Effects of trehalose on gene expression in "Arabidopsis thaliana" seedlings : a genome-wide analysis

Trehalose (α-D-glucopyranosyl-[1,1]-α-D-glucopyranoside), a non reducing disaccharide
consisting of two glucose units is present in a large variety of organisms such as bacteria,
fungi and invertebrate animals where it may serve as a storage compound or stress
protectant. Trehalose also accumulates in a few desiccation-tolerant ‘resurrection plants’
and may have similar functions there. However, in almost all higher plants, including the
model plant Arabidopsis thaliana, trehalose is only present in hardly detectable amounts.
Nevertheless, plants contain several genes for each of the two enzymes required for
trehalose synthesis, and an Arabidopsis mutant lacking one of these genes is embryolethal.
It is therefore interesting to investigate the possible physiological roles of trehalose
in plants. For this purpose A. thaliana seedlings were incubated with trehalose, and the
effects on gene transcript levels were studies at the whole-genome scale. Some of the
transcripts most strongly induced by trehalose were selected for a detailed analysis, and
their expression was studied by quantitative PCR.
It has recently been shown by others, working also with A. thaliana, that several effects
of exogenous application of trehalose can be mimicked by genetic manipulations leading
to enhanced endogenous trehalose-6-phosphate (T6P) levels, and that trehalose
application indeed rises the endogenous T6P level. (This effect might result from a
feedback inhibition of trehalose-6-phophate phosphatase when intracellular trehalose
levels are high.) Thus, the changes in gene expression triggered by trehalose application
may be due to an increase in intracellular T6P concentrations rather than to an increase in
trehalose levels per se.
A problem of trehalose application, as used by others and initially also in this thesis, is
the ubiquitous occurrence of trehalase activity in plants, an enzyme most likely present in
the cell wall. This enzyme reduces exogenous trehalose levels and generates glucose,
making it difficult to interpret data on exogenous trehalose application. To prevent this, in
the main part of the thesis, trehalose was applied in combination with the potent trehalase
inhibitor validamycin A (val). In the presence of validamycin A, application of 2-4 mM
trehalose was sufficient to alter gene expression in a similar way as application of 25 mM
alone.
In this work it is shown that 25 mM trehalose added in combination with val (tre/val)
caused a two fold or more up or down regulation of 2277 genes of which more than a
third was also regulated by abscisic acid (ABA), linking trehalose metabolism to ABA
metabolism or ABA signalling. In the ABA deficient Arabidopsis mutant aba1, selected
genes inducible by tre/val and ABA still could be induced by tre/val, indicating ABA and
ABA metabolism not to be necessary to induce ABA regulated genes upon tre/val
treatment. The qualitative difference of the kinetic induction curve of gene expression
over time after tre/val application for the genes inducible by tre/val and ABA compared
to the induction curve after ABA treatment further supports the hypothesis the tre/val
induction is independent of ABA. Interestingly, the induction of some of these by tre/val
and ABA inducible genes was suppressed in abi2-1, a mutant impaired in the ABA
response. Therefore, it can be suggested that ABI2 is necessary for the regulation of at
least some genes induced by tre/val. The results obtained by the two ABA mutants
suggest the signalling cascade triggered by tre/val affects the ABA signalling pathway
downstream of the ABA binding site but upstream of ABI2 activity.
Classification of the genes up regulated by tre/val but not by ABA revealed a set of genes
involved in disease resistance and secondary metabolism indicating trehalose in
combination with validamycin A may act as an elicitor in plants.
Sugars such as sucrose (suc), glucose and fructose serve in plants not only as important
intermediates in the primary metabolism, carbon sources or substrates for storage
compounds like starch and cellulose, but can also act as signalling molecules in a similar
way as trehalose/trehalose-6-phosphate. Plant cells can sense these sugars via either a
hexokinase-dependent, or -independent system. The signal transduction pathways of both
these systems include protein phosphatases (PPs) and protein kinases (PKs).
One gene that is readily induced by suc is the one encoding sucrose:fructan 6-
fructosyltransferase (6-SFT) in barley, a key enzyme in fructan synthesis. The promoter
of this gene was studied in excised barley leaves and transformed Arabidopsis bearing a
GUS reporter gene driven by a region of the barley 6-SFT promoter. The broad-spectrum
kinase inhibitor K252a as well as genistein (GEN), an inhibitor thought to be specific for
protein tyrosine kinases in animal systems, were able to reduce the Suc induced
activation of the 6-SFT promoter in both systems, indicating that PKs are involved in Suc
mediated regulation. Interestingly, staurosporine (STAU), an inhibitor of PKs similar to
K252a did not affect Suc induction, indicating a degree of specificity of these inhibitors.
A strong reduction of Suc triggered induction of 6-SFT expression was caused by 1 μM
of the potent PP inhibitor okadaic acid (OK). This suggests that PP2A activity is also
involved in the Suc mediated regulation of the 6-SFT promoter.