QTL Analyses
Dormancy is a quantitative trait, involving
many genes and is influenced substantially by environmental factors. Within a
given plant species, different accessions of wild plants and different
varieties of cultivated plants exhibit genetic variation in seed dormancy.
Quantitative traits are becoming more amenable to genetic analysis because the
position of quantitative trait loci (QTL) and the relative contribution of
these loci can now be determined (reviewed in Koornneef et al., 2002).
Recombinant inbred lines are best for QTL analysis of dormancy and allow the
testing of a large number of genetically identical seeds in different
environmental conditions. This type of analysis has been reported for
Arabidopsis thaliana (Van Der Schaar, et al., 1997), barley (Han et al., 1996),
rice (Lin et al., 1998) and wheat (Kato et al., 2001). Many of the QTL of wheat
co-locate with those of barley, but not with those of rice (Kato et al., 2001).
Crosses between wild and cultivated genotypes are useful for QTL analysis
because of the deeper dormancy associated with the former (Cai and Morishma,
2000; Fennimore et al., 1999). This type of analysis in barley and Arabidopsis
is being followed by the study of individual genes (or chromosome regions)
containing specific dormancy QTL and by fine mapping (Koornneef et al., 2002).
Ultimately these approaches may allow the molecular identification of genes
that affect dormancy in these species by mapped-based cloning.
Metabolite Profiling
Metabolites are the end products of
cellular regulatory processes, and their levels can be regarded as the ultimate
response of biological systems to genetic or environmental changes. Thus,
metabolomics is the link between genotype and phenotype (Fiehn, 2002) and this functional
genomics approach will contribute to the understanding of complex developmental
processes in plants, especially the regulation of metabolic networks and its
perturbation by genetic or environmental means. The term ‘metabolomics’
encompasses many approaches including: (1) an unbiased identification and
quantification of all metabolites in a given biological sample from an organism
grown under defined conditions; (2) quantification of several pre-defined
targets e.g. of all metabolites of a specific pathway or a set of metabolites
typical for different pathways; (3) metabolic fingerprinting—the collection and
analysis of crude metabolite mixtures to rapidly classify samples without
separation of individual metabolites; and (4) metabolite target analysis—involving
the development of specialised protocols for the analysis of a specific set of
metabolites (e.g. hormones and hormone metabolites that can be present at low
quantities and hence represent ‘difficult analytes’) (Fiehn, 2002).
The number
of metabolites present in plant kingdom is estimated to exceed 200 000 and a
fundamental lack of biochemical and physiological knowledge about network
organization in plants, may impede progress on some metabolomic initiatives
(reviewed in Fiehn, 2002). However, regarding metabolite target analysis, a
liquid chromatography electrospray tandem mass spectrometry method has been
developed to quantify simultaneously all of the major plant hormone classes and
hormone metabolites in seeds. Further, the technology has been applied to
characterise hormone flux associated with secondary dormancy and germination of
lettuce seeds (Chiwocha et al., 2003). Hormones and hormones metabolites
targeted in this study included ABA and its metabolites (ABA-GE, 7′-OHABA, PA
and DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin,
zeatin riboside, isopentenyladenine, isopentenyladenosine, and gibberellins 1,
3, 4 and 7.
Thermodormancy was achieved by incubating imbibed seeds at a
non-optimal temperature for germination (33°C instead of 23°C). The state of
secondary dormancy induced in this manner was associated with surprisingly
active hormone flux. Moreover, the hormone and hormone metabolite profiles of
germinating and thermodormant lettuce seeds were distinct. This was
particularly true for ABA and its metabolites, in which thermodormant seeds
accumulated high levels of DPA, while germinating seeds accumulated high
amounts of ABA glucose ester. Thermodormant seeds were further distinguished
from germinating seeds by exhibiting major accumulations of IAA and zeatin
which were not accompanied by any significant increases in the levels of their
conjugates IAAsp and zeatin riboside, respectively. The most striking changes
potentially reflective of hormonal cross-talk included a marked accumulation of
auxin (IAA) levels in thermodormant seeds, that was coincident with a major
increase in the level of DPA (Chiwocha et al., 2003). Whether there is a key
interaction between auxin biosynthesis and ABA catabolism remains to be determined.
Targeted metabolic profiling of plant
hormones can be expanded to include a greater number of signalling compounds to
give a more comprehensive view of hormone levels, hormone metabolism and
changes in intracellular mediators (secondary messengers). Although these
studies have not been performed yet on the seeds of a range of species,
understanding metabolic flux during dormancy maintenance, termination of
dormancy, germination and early post-germinative growth will generate
information about how hormone-induced signalling networks control these complex
processes in seeds. It will in essence provide a ‘snapshot’ of the hormone
signalling status of the seed during key stages and transitions.
Transcriptomics and Proteomics
Genes and proteins whose patterns of
expression/synthesis coincide with dormancy maintenance and its termination
need to be functionally characterised. Further, the lack of sensitive screens
for mutants and the high degree of gene redundancy in plant genomes has
contributed to a reduced effectiveness of ‘forward genetic’ approaches to
identifying the genes that play regulatory roles in the dormancy-to-germination
and germination-to-growth transitions.
Proteomics approaches are being conducted
to enhance our understanding of germination (Gallardo et al., 2001, 2002;
reviewed in Bove et al., 2001). This type of analysis has been undertaken in
Arabidopsis (ecotype Landsberg erecta). The basic approach involved
two-dimensional gel electrophoresis separation of proteins derived from seeds
at different stages (mature-dry, and mature seeds imbibed for 1, 2 and 3 days);
about 1,300 proteins were resolved on the 2D gels and were classified with
respect to their accumulation patterns. Several of the separated proteins were
identified by matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) spectrometry (Gallardo et al., 2001; reviewed in Bove et al.,
2001).
Germination sensu stricto (1 day imbibed seed) was characterised by
changes in the abundance of ~40 proteins. Correlating with the resumption of
cell elongation and cell-cycle activity, was the accumulation of an actin 7
(potentially required for germination and/or hypocotyl elongation), tubulin subunits
and a WD-40-repeat protein (containing a repetitive segment of 40 amino acids
ending in Trp-Asp). This latter protein resembles receptors of activated
protein kinase C (having ~80% homology) and it may play a role in signal
transduction and hormone-controlled cell division. Confirming several earlier
studies (reviewed in Bewley, 1997), many of the proteins accumulated during
very early hydration were derived from components of the mature dry seed (e.g.
conserved mRNAs or proteins). The post-germinative phase was characterised by
changes in the abundance of 35 proteins. Many were linked to the establishment
of photosynthesis, the mobilisation of reserves or protective mechanisms (i.e.
pathogen and herbivore-defence-related) (Gallardo et al., 2001). Unfortunately
many of the changes in abundance of proteins were likely undetectable using
these methods, in part due to the higher abundance of seed storage proteins and
limitations associated with 2D gel techniques (e.g. hydrophobic proteins do not
separate well). The ecotype of Arabidopsis used in the above-noted study
exhibits little or no dormancy and it is likely that these studies will be
followed up using the far more dormant Arabidopsis ecotype, Cape Verde Island
(cvi) (Bove et al., 2001).
‘Phenomic’ approaches (functional genomic
analyses of entire mutant collections) will aid in the unequivocal assignment
of functions to unknown plant genes. Some of these studies have been initiated
on mutants that are disrupted in hormone biosynthesis/response (Gallardo et
al., 2002; Hoth et al., 2002). For example, a proteomics analysis to compare
wild-type Arabidopsis seeds with seeds of the GA-deficient mutant (in which
radicle emergence is completely dependent on exogenous GA) was undertaken
(Gallardo et al., 2002). This study revealed that of the 40 or so proteins
whose abundance changes during germination, only one, the cytoskeleton
component αα-2,4 tubulin, appeared to require GA. The abundance of several
proteins associated with later stages including metabolic control of seedling
establishment was dependent on GA, and this included an increase in
S-adenosyl-methionine synthetase, a housekeeping enzyme that catalyses the
formation of S-adenosyl-methionine from ATP and methionone. Notably, GAs also
appeared to control the abundance of the cell wall hydrolase, ββ-glucosidase,
that possibly mediates embryo cell wall loosening involved in cell elongation
and radicle extension (Gallardo et al., 2002).
Using a transcriptional profiling approach
(massively parallel signature sequencing, or MPSS), Hoth et al. (2002) analysed
gene regulation in seedlings of the abi1 mutant of Arabidopsis. Of the 1,354
genes that are responsive to exogenous ABA in wild-type seedlings, only ~9% of
these ABA-responsive genes show a wild-type pattern of expression in the abi1
mutant seedlings; most exhibited reduced or strongly diminished expression.
What is particularly interesting about the study is that several novel gene
families were uncovered whose expression is ABA-regulated (Hoth et al., 2002).
These included (1) About 100 genes encoding transcription factors or
DNA-binding proteins; (2) Genes encoding ribosomal proteins that stabilise the
tertiary structure of ribosomes and control the dynamics of protein synthesis;
the majority of these (15 out of 21 identified) were down-regulated by ABA. (3)
Genes encoding several proteins involved in regulated proteolysis. Most of
these were up-regulated (~75%), and, in some cases, greater than 10-fold. Some
of the encoded proteins had sequences that would predict RING finger motifs,
F-boxes, or U-boxes; thus they may interact with target proteins of the
ubiquitin-proteosome pathway (Hoth et al., 2002 and references therein). The
ABA-repression of some the genes encoding these proteins further strengthens
evidence from other studies showing that ABA blocks the degradation of certain
proteins in seedlings (e.g. ABI5) (Lopez-Molina et al., 2001). (4) Genes
encoding kinases and phosphatases, illustrating the importance of reversible
protein phosphorylation as a key element of ABA signalling. About an equal
number of genes encoding kinases (~40 in each case) were up- or down-regulated
by ABA. However, the genes encoding phosphatases (most of which were type 2C
phosphatases) were almost exclusively up-regulated by ABA. In the presence of
ABA, dephosphorylation events may directly activate or inhibit transcription
factors; alternatively, these may mediate oscillations in cytosolic Ca2+
concentrations that in turn regulate gene transcription (Hoth et al., 2002).
Although the study did not focus on seed dormancy or germination, it
illustrates the wealth of information regarding signal transduction pathways
that can be derived from targeted functional genomics approaches.
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