Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

Functional Genomics of Stress Tolerance
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Positional cloning has helped identify several genes in the stress signaling
pathway. These include SOS1, a Na + /H + antiporter (Shi et al., 2000), SOS2, a Ser/Thr
protein kinase (Liu et al., 2000), SOS3, a calcium binding protein (Liu and Zhu, 1998),
SOS4, a pyridoxal kinase (Shi et al., 2002), SOS5, a cell surface adhesion protein (Shi et
al., 2003), HOS1, a ring finger protein which negatively regulates low-temperatureresponsive
gene transcription (Ishitani et al., 1998; Lee et al., 2001), SpI7, a heat stress
transcription factor (Yamanouchi et al., 2002), STT3, subunit of oligosaccharyltransferase
complex which controls adaptive response to salt/osmotic stress (Koiwa et al., 2003),
FRO1, a NADH dehydrogenase subunit of mitochondrial respiratory chain complex I
which is involved in regulation of nuclear gene expression in response to cold (Lee et
al., 2002) and LOS5/ABA3, a molybdenum cofactor sulfurase that modulates cold and
osmotic stress responsive gene expression (Xiong et al., 2001). Several genes involved
in biotic stress signaling have also been identified through this approach; these include
Xa21, which confers resistance to bacterial pathogen Xanthomonas Oryzae (Song
et al., 1995), Pib, which confers rice blast resistance (Wang et al., 1999), Pita2 (Bryan et
al., 2000), Pi5(t) (Jeon et al., 2003), Xa1 (Yoshimura et al., 1996) and Xa26 (Yang et al.,
2003).
Several mutagens produce sizable deletions like diepoxybutane, fast neutron
and gamma rays. A collection of 40,000 deletion mutants has been made in rice in the
IR64 background using these mutagens (Leung et al., 2001). Analysis of 10,000 of these
mutant lines for alteration in stress signaling revealed at least 0.3% of the mutants to be
altered in the disease response pathway. Abiotic stress tolerance was also assessed in
the mutant populations. A total of 3,000 lines were studied for submergence stress, of
which six mutants were identified which showed ~75% recovery after submergence
stress. Under drought conditions, 26 out of 1200 plants showed better response compared
to wild type. These mutants have only been studied at the physiological level but
the molecular basis of these alterations in stress response is still to be determined.
Apart from deletion mutants, a high throughput technique known as TILLING
(Targeting Induced Local Lesions In Genomes), for reverse genetics is available which
can detect single base changes. The approach makes use of high-density point mutations
generated using chemical mutagenesis. These populations are screened for mutations
using denaturing high performance liquid chromatography (DHPLC) (McCallum
et al., 2000) or endonuclease, which would specifically cleave mismatches in a heteroduplex
between wild type and mutant (Colbert et al., 2001). Users can make use of the
TILLING facility through an interactive web based system; http://www.proweb.org/
coddle and the mutant collection consisting of about 10,000 lines can be accessed
through Arabidopsis Biological Research Center (ABRC, Ohio State University). TILL-
ING has been recently used to discover polymorphism in natural populations. This
approach called Ecotilling, was used to study variation in 192 accessions of Arabidopsis.
Study of different ecotypes through TILLING may help identify gene function, e.g. a
null allele was identified by Ecotilling amongst the different accessions for DRM1
(DOMAINS REARRANGED DNAMETHYLASE1) gene showing that the gene is dispensable
(Comai et al., 2004).