The present study was undertaken with the hope of identifying the genetic loci encoding the anthocyanin biosynthetic enzymes UDPglucose 3-0-glucosyltransferase (UFGT) and chalcone synthase (CHS) . Mutations in the genes encoding these enzymes in maize, petunia, and other species result in a recessive anthocyaninless phenotype. Since these genes have been molecularly cloned from maize and petunia, it is possible to introduce them to tomato by transformation and test for complementation of the homologous tomato genes. As a prerequisite to performing this test, we set out to identify mutants of these enzymes in tomato by enzyme assay. There are eight known genetic loci in tomato with alleles conditioning a "completely anthocyaninless" phenotype (a, aa, ae, af, afr, ah, aw, and bls; obtained from C. Rick); afr and bls have pleitropic effects on- growth, the other six "completely anthocyaninless" mutants have not been reported to possess any other altered characteristics besides loss of anthocyanin pigmentation, and so are clearly candidates for anthocyanin biosynthetic enzyme genes. We assayed UFGT and CHS activity in leaves from seedlings that had been grown for 13 days under high intensity fluorescent lighting (16h. days, 8h nights) at 16C, conditions that induce high levels of anthocyanin production. UFGT was assayed in extracts prepared from 50 mg leaf tissue (not fully expanded) homogenized in 100 ul cold 35mMtris8.2 and diluted to 200 ul. 25 ul of each extract was assayed for UFGT as described by Dooner (1981). Reactions were 50ul and were extracted with 300 ul ethyl acetate. 200 ul of the ethyl acetate phase was counted in a scintillation counter (see Table 1). Controls were run without quercetin. CHS was assayed in a similar homogenate except for the use of 0.1 M KPO4, pH 7.6, 1.42 mM B-mercaptoethanol and the addition of BSA to 5 mg/ml immediately after homogenation. 40 ul homogenate was assayed as described in Dooner (1983) in 50 ul. The reaction was stopped with 20 ul methanol and extracted with 300 ul ethyl acetate. Controls were run without p-coumaroyl CoA. 150 ul ethyl acetate phase was counted (Table 1) and 100 ul was spotted on Whatman #1 paper and chromatographed in 30% HOAC to confirm the identity of the naringenin reaction product.
Table 1. Activities (in cpm) of UFGT and CHS.
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Genotype Accession UFGT Control CHS Control
_______ _________ ____ _______ ___ _______
Control "Red Cherry" Herbst 3105 47 1282 77
a/a LA784 6518 10 876 136
aa/aa LA1194 3325 30 183 98
aa/aa LA1525 5195 25 289 134
aa/aa LA1700 5655 40 4495 215
ae/ae LA1665 6810 40 694 115
af/af LA1444 2078 10 998 117
afr/afr LA1784 7458 38 1414 119
ah/ah LA1164 2490 33 1840 133
aw/aw LA271 3655 35 1965 191
bls/bls LA1004 1295 33 430 108
We find no evidence that any of these anthocyanin deficient mutants
lacks either UFGT or HS activity. Although some genotypes possess lower
levels of enzyme activity than the "Red Cherry" control, these differences
are not sufficient to account for the lack of anthocyanin in these plants.
They probably result from the various genetic backgrounds these mutants are
in. For example, 2 of the 3 aa/aa accessions showed low CHS activity, but
the third one was even higher than the control. We also conclude that none
of these genes can be regulatory loci like R and B in maize since none
results in null activities for both enzymes.Literature Cited:
Dooner, H.K. 1981. CSHSQB. 45:457-462.
Dooner, H.K. 1983. Mol. Gen. Genet. 189:136-141.