Cloning of a P-regulated chalcone-flavanone isomerase gene from maize
--Erich Grotewold and Thomas Peterson
Chalcone synthetase (CHS) is the first enzyme of flavonoid biosynthesis and converts malonyl-CoA and 4-coumaroyl-CoA into chalcone. The similarity of flavonoid biosynthesis in maize and other plants (petunia, bean, etc.) suggests that maize should contain a second enzyme, chalcone-flavanone isomerase (CHI), responsible for the isomerization of the chalcone into the corresponding flavanone. Flavanone is the substrate for the synthesis of 3-deoxy and 3-hydroxy flavonoid derived pigments. The chalcone-flavanone isomerase enzyme should act between the C2 and A1 encoded products, which opened the possibility that its expression would be regulated by the P gene in the pericarp, as are C2 and A1 (Grotewold et al., submitted). However, CHI activity has not been reported in maize, nor has any mutant in this step been isolated.
To clone a maize CHI gene, we used highly degenerate primers made from sequences conserved between CHI proteins from several plants (we appreciate very much the suggestions provided by Dr. Arjen van Tunen regarding the sequences of the primers). We obtained a 210 bp cDNA by PCR, which was used as probe for screening a cDNA library from pericarps carrying a functional P gene (P-rr) (Grotewold and Peterson, 1990; MNL 64:37). We isolated nine independent clones about 650 bp long which showed identical sequences between them.
The translated sequence of the cDNA clones showed about 65% identity at the amino acid level with the sequence of two chalcone-flavanone isomerase proteins described in Petunia hybrida. The first amino acid encoded by the longest cDNA clone corresponds to amino acid 47 of the sequence of the petunia CHI-B protein (van Tunen et al., 1989; Plant Mol. Biol. 12:539-551), indicating that the obtained cDNA clones are not full-length.
Northern blot hybridizations of pericarp poly A RNA showed a single transcript of about 1 kb hybridizing with these cDNA clones. No transcript could be detected in pericarp RNA lacking a functional P gene, suggesting that the maize CHI gene could be regulated by P in the tissues in which P is being expressed.
We don't know yet how many CHI genes are present in maize, although our results suggest that a single gene would be transcribed in the pericarp under the control of P. In petunia, different CHI genes are transcribed in different tissues (van Tunen et al., 1988; EMBO J. 7:1257-1263). It remains to be shown if this is the case with the maize CHI gene. Since the step catalyzed by the product of the CHI gene is common to the synthesis of all flavonoid derived products, including the anthocyanins, it would be expected that other regulators of the pathway (for example R, B, C1 and Pl) would also be able to regulate the expression of CHI in other tissues.
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