| Summary: | Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. === Despite numerous attempts involving a variety of target genes, the identity of the key
regulatory genes of sucrose metabolism in sugarcane is still illusive. To date,
genomic research into sucrose accumulation in sugarcane has focused on genes
that are expressed in association with stalk development/maturation, with the aim of
identifying key regulatory steps in sucrose metabolism. The identification of possible
controlling points, however, is complicated by the polyploid nature of sugarcane.
Although these studies have yielded extensive annotated gene lists and correlative
data, the identity of key regulatory genes remains elusive. A close relative of
sugarcane, Sorghum bicolor, is diploid, has a small genome size and accumulates
sucrose in the stalk parenchyma. The main aim of the work presented in this thesis
was to use S. bicolor as a model to identify genes that are differentially expressed
during sucrose accumulation in the stalk of low and high sucrose genotypes.
In the first part of the study, a macroarray protocol for identification of differentially
expressed genes during sorghum development was established. Firstly, the
macroarray sensitivity of probe-target hybridisation was optimised with increasing
amounts of target DNA i.e. 0.005-0.075 pmol. The hybridisation signal intensity
increased as expected with increasing amounts of probe until the hybridisation
signals reached maximum levels at 0.05 pmol. As a result, to ensure quantitative
cDNA detection, probes were arrayed at 0.05 pmol when 1 μg target cDNA was
used. Secondly, intra-array and inter-array membrane reproducibility was found to be
high. In addition, the protocol was able to detect species of mRNA at the lowest
detection limit tested (0.06%) and permits the detection of an eight-fold variation in
transcript levels. The conclusion was therefore that the protocol was reproducible,
robust and can reliably detect changes in mRNA levels.
In the second part of the study, sugar accumulation levels in the immature and
maturing internodal tissues of sorghum GH1 and SH2 genotypes were compared
during the boot and softdough stages. Sugars (i.e. fructose, glucose and sucrose)
accumulated differently in the immature and maturing internodes in both sorghum
genotypes during the boot and softdough stages, with sucrose being the dominant
sugar in both stages. Based on these differences in sugar accumulation patterns,
immature and maturing internodal tissues of sorghum genotypes were compared for differentially expressed genes. A number of genes were found to be significantly
differentially expressed during both stages.
In order to validate the reliability of the macroarray analysis, fourteen genes were
arbitrarily selected for semi-quantitative RT-PCR. Seven genes (50%) revealed a
similar pattern of transcript expression, confirming the macroarray results. The other
seven genes, however, showed a different expression trend compared with the
macroarrays. In this study, ESTs from rice and sugarcane were used for probing
sorghum. The probability of cross-hybridisation between the probes and various
isoforms of the homologous sorghum sequences is thus high, potentially leading to
the identification of false positives. In addition, variation in expression patterns could
have been introduced by technical and biological variation.
Lastly, to verify that changes in the levels of a transcript are also reflected in changes
in enzyme activity, seven candidates were tested for enzyme activity. Only three i.e.
soluble acid invertase (SAI), sucrose synthase (SuSy) and alcohol dehydrogenase
(ADH), out of these seven genes showed enzyme activity levels reflective of the
relative transcript expression. We concluded that changes in transcript levels may or
may not immediately lead to similar changes in enzyme activity. In addition, enzyme
activity may be controlled at transcriptional and at posttranscriptional levels.
In conclusion, sugar accumulation in low (GH1) and high (SH2) sucrose sorghum
genotypes is influenced by differences in gene expression. In addition, the power of
macroarrays and confirmation with semi-quantitative RT-PCR for identification of
differentially expressed genes in sorghum genotypes was demonstrated. Moreover,
the transcript and enzyme activity patterns of SAI, SuSy and ADH genes showed
expression patterns similar to those of sugarcane during sucrose accumulation.
Therefore, using sorghum as a model promises to enhance and refine our
understanding of sucrose accumulation in sugarcane.
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