protein spots is achieved by use of visible staining techniques. Proteins within the spots of interest
are then identified by firstly digesting to peptides, typically with trypsin, and subsequently analysed
by MS. The utility of this method is hampered for being low throughput, costly and a labour and
time consuming, and can also be tedious due to gel-to-gel variations that can complicate the image
analysis process. Difference gel electrophoresis (2D-DIGE) circumvents many of these issues and
allows for more accurate determination of both qualitative and quantitative variations of samples
(Ünlü et al., 1997). DIGE is a prelabelling technique using separate spectrally resolvable fluorescent
dyes and allows multiple samples to be co-separated and visualized on one 2-DGel (Tongs et al.,
2001). Currently, the most commonly used methods for the study of complex mixtures are based on
mass spectrometry (Aebersold and Mann, 2003) according to the following outline: Proteins in
more or less complex samples derived from 2-D gels are proteolytically cleaved into smaller peptides.
The resulting peptides are then analyzed using MS based automated matrix assisted laser desorption/
ionization time of flight mass spectrometric (MALDI TDF-MS) peptide mapping followed by
extensive data based searches (Henzel et al.,1993). An alternative method to analyze proteins directly
by MS, without gel separation, has been developed and is referred to as multidimensional protein
identification technology (MudPIT) or liquid chromatography (LC)-MS/MS that couples capillary,
high-performance liquid chromatography (HPLC) to MS/MS and allows automated analyses of peptide
mixtures that are generated from complex protein samples (Appella et al., 1995; Washburn et al.,
2001). Further, quantitative proteomics becomes feasible using an innovative reagent, termed isotope-
coded affinity tag (ICAT), in the LC-MS/MS system (Han et al., 2001). The development of the
yeast two-hybrid (Y2H) system allows identification of protein-protein interactions (Fields and Song,
1989). This genetic procedure allows the rapid identification of in vivo protein-protein interactionsd
and the simple isolation of corresponding nucleic acid sequences encoding the interacting partners
(Kersten et al., 2002). Recently, protein microarray technology has emerged following successful
applications of DNA chip methods for large-scale studies on protein profiling and function
characterization (MacBeath, 2002; Mitchell, 2002). Quantitative non-2 D gel based methods, especially
those centred on stable isotope labelling and multidimensional chromatography, such as clearable
isotope code affinity tagging (cICAT) and isobaric tagging for relative and absolute quantification
(iTRAQ) will hopefully increase the scope and power of future proteomics in vivo metabolic labelling
and in vitro chemical derivation experiments in plants (Wu et al., 2004; Gruhler et al., 2005;
Rampitsch and Srinivasan, 2006; Lilley and Dupree, 2006). The current and developing proteomics
approaches have allowed large scale determination of protein patterns, structure and function in
plant organs, tissues and cells.
Recent years have also seen an explosion in the development and employment of organelle
proteomics (Millar, 2004; Warnock et al., 2004). These methods depend primarily on subcellular
fractionation by MS for protein identification (Dreger, 2003); however, new microscopy-based
approaches for determination of protein localization include immunoelectron microscopy, in situ
labelled with fluorescently-tagged antibodies, and pluorescent protein fusions (e.g., green fluorescent
protein – GFP) coupled with confacal microscopy (Pan et al., 2005). Assuming that the members of
large protein families may share significant identities, their function can widely vary (Dunwell et
al., 2004). Knowing the ultimate localization of these proteins, as well as the pathways used for
getting these, may give clues to their definite functions (Nair and Rost, 2005). Several studies of
plant proteomics have been reported on plant tissues and purified cellular compartments including
 The ‘-OMICS’ Technologies and Crop Improvement 11

nucleus, plastids, mitochondria, ER, Golgi apparatus, cell wall, plasma membrane, peroxisomes and
vacuoles (see Pan et al., 2005). The identification of those proteins recruited to fulfil the specific
function of subcellular compartments has given an additional dimension to the proteome analysis
(Canovas et al., 2004).

Applications
Proteome analysis is becoming a powerful tool to monitor developmental changes or influence of
environmental stimuli on protein patterns, so as to gain insight into the functioning of plants at
molecular level. Detailed information on the application of proteomics has been published in excellent
reviews by Canovas et al. (2004) and Rampitsch & Srinivasan (2006). A brief account of some of
the major applications of proteome studies is given here. In Arabidopsis, while studying the role of
GAs during initial stages of seed germination, and the impact of scarification on seed germination,
application of proteome analysis resulted in better understanding of the complex cellular events.
Similarly, in barley, the proteome analysis revealed new insights into cellular mechanisms underlying
seed development during grain filling and seed maturation phases. In rice, proteome studies have
helped in detecting novel traits useful for breeding (Yu et al., 2002). Mutants are generally subjected
to proteome analysis to compare their responses to different hormonal treatments, nutritional factors,
and photosynthetic traits. In maize, a number of previously unknown novel genes coding for enzymes
in metabolic pathways were identified during grain development following proteome analysis.
Proteomics has been widely used to assess genetic variability at the level of expressed proteins
(Canovas et al., 2004). Accordingly, closely related lines were successfully differentiated in wheat
cultivars, barley and rice lines, maize genotypes, and a number of other crop species.
Both abiotic and biotic stresses can bring about dramatic changes to the plant proteome, and
these are manifested as the up- or down- regulation of proteins, or their posttranslation modification
(Rampitsch and Srinivasan, 2006). Salinity stress results in change in the proteome, as the plant
attempts to restore homeostasis in osmolarity to resume growth and development. Detailed analysis
indicated that during salt stress, plant diverts carbon to glycolysis to provide the energy required to
return the plant to homeostasis (Yan et al., 2005; Zhu, 2001; Kleczowski et al., 2004). In case of
pathogen attack (biotic stress), the proteome analyses have indicated involvement of defense and
stress related proteins, metabolic enzymes, translocation and protein turnover proteins, and proteins
of unknown functions in the defense response (Campo et al., 2004; Kim et al., 2004 a,b; Rampitsch
and Srinivasan, 2006). The application of proteomics has also been used to decipher the highly
complex genetic interactions involved in plant-microbe interactions and for studying symbioses
(nitrogen symbiosis, ecto- and endo-mycorrhizal symbiosis) in plants. Proteome approaches have
also provided new insights into molecular controls regulating wood formation in plants, especially
pine species (for details refer the two reviews mentioned above).

METABOLOMICS
The quality of crop plants and their derived products is the direct function of their metabolites (i.e.,
the biochemical component of cells and tissues). The metabolites determine the flavour, aroma,
colour and texture of crops, their storage properties and performance in field (Memelink, 2005).
The metabolite content of a plant constitutes its metabolome. Metabolomics is a technology that
deals with comprehensive analysis in which all the metabolites of an organism are identified and
quantified at a given time (Fiehn, 2002). It has emerged as a genome functional methodology that
12 Crop Improvement: Strategies and Applications

contributes to our understanding of the complex molecular interactions in the biological systems
(Hall et al 2002). As such, metabolomics represents the logical progression from large scale analysis
of RNA and proteins at the system level (Weckwerth, 2003).
The metabolites are the end products of cellular regulatory processes, and their levels can be
viewed as response of biological systems to environment or genetic manipulations (Maloney, 2004).
The metabolome is very diverse. It includes lipid soluble chemical that are normally found in
membranes, polar chemicals for aqueous parts of the cell, acidic and basic ions, stable structures
and structures that oxidize at the slightest mistreatment (Maloney, 2004). The total number of
metabolites, which are produced within the plant kingdom, including secondary metabolites, is
estimated to range between 100000 to 200000 (Oksman-Caldentey and Inze, 2004). The quantitative
and qualitative measurements of such a large number of cellular metabolites provides a broad view
of the biochemical status of an organism, which can then be used to monitor and assess gene
function (Fiehn et al., 2000). Further, the importance of metabolites in control, communication, and
as building blocks and energy transporters within the biological systems provides metabolomics a
unique opportunity for phenotypic and genotypic profiling (Tolstikov et al., 2003). Plants react to
any change in their surroundings. The metabolites are reported to function in many resistance and
stress responses in plants (Bino et al., 2004). The biochemical response of an organism to a conditional
perturbation can be characterized by its effect on the differential accumulation of individual metabolites
(Raamsdonk et al., 2001). As metabolites represent the catabolic and anabolic activities being
performed by proteins at any given time (Maloney, 2004), it is increasingly understood that they
(metabolites) themselves modulate macro-molecular processes through, for example, feedback
inhibition and signaling molecules (Dixon et al., 2006).The cascading effect begins with a modified
genome leading to modified proteins, and consequently, change in the pattern and/or concentration
of metabolites. Therefore, change in genotypes will be manifested through an observed change in
metabolome, which helps in better understanding of the correlation between genes and functional
phenotype of an organism (Sumner et al., 2003; Bino et al., 2004).
Metabolomics research is proving an invaluable tool for generating information of use in many
fields. For functional genomics strategies, potentially fast track methods exploiting metabolomics
analyses of tagged lines or known mutants are likely to prove invaluable. Further, metabolomics
information is not only assisting in the establishment of deeper understanding of the complex
interactive nature of plant metabolic network and their responses to environmental and genetic change,
but also is providing unique insights into the fundamental nature of plant phenotypes in relation to
development, physiology, tissue identity, resistance, biodiversity etc.

Metabolomics Techniques
Metabolites have a much greater variability in the order of atoms and subgroups compared to
genes and proteins. Therefore, there is no single analytical technique that can be used in metabolomics
to define, identify and quantify all metabolites in a biological sample. Nevertheless, metabolomics
is driven primarily by advances in mass spectrometry (MS), nuclear magnetic resonance (NMR) and
fourier transform infrared (FT-IR) spectroscopy coupled with chromatographic separation
procedures. The success of metabolome analysis is dependent on a few key aspects—production of
the biological materiall/sample and sample extraction/metabolite detection (Hall 2006). Several
technical and review reports are available on appropriate strategies for the design of metabolic
experiments (Fiehn, 2002; Gulberg et al., 2004; Hall, 2006). Bino et al. (2004) have suggested
 The ‘-OMICS’ Technologies and Crop Improvement 13

minimum information about a metabolomics experiment (MIAMET), which incorporates experimental

design; sampling; preparation, metabolite extraction and derivation; metabolite profiling design, and
metabolite measurement and specifications. Due to convoluted nature of plant metabolism, the
metabolic data is quite complicated. Different analytical approaches have been, therefore, designed
for specific analytes. These approaches include target analysis, metabolite (or metabolic) profiling,
metabolomics and metabolic fingerprinting. Metabolite target analysis involves a combination of
techniques to prepare and analyze samples for one or a small number of compounds from complex
mixtures. It is the most wide-spread technique, and is applied in all areas of research such as the
monitoring of phytohormones and also to directly study the primary effect of a genetic alteration
(Fiehn, 2002). Metabolite profiling is the measurement of hundred or thousands of metabolites. It
requires streamlined extraction, separation and analysis in highly throughput manner, so as to measure
large number of metabolites in the presence of extraordinarily complex mixture of chemicals (matrix),
which are found in cellular extracts (Kopka et al., 2004). Metabolic fingerprinting looks at a few
metabolites to help differentiate samples according to their genotype, phenotype or biological relevance
(Shanks, 2005). Metabolomics in the strict sense is the measurement of all metabolites in a given
system. It is not yet technically possible, and will probably require platform of complementary
technologies, because no single technique is comprehensive, selective, and sensitive enough to measure
them all (Weckwerth, 2003). For target compound analysis and metabolic profiling, main techniques
are gas chromatography (GC), high performance liquid chromatography (HPLC) and NMR. These
approaches rely on chromatographic separation, often coupled with well developed calibrations for
specific analytes. In metabolic fingerprinting, samples are analyzed as crude extracts without any
separation step using NMR, direct injection mass spectrometry (MS), or FT-IR spectroscopy. These
fingerprinting approaches are often combined with multivariate analyses to get most out of the data.
For the widest coverage of metabolomics, the hyphenated complementary analytical techniques of
liquid chromatography (LC)/MS, LC/MS, LC/MS/MS and LC/NMR are likely to make increased
impact in the future (cf.www.metabolomics-nrp.org.uk, also see for technological details). A reference
may be made to a review by Hall (2006) for details regarding various metabolomics techniques,
their applications and limitations.

Applications
Plant metabolomics is still a field in its infancy, but has a potentially broad field of applications
aimed at facilitating an improved understanding of dynamic biochemical composition within the
living systems. This knowledge will prove to be fundamental in monitoring crop quality characteristics
(Hall et al., 2005), identifying potential biochemical markers to detect product contamination and
adulteration (Reid et al., 2004), and optimizing trait development in agricultural products and in
biorefining (Dixon et al .,2006). Metabolomics offers the unbiased ability to characterize and
differentiate genotypes and phenotypes based on metabolic levels. In a detailed review, Hall (2006)
has discussed applications of plant genomics in six major areas—genotyping and phenotyping;
population screening, understanding physiological processes, biomarkers and bioactivity, quality and
breeding, and substantial equivalence. He has included several examples where metabolite data
have been found useful in differentiating various genotypes and understanding plant responses to
biotic and abiotic stresses, characterization of the novel plant products, breeding of crops based on
specific biochemical composition and assessing the substantial equivalence i.e., comparison between
transgenic and wild-type plants.
14 Crop Improvement: Strategies and Applications

The knowledge of metabolomic also has great potential for application in efficient engineering
of crops that combine an attractive appearance and taste with improved levels of phytonutrients
such as flavonoids and carotensids (Bino et al., 2004). Plant properties can be improved in various
ways, such as by increasing metabolic fluxes into valuable biochemical pathways using metabolic
engineering (e.g., enhancing the nutritional value of foods, decreasing the need for pesticide or
fertilizer application etc.), or into pathways needed for the production of pharmaceuticals in plants
(Giddings et al., 2000). Similarly, metabolic shortcuts can be created by introducing foreign set of
enzymes(s) that lead to the production of desired end products from other or more upstream
precursores, and the foreign enzymes can also lead to the production of new metabolites (Memelink,
2005).

BIOINFORMATICS
Advances in the field of genomics technologies are generating biological information in the form of
digital data, which is too much for human brain to comprehend and analyze. Bioinformatics is a
knowledge based theoretical discipline that allows the scientists to store, analyze, and compare the
otherwise overwhelming amounts of genomic data. Bioinformatics involves biology, computer science
and information technologies to form a single discipline (Rhee et al., 2006; Vassilev et al., 2005).
It uses computers and sophisticated software to search and analyze databases accumulated from
genome sequence projects and other sources. Bioinformatics has facilitated the analysis of genomic
and post-genomic data, and the integration of data from the related fields of transcriptomics,
proteomics and metabolomics (Varshney et al., 2005). Several bioinformatics tools and databases
have been developed for DNA sequencing analysis, transcriptomics, proteomics and metabolomics,
and a comprehensive list of the same has been provided separately by various workers (Bino et al.,
2004; Varshney et al., 2005; Rhee et al., 2006). The integration of knowledge from bioinformatic
tools, databases and other different fields enables the identification of genes and gene products, and
helps in elucidating the functional relationships between genotypes and phenotypes (Edwards and
Batley, 2004). Using bioinformatics tools, scientists can search genomic data and identify a region
important for a desired trait. Then, through biotechnology methods, they can transfer that trait to
another organism to create a useful product or outcome, e.g., converting a drought-sensitive crop to
a drought-tolerant crop. The computational approaches facilitate the understanding of various
biological processes by providing a more global perspective in experimental design, and ability to
capitalize on the emerging technology of database mining by which testable hypothesis are generated
regarding the function or structure of a gene or protein of interest by identifying similar sequences
in characterized organisms. Bioinformatics will continue to play an important role to glue basic
research with applied research, and biotechnology will play an essential role in solving urgent
problems of our society, such as developing renewable energy, reducing world hunger and poverty,
and preserving the environment (Rhee et al., 2006).

CONCLUSION
Considerable progress has been made building infrastructure for applying knowledge and tools of
genomics (and other ‘omics’) to allow the characteristics of crop plant to be altered for improved
actual and potential yields, increased resource use efficiency and enhanced crop system health.
However, there is a continuing need to coordinate disciplines such as structural genomics,
transcriptomics, proteomics and metabolomics with plant physiology, biochemistry and plant breeding
 The ‘-OMICS’ Technologies and Crop Improvement 15

for formulating strategies to work on problem-oriented and process-oriented goals leading to crop
improvement. Recently, a new scientific discipline—crop system biology—has been proposed by
yin and Struik (2007) as a complementary approach to connect functional genomics and crop modeling
for assisting plant breeding programmes to omprove the yield related characteristics of major crops.
It is envisaged that this science will develop into a highly computer-intensive discipline, which
should enable in silico assessment of crop response to genetic fine-tuning under defined environmental
scenarios, thereby becoming powerful tool in supporting breeding for complex crop tyraits. The
genomics revolution is creating its own revolution in plant breeding that cannot be ignored. The
promotion of all the innovative approaches is fundamentally important in addressing the need for
enhancing agricultural productivity and sustainability, and to put them to use for the public good.

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