Biology Reference
In-Depth Information
If ascorbate is affecting gene expression in the nectary, then we might
expect to observe differences in ascorbate-regulated gene expression at
stages of nectary development when ascorbate levels differ significantly.
Ascorbate is generally low in the nectary until late in development (Horner
et al., 2007), when ascorbate is rapidly synthesized to serve as the extracellu-
lar antioxidant functioning in the nectar redox cycle. Analysis of our
macroarrays (Table 4, Thornburg et al., 2003) reveals that a number of genes
appear to show such ascorbate-mediated regulation. In presecretory nectaries
(stage 6) ascorbate is relatively low. At this time, a number of stress and de-
fence genes are highly expressed. Later, at anthesis (stage 12), when
ascorbate production is very high, these same genes are now downregulated.
This group of genes includes the same genes identified by Pastori et al.
(2003). Further, Kiddle et al. (2003) showed that metallothionein was
upregulated in response to ascorbate application. Our analysis (Table 4) also
shows this upregulation for metallothionein. Thus, ascorbate also appears to
have a role in gene regulation during nectary development.
Table 4.
Gene expression levels from macroarray analysis.
Percentage of leaf*
Gene
Stage 6
Stage 12
PR-1
419 ± 40
192 ± 101
PR-5
2,873 ± 735
214 ± 118
Chitinase
752 ± 410
387 ± 125
CHS
452 ± 128
211 ± 100
Subtilisin-like
634 ± 78
465 ± 84
Metallothionein
952 ± 248
1,831 ± 489
*mRNAs from stage 6 or stage 12 nectaries were labelled with [
32
P] and the labelled cDNAs
were hybridized to a macroarray containing approximately 500 tomato defense genes. Results
are average amounts of hybridization as a percentage of that in the control leaf ± SE.
(Thornburg et al., 2003.)
5.2
EST analysis
We have sequenced 12,534 nectary-expressed ESTs from three different
stages of nectary development: stage 6 (presecretory developing nectaries),
stage 12 (mature secretory nectaries at anthesis) and stage PF (nectaries
44 h post-fertilization). These have been clustered using the TIGR clustering
program (tigrcl) to identify groups of overlapping sequences that are referred
to as tentative contigs (TCs) or unigenes. This clustering permitted the iden-
tification of 6,158 nectary-expressed unigenes. Following annotation of
