--Juliana Nash and Virginia Walbot
The product of the Bronze-2 (Bz2) gene is required last in the
sequence of known anthocyanin biosynthetic structural genes (C2 ->A1->A2->Bz1->Bz2->),
yet its function remains unknown. To look for clues that might lead us
to the biochemical activity of Bz2, we performed nucleotide and
amino acid sequence searches against the GenBank and EMBL DNA and protein
sequence databases. We found that the primary amino acid sequence of the
soybean Gmhsp26-A protein (Czarnecka, E et al., Mol. Cell. Biol.
8:1113-1122, 1988) is very similar to the Bz2 protein sequence (Nash,
J et al., Plant Cell 2:1039-1049, 1990); both encode a 26kDa gene product.
The first 85 amino acids of exon 1 share a 44% identity and a 68% similarity,
while there is no significant similarity between the second exons (see
Figure 1). Transcripts produced from Bz2 and Gmhsp26-A are
susceptible to splicing failure, and the resultant unspliced messages encode
an approximately 14kDa protein in each case because of an in-frame stop
codon within the intron sequence (Czarnecka, E et al., 1988; Nash, J et
al., 1990). No gene sequences in the databases shared significant similarity
to the Bz2 nucleotide sequence
2 TAGTMRVLGGEVSPFTARARLALDLRGVAYELLDEPLGPKKSDRLLAANP 51
|.:.:::|| .|||..|..:||.|:||.|.:|:| || .||| || ||
4 TQEDVKLLGIVGSPFVCRVQIALKLKGVEYKFLEENLG.NKSDLLLKYNP 52
. . . . .
52 VYGKIPVLLLPDGRAICESAVIVQYIEDVARESGGAEAGSLLLPDDPYER 101
|. |:|| ::.::.:|.|| |||:||::. ::.. ..:.. : :..|
53 VHKKVPV.FVHNEQPIAESLVIVEYIDETWKNNPILPSDPY...QRALAR 98
. . . . .
102 AMHRFWTAFIDDKFWPALDAVSLAPTPGARAQAAEDTRAALSLLEEAFKD 151
:| .. | : ....: .|. . . . .:. |. . . |.: |.
99 FWSKFIDDKIVGAVSKSVFTVDEKEREKNVEETYEALQFLENELKD..KK 146
. . . . .
152 RSNGRAFFSGGDAA.......PGLLDLALGCFLPALR...ACERLHGLSL 191
.| .| .: || | : ::| ::.. : :.: ::: |
147 FFGGEEFGLVDIAAVFIAFWIPIFQEIAGLQLFTSEKFPILYKWSQEF.L 195
. . .
192 IDASATPLLDGWSQRFAAHPAAKRVLPDTE 221
.: . .:|..:.. || .| |....
196 NHPFVHEVLPPRDPLFAYFKARYESLSASK 225
Figure 1. Amino acid sequence similarity between BZ2 and Gmhsp26-A proteins. Identical, very similar, and remotely similar residues are indicated by |, :, and ., respectively. Similarity calculations made in the text utilized only the very similar residues. BZ2 amino acid sequence is on the upper side of the alignment while Gmhsp26-A is on the lower: residue positions are noted in the margins. The intron position for Bz2 follows residue #114 and for Gmhsp26-A follows residue #102. Sequence alignment was performed using GCG software (Genetics Computer Group, Madison, WI).
While the structural similarity of the first exons of these proteins
is very high, the function of neither protein has been determined. The
Gmhsp26-A protein was named a heat-shock protein because of a weak
similarity to the set of small heat-shock proteins of animals, but the
protein lacked any significant similarity to plant small heat-shock proteins
(Czarnecka, E et al., 1988). Transcripts from Gmhsp26-A are induced
as a result of many different stress conditions including, heat-shock,
heavy-metal stress, oxidative stress, and ABA treatment (Czarnecka, E et
al., Plant Mol. Biol. 3:45-58, 1984). Walbot, V et al. (this issue) show
that Bz2 expression may also be affected by ABA levels in kernel
tissue. The expression patterns of Bz2 have not been determined
for each of the above mentioned stress conditions yet, but we have shown
that the Bz2 gene is not a heat-shock protein gene: its transcript
abundance decreases upon exposure of maize seedlings to 41 C treatments
(J. Nash and V. Walbot, submitted). In fact, the heat-shock protein homology
of Gmhsp26-A is within the second exon of this protein indicating
that any related function of this soybean protein to heat-shock would be
contained in a region of the protein with which Bz2 does not share
similarity. These findings, along with the observation of the similar intron-splicing
behaviors of Gmhsp26-A and Bz2, are suggestive that these
proteins may have been constructed by the process of exon-shuffling. Perhaps
the first exon shares a function important to both of these proteins, such
as metal-binding or a role in hormone-stress physiology, but the second
exons are derived from different ancestral histories.
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