ALKALOID BIOSYNTHESIS IN PLANTS: Biochemistry, Cell ... - UFV

50 FACCHINI
enhancer for basal expression, and two separate elicitor-responsive elements
were found between −99 and −87, and between −87 and −37. In vitro binding
of nuclear factors to the −572 to −37 region of the TDC promoter has also
been described (126). Two binding activities that interact with multiple TDC promoter
regions were identified as GT-1 and 3AF1 in tobacco and C. roseus nuclear
protein extracts. Mutagenesis of the GT-1 binding sites did not affect basal or
elicitor-induced expression, but did reduce TDC promoter activation by UV light.
Only UV-B wavelengths were shown to induce TIA accumulation and TDC expression
in C. roseus, suggesting the involvement of a specific receptor (123).
Loss-of-function analysis revealed redundant UV-responsive elements in the TDC
promoter between −99 and +198 (123).
The coordinate accumulation of transcripts suggests that TDC, STR, and CPR
genes are regulated by common nuclear factors in response to elicitor treatment
and UV light (108, 132). Deletion of the CPR promoter to −366 eliminated the
elicitor-inducible expression observed with a 632-bp promoter (90). The −632 to
−366 region of the CPR promoter also contains strong GT-1 binding sites. The
main elicitor-responsive sequences of the STR1 promoter were identified in the
−339 to −145 region (131). Again, GT-1 was shown to bind to this and other
regions of the STR1 promoter. Despite the strong interaction of GT-1 with TDC,
STR1, and CPR promoters, it remains to be determined whether GT-1 binding sites
play a role in gene expression in vivo.
Other transcription factors are undoubtedly involved in the basal expression
of the TDC, STR1, and CPR genes. A G-box motif at −105 was shown to bind
G-box binding factors (GBFs) in vitro, but was not essential for the elicitor-induced
expression of STR1 in vivo (131). This G-box element also interacts with tobacco
nuclear factors and the G-box-binding factor TAF-1 (124). Mutation of the G-box
motif prevented binding of these factors and reduced the functional activity of
constructs containing tetramers of the STR1 G-box sequence. A G-box tetramer
fused only to a TATA-box conferred seed-specific expression in transgenic tobacco,
but required the enhancer region from the CaMV promoter for expression in leaves.
These results suggest that sequences flanking the G-box motif determine STR1
promoter activity in different tissues (124).
A GCC-box-like element in the −100 to −58 region of the STR1 promoter is
necessary and sufficient for JA- and elicitor-responsive expression (109). Two
cDNAs encoding C. roseus AP2-domain (ORCA) proteins that bind the JA- and
elicitor-responsive elements of STR1 were isolated using a yeast one-hybrid screen.
The ORCA2 protein trans-activates the STR1 promoter, and expression of the
ORCA2 gene is induced by elicitor and JA treatment of C. roseus cell cultures. In
contrast, the ORCA1 gene is constitutively expressed. These results specifically
identify a GCC-box-like element and ORCA2 in the JA- and elicitor-responsive
expression of the STR1 gene.
Deletion analysis has also revealed the location of regulatory domains necessary
for TYDC7 and BBE1 promoter activities in a transient GUS assay system based
on the microprojectile bombardment of cultured opium poppy cells (130). The