The protein encoded by the Bronze2 gene in maize performs the last genetically defined step in the anthocyanin biosynthetic pathway (C2 -->A1-->A2-->Bz1-->Bz2), resulting in the purple pigmentation seen in various plant tissues such as the leaves, husks, and aleurone. While its biochemical function in this pathway has not yet been shown, several functions for the BZ2 protein can be envisioned: the BZ2 protein may function as a malonyltransferase to catalyze the addition of malonic acid to the precursor cyanidin-3-glucoside to produce the cyanidin-3-malonylglucoside found in the maize vacuole (Harborne and Self, Phytochemistry 26:2417-18, 1987), or as a transporter to ensure that the anthocyanin is delivered into the vacuole, or it may stabilize the anthocyanin in the vacuole by complexing with metal ions. This lab has previously shown (Nash and Walbot, MNL 66:104, 1992; Nash and Walbot, Plant Physiol. 100:464-71, 1993) that the BZ2 amino acid sequence is highly similar to the amino acid sequence of the soybean small heat shock protein (HSP)-like hsp26A gene; there is 66% similarity between the first exons of these genes. In addition, Schmitz and Theres (Mol. Gen. Genet. 233:269-77, 1992) also reported the similarity of Bz2 with a number of other plant genes. However, only the percent similarity, and not the actual alignment, was reported. We have aligned the first exon sequences of all genes related to Bz2, shown in Figure 1.

In addition to the high amino acid homology only within the first exons of all these genes (amino terminus of protein), there are several other striking characteristics suggesting that these proteins are related:

• Structurally, the organization of all the genes is highly conserved: each gene for which the exon:intron structure was reported contains a single intron in the exact same position in each message. Beyond the first exon, the similarity of Bz2 with the other genes is extremely low. This conservation of exon:intron boundaries supports the suggestion (Nash and Walbot, MNL 66:104, 1992) that this class of proteins may have been constructed by the process of exon-shuffling.
• Functionally, all are involved in responses to environmental stress conditions, particularly auxins and heavy metals, although the biochemical function of most is poorly understood (the exception being the glutathione-S-transferases or GSTs):

• Transcripts of both the Bz2 and the hsp26A genes are unusual in that high amounts of unspliced, intron-containing message have been reported, particularly as a result of heavy metal stress as well as a generalized "field" stress (Czarnecka et al., Mol. Cell. Biol. 8:1113-22, 1988; Nash et al., Plant Cell 2:1039-49, 1990; Marrs and Walbot, in preparation). Both the Bz2 and the hsp26A unspliced transcripts, if translated, would encode highly related, truncated proteins of about 14 kD (vs. the "full length" protein of ~26 kD) because of a stop codon in the intron of each gene. The production of the 14 kD protein was observed by Czarnecka et al. after hybrid-release translation using RNA from cadmium-stressed seedlings. While the presence of unspliced transcripts of the other genes in this class is not mentioned in the literature, their presence would also encode for truncated proteins of 14-17 kD because of stop codons in the introns.

Another possibility is that the proteins encoded by these genes could have dual roles in the cell both during normal cell metabolism and during stress. Several of these proteins share significant homology with authentic plant and animal GSTs, including the BZ2 protein sequence to some extent. GSTs are involved in the detoxification of a wide variety of xenobiotic compounds and herbicides (and heavy metals indirectly, which are detoxified by the synthesis of phytochelatin from glutathione). In addition, GSTs also play a role in normal plant secondary metabolism, and are also thought to protect cells against oxidative damage. Malonyltransferase enzymes are another class of enzymes in plants that have roles in both detoxification of xenobiotic compounds as well as during normal secondary metabolism (Sandermann, TIBS 17:82-84, 1992). Genes for known malonyltransferases have yet to be cloned. The BZ2 protein could have a dual function as a malonlytransferase, malonating either cyanidin-3-glucosides during anthocyanin production or other xenobiotic substrates for detoxification during stress. Alternatively, BZ2 could function as a metal binding protein either to stabilize anthocyanins or to chelate heavy metals during stress.

Our current findings concerning Bz2 regulation during cadmium stress as well as previous results from this lab imply that alternative forms of Bz2 may play a role in the cell during stress. We are currently testing whether the BZ2 protein functions biochemically as a malonyltransferase or as a glutathione-S-transferase both during anthocyanin production and during stress. Positive results would provide the first evidence of the biochemical function of Bz2.

Figure 1: Amino acid sequence alignment between the first exon of Bz2 and the first exon (or amino terminal "half") of other related genes: soybean hsp26A (Czarnecka et al., Mol. Cell. Biol. 8:113-22), tobacco parA (Takahashi et al., PNAS 86:9279-83), tobacco NT103 and 107 (van der Zaal et al., Plant Mol. Biol. 16:983-98; Droog et al., Plant Mol. Biol. 21:965-72, 1993), potato PRP1 (Taylor et al., Mol. Plant-Microbe Interact. 1:157-60), and maize GSTIII (Grove et al., Nucl. Acids Res. 16:425-38). The paper by Droog et al. (Plant Mol. Biol. 21:965-72, 1993) reports the complete alignment (both first and second exon) for all these genes with the exception of Bz2. Residues identical and very similar to Bz2 are indicated by (|) and (:), respectively. Similarity groups used are I,L,V,F,W,Y; K,H,R; D,E,N,Q; S,T. Hyphens indicate gaps introduced to optimize alignment. Asterisks (*) indicate residues identical in all seven sequences. A slash (/) indicates the position of the exon/intron boundary, where known.

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