3. Introduction
The first completed plant genome sequence, that of Arabidopsis thaliana, was
published in the year 2000. Genome mapping and sequencing projects are on
going in a number of other plant species, including many commercially
important crops.
These projects are complemented by the availability of increasing numbers of
plant ESTs, currently over 250 000, representing a wide variety of species.
For the first time, therefore, it is possible to begin to look at all aspects of plant
biology on a global scale. This includes aspects that are of particular interest in
agriculture, such as growth and development, tolerance or resistance to stress
and disease, and metabolic activity.
4. Traditionally, there have been two approaches to improving the quality of
cultivated plants. The first is by conventional breeding, which involves
exploiting varietal or specific differences and using crosses and tissue-
culture-based techniques to concentrate favourable traits in particular
plant lines.
One problem with this approach is that many of the most valued traits in
agriculture – disease resistance, stress resistance, increased yield etc. – are
controlled by QTLs, so complex and extensive breeding programmes are
reguired.
5. The alternative is genetic engineering, because single genes from other
species can often confer desirable traits on plants. Although this approach
to agricultural improvement is comparatively rapid, the behaviour of the
transgenes is unpredictable and there is much public concern about the
long-term effects of such plants on and the environment.
Genomic resources now provide an additional route to the improvement
of plants, through the rapid identification of genes and pathways
controlling important plant traits.
The technology and application of genetic engineering in plants is
discussed in detail in our sister text, Principles of Gene Manipulation.
Some of the issues of public concern over the use of transgenic plants and
food products derived therefrom have been discussed.
6. Global Gene Expression Profiling
Gene expression profiling is the determination of the pattern of genes
expressed, at the level of transcription, under specific circumstances or in a
specific cell to give a global picture of cellular function.
Initial studies of global gene expression in plants were small in scale. The
random sampling of EST sequences from rice suspension cells and
Arabidopsis plants exposed to osmotic stress helped to reveal a number of
stress-induced genes . The first array-based study involved a glass
microarray containing 48 Arabidopsis ESTs and compared gene expression
in two tissues – roots and leaves.
7. A similar study with a larger number of ESTs was performed by Ruan et al.
(1998) and, in the same year, Arabidopsis microarrays containing about
800 ESTs were used to study light-induced gene expression. Interestingly,
the first plant microarray experiment with direct agricultural implications
involved not Arabidopsis but strawberry, where 200 genes were shown to
undergo changes in expression during ripening.
Over the last 2 years, an increasing number of expression profiling
experiments have been carried out to investigate gene expression changes
associated with traits of agricultural value.
8. EST sampling projects in two naturally salt-tolerant species
(Mesembryanthemum crystallinum and Dunaliella salina) and two species
resistant to desiccation (Tortula ruralis and Craterostigmata plantagineum)
have been used to identify drought stress-related genes. Sampling
differences between well-watered and salt-stressed or desiccated plants
were used to identify ESTs either induced or repressed by these extreme
conditions.
Large-scale microarray-based screening projects have been undertaken to
identify drought stress-related genes in Arabidopsis, rice and M.
crystallinum and oxidative stress-related genes in Arabidopsis.
9. They found that over 700 transcripts showed significantly increased or
reduced expression levels, some of which were specific to particular
treatments (e.g. exposure to chemicals such as methyl jasmonate and
salicylic acid which are known defence-related signalling molecules) and
some of which were general (i.e. affected by multiple treatments).
These data suggested that there was considerable cross-talk between
different defence signalling pathways.
Microarrays have also been used to study the response of the plant to biotic
stresses. Schenk et al. (2000) have recently investigated disease defence
responses in Arabidopsis thaliana using microarrays containing about 2400
cDNAs.
10. Reymond et al. (2000) studied the effect of mechanical wounding and
insect feeding on gene expression profile in Arabidopsis.
The phenomenon of cross-talk may represent a problem when devising
strategies to combat disease, because artificially modifying the response
to one stimulus could alter the response to several others in an
undesirable way.
However, as well as identifying common themes in transcriptional profiles,
microarray analysis can also separate individual responses.
11. Thus, strategies to counteract the effects of insect feeding could be
targeted to a specific subset of genes so as not to interfere with water
stress tolerance. As well as local defence responses, the transcriptional
changes accompanying systemic acquired resistance have also been
monitored at the global level in Arabidopsis.
They found that mechanical wounding induced a number of genes also
known to be involved in the response to water stress, while such genes
were only minimally affected by insect feeding.
12. Proteomics and Plant Breeding
Two-dimensional gel electrophoresis (2DE) is a technique for the high-
resolution separation of proteins, allowing systematic protein
characterization and the identification of proteins that are differentially
expressed in alternative sample.
The technique has also been used to characterize proteins that are
involved in stress response pathways, e.g. the response to drought, cold
and heat shock.
14. 2DE has also been used as a diagnostic tool in plant breeding because
different lines or cultivars often show polymorphisms in terms of the
spots generated on two-dimensional gels. Such differences have allowed
unambiguous genotyping in many plant species, including rice, wheat and
barley .
Polymorphism occurs at three levels. Firstly position shifts (PS), which often
correspond to mutations that change the mass and/or charge of the protein.
Similarly, 2DE has been used to distinguish between intraspecific variants
and to investigate the taxonomy of closely related species, e.g. in the genus
Triticum.
15. The first two types of polymorphism are generally Mendelian characters
and represent useful genetic markers. These have been used in wheat,
maize and pine, for example, to generate comprehensive genetic maps also
containing DNA markers such as RFLPs, RAPDs and AFLPs.
Quantitative polymorphisms are useful for identifying QTLs that cannot be
pinned down by traditional map-based cloning or functional candidate
approaches . The basis of this method is that PQLs that map co incidentally
with QTLs can be used to validate candidate genes.
Secondly presence/absence polymorphisms (P/A), where a protein is present
in one sample but absent in another. Finally quantitative variants,
corresponding to so-called protein quantity loci (PQLs).
16. An example is provided by the study of de Vienne et al. (1999) who
identified a candidate gene on chromosome 10 of maize for a QTL
affecting drought response. The candidate gene was ASR1, known to be
induced by water stress and ripening.
Verification of the association was made possible because a PQL found by
the comparison of 2DE gels from control and drought stressed maize
plants mapped to the same region; the PQL controlled levels of the ASR1
protein under different drought stress conditions.
