Abstract
M.Sc.
As part of an investigation into differential gene expression in response to abiotic and
chemical inducers of acquired resistance in tobacco, a PCR fragment of 660bp was
repeatedly found in RNA preparations from treated cell suspensions by differential
display analysis. The fragment (D1B) was isolated, purified, cloned and sequenced. The
nucleotide sequence of the fragment was compared with sequences in the BLAST
sequence database and was found to be homologous to the mitochondrial orfx genes
from Arabidopsis thaliana, Beta vulgaris, Oenothera berteriana, Oryza sativa and
Marchantia polymorpha. In order to obtain the full sequence of the gene specific primers
were designed using the Arabidopsis sequence as template. The primers were
designed to complete the 5’-end of the gene and were designed to overlap the D1B
fragment previously found. A fragment (C3Y) of 460bp was isolated, purified, cloned
and sequenced. The complete sequence (D1B and C3Y combined) was 851bp long and
showed 96% homology with the Arabidopsis orfx gene on the nucleotide level and 87%
homology on the translated amino acid level. The sequence was submitted to the Basic
Local Alignment Search Tool (BLAST) database as accession gi: 24209907.
In plant genomes, the orfx gene is closely linked to important structural genes such as
the nad subunits of complex I (NADH: ubiquinone oxidoreductase). Orfx codes for a
hypothetical protein that shows homology to the mttB (membrane targeting and
translocation) gene found in E. coli. In bacteria the gene is essential because if deleted,
the organism was no longer viable. Functional analysis of the bacterial gene revealed a
novel pathway specific for membrane targeting and secretion of cofactor containing
proteins, such as iron-sulphur (Fe-S) clusters, of which the mttB gene encodes one
subunit. It is thought that a similar pathway might be responsible for the correct
localisation and assembly of such Fe-S containing protein complexes in the inner
mitochondrial membrane of higher plants. The differential display result may be
indicative of a general up-regulation of mitochondrial gene expression in response to the
triggering of plant defences or a possible specific effect on the expression of the orfx
gene.
A hypothesis was formulated that chemical inducers of plant defences affect the
mitochondria of treated plant cells to result in increased production of reactive oxygen
intermediates (ROI), similar to the oxidative microbursts proposed to be involved in
systemic required resistance. Using a dichlorodihidrofluorescein (H2DCFDA) assay, it
was found that salicylic acid (SA), benzo (1,2,3) thiadiazole-7-carbothioc acid S-methyl
ester (BTH) and isonitrosoacetophenone (INAP) increased ROI production within cells in
a dose dependant manner. The biochemical basis of this effect could possibly be
related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of
the mitochondrial electron transport chain by SA, BTH and INAP.
Prof. I.A. Dubery