The Bronze-2 protein has strong sequence similarity to a soybean stress protein, Gmhsp26-A

--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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