DISCUSSION

Single effects and epistasis: What is clear from our results is that the flavone pathway is not nearly as well defined as originally proposed or as simplistic as the anthocyanin pathway has been depicted. Structurally, apimaysin and maysin are highly related compounds, differing only by a 3′-hydroxyl group (apimaysin 3′-H, maysin 3′-OH). Based on the anthocyanin synthesis model, in which all anthocyanins are synthesized from a common pathway, we had assumed that the synthesis of apimaysin and maysin would also occur in a common pathway. We assumed the same structural enzymes, except flavonoid 3′-hydroxylase, and the same pools of metabolic precursors would be required. Instead, the syntheses of apimaysin and maysin appear to be independent. This population was segregating at pr1, which is known to affect 3′-hydroxylation of anthocyanins. The genomic region containing pr1 was detected as the major QTL affecting apimaysin levels. Apimaysin was detected only in individuals homozygous for the nonfunctional NC7A pr1 allele, the expected consequence of homozygosity for a nonfunctional pr1 allele. This QTL, however, did not affect maysin levels, which was an unexpected outcome. Apimaysin was not made at the expense of maysin, but rather, silks with apimaysin had increased total flavone levels.

The major QTL for maysin in this population is consistent with the genomic region previously identified as containing the maysin QTL, rem1. As in previous studies, a twofold increase in maysin levels was observed in individuals from one of the homozygous rem1 genotypic classes (Byrne et al. 1996, 1998). However, rem1 did not affect apimaysin levels, again demonstrating an independence of the pathway leading to maysin synthesis from the pathway leading to apimaysin synthesis. The apparent independence of apimaysin synthesis from maysin synthesis suggests that perhaps different precursors are being used or that highly related, yet distinct sets of enzymes are involved.

The two major single effects, pr1 and rem1, are also of interest because of their significant epistatic interaction. Each of the effects alone increased total flavone levels: rem1 by increasing the amount of maysin, and pr1 by permitting the synthesis of apimaysin. Individuals homozygous for NC7A alleles at both rem1 and pr1 should simultaneously be capable of producing apimaysin and synthesizing additional maysin. However, total flavone levels in the double homozygous class were no higher than with either individual single homozygote effect (Table 1). Even though apimaysin and maysin syntheses appear to be independent of one another, it appears that an upper limit exists that governs how much total flavone can be produced by the pathway or tolerated by the cell. It should be noted that total flavone levels in some inbred lines are considerably >0.8%. Maysin levels between 1.5 and 2.0% have been observed in some backgrounds (E. Lee, unpublished data), suggesting that the mechanism regulating the ceiling level may be background specific.

B-ring substitution: Maysin is hydroxylated at the 3′-position, and functional Pr1 is required for 3′-hydroxylation of anthocyanins in maize aleurone tissues (Larsonet al. 1986). However, allelic constitution differences at pr1 have no effect on maysin levels. How can maysin be synthesized if pr1 is not involved? There are several possibilities. First, it is possible that pr1 is not the QTL, but rather, the apimaysin QTL is tightly linked to pr1. It has not been demonstrated that pr1 encodes flavonoid 3′-hydroxylase, only that anthocyanin synthesis in aleurone tissues requires pr1 for hydroxylation at the 3′ position (Larsonet al. 1986). We have worked with other inbred lines that contain functional p1 alleles in silks and nonfunctional pr1 alleles. These lines, when grown in the same environment, tend to accumulate a higher proportion of dihydroxy flavones (i.e., maysin) to monohydroxy flavones (i.e., apimaysin) than the nonfunctional pr1 parent (NC7A) used in this study (NC7A 1.28:1; Tx601 35.3:1; Mp708 3.7:1; PI340853 5.3:1) (E. Lee, unpublished data).

A second possibility is that pr1 may encode a flavonoid 3′-hydroxylase, but there may be another gene homologous to pr1 that is also used in maysin synthesis. Maize has many duplicate loci with similar functions, tissue specificities, and/or developmental expression patterns [e.g., c2 and whp1 encode chalcone synthase (Frankenet al. 1991), r1 and b1 encode homologous myc-like transcription factors (Chandleret al. 1989), and c1 and pl1 encode homologous myb-like transcription factors (Coneet al. 1993)]. Larson et al. (1986) found that sheath tissues of nonfunctional pr1 plants still retained appreciable amounts of F3′H activity, suggesting the presence of another F3′H. When pr1 is nonfunctional, maysin is made. Our expectation was that a functional pr1 would increase maysin. This is not the case. Maysin levels in this population remained unchanged regardless of the constitution at pr1.

Finally, it is possible that the B-ring substitution patterns for flavones may occur at the 9-carbon stage rather than the 15-carbon stage. The flavonoid B-ring arises from a common phenylpropanoid pathway intermediate that is generally depicted as being 4-coumaryl-CoA (4-OH). However, chalcone synthase and chalcone isomerase from other species can use caffeoyl-CoA (3,4-diOH) and other 9-carbon substrates in addition to coumaryl-CoA (see Heller and Forkmann 1988; Luiet al. 1995). Perhaps caffeoyl-CoA is the precursor used for maysin synthesis, not 4-coumaryl-CoA. If true, this would explain why pr1 does not behave as a QTL for maysin synthesis and why apimaysin synthesis is independent of maysin synthesis. Two different substrates would be used, caffeoyl-CoA for maysin and 4-coumaryl-CoA for apimaysin. The B-ring hydroxylation would be carried out by caffeic acid-3-hydroxylase (C3H) converting 4-coumarate to caffeic acid. In this scenario, F3′H would function in an alternate pathway that requires 4-coumaroyl-CoA as its substrate. When F3′H is functional, an unknown nonflavone compound is produced. Apimaysin is synthesized only when F3′H is nonfunctional.

Antibiosis: Regardless of allelic constitution at either rem1 or pr1, there was a substantial reduction in larval weight of the experimental group, compared to the larval weight of the control diet group (Table 3). Both the pr1 and rem1 regions are QTLs for corn earworm antibiosis. However, the genotypes at pr1 and rem1 that result in lower maysin, flavone, and/or apimaysin levels are the genotypes that have lower larval weights (i.e., less flavone, more antibiosis). This is further reflected in the rather poor correlations between the flavones and larval weight. Maysin levels were negatively correlated with larval weight (r = −0.38), total flavone levels were not significantly (P < 0.01) correlated with larval weights, and apimaysin levels were positively correlated with larval weights (r = 0.48).

How can increasing the levels of an antibiotic compound result in apparently less antibiosis? First, total flavone levels of ~0.3% reduce larval growth to near zero. Higher flavone levels show no additional effects because many larvae are already dead. In other populations where rem1 behaves as a QTL for maysin, it does not behave as an antibiosis QTL, again suggesting that the baseline maysin levels in those populations may be in excess of what is necessary for antibiosis (Byrne et al. 1996, 1998). In those populations, however, other maysin QTLs were also QTLs for antibiosis (Byrne et al. 1997, 1998). Another possibility is that the additional maysin made through rem1 and the apimaysin made when pr1 is recessive comes at the expense of other related antibiotic compounds. The high correlation between maysin levels and larval weights observed by Byrne et al. (1996) in a population segregating for a functional p1 allele indicates that the compounds involved in corn earworm antibiosis are under p1 control. The initial study that identified maysin as an antibiotic factor in silks also found evidence that other nonflavone compounds were involved in corn earworm larval antibiosis (Waisset al. 1979). After the chemical removal of the flavones from silk tissues, Waiss et al. (1979) found that compounds extracted in a “hot water” fraction were as effective in larval bioassays as the flavone fraction. The silk residue remaining after removal of both the flavones and the “hot water” fraction retained antibiotic levels equal to the flavone and “hot water” fractions. Other flavonoid-like compounds with a 3′,4′-dihydroxy constitution on the B-ring and a 5,7-dihydroxy constitution on the A-ring have antibiotic activity towards corn earworm larvae, including flavonols, flavanones, and dihydroflavonols (Elligeret al. 1980b). These findings suggest that it is not the particular flavonoid class that determines antibiotic levels, but rather the A- and B-ring hydroxylation patterns. One explanation of our larval weight results is that synthesis of additional flavones comes at the expense of one of the other flavonoid compound(s). Furthermore, the positive correlation between apimaysin levels and larval weights may reflect lack of 3′ hydroxylation on the B-ring of flavonols, flavanones, and dihydroflavonols, rendering them less effective against corn earworm larvae.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Composite MAPMAKER/QTL scans of chromosomes 5 (a) and 9 (b) for apimaysin levels, maysin levels, and total flavone levels. Likelihood ratios are on the y axis and the framework maps are on the x axis.

Conclusions: Although the genetic basis of the variation in synthesis of maysin and apimaysin for this population was superficially simple, one major QTL explaining the majority of the variation for each chemical, this study revealed a number of important points about flavonoid synthesis and the biological interpretations of QTL analyses. First, the model for anthocyanin/flavonol synthesis does not necessarily fit C-glycosyl flavone synthesis. The anthocyanin/flavonol synthesis model depicts B-ring substitutions occurring at the 15-carbon stage, as well as the sharing of substrates and enzymes between pathways. For C-glycosyl flavone synthesis, this is not the case. Second, synthesis of highly related compounds within a pathway, and presumably similar effects on traits, can appear to have independent genetic control. Therefore, identification of different QTLs in separate populations cannot be interpreted to mean that different genetic systems or pathways affect trait expression.

The third point reinforced in this study is the importance of considering related pathways in explaining QTL effects. Is the increase in maysin through rem1 and the synthesis of apimaysin through pr1 coming at the expense of other antibiotic compounds? The genetic mechanism underlying the increase in maysin by rem1 is unknown, and, at best, the role of pr1 in maysin synthesis is not entirely clear. However, the lack of correlation between the increased flavone levels through rem1 and pr1 and larval weight suppression suggests that either the additional flavones are made at the expense of other antibiotic compounds, or that the population's baseline flavone level is sufficient to cause larval death. Because of the very high correlation between maysin levels and larval weight when variation for maysin levels results from segregation of a functional vs. nonfunctional p1 allele (Byrne et al. 1996, 1997), we suspect that if there are “unknown antibiotic compound(s)” involved, that they are also under p1 control.

Finally, epistatic interactions may represent at least two distinct mechanisms: first is the more commonly considered complementary gene action affecting a single process (trait), and second is the specification of conflicting processes that cannot be simultaneously accomplished by cellular metabolic systems. A surprising result of this study was the nature of the epistatic interaction involving the two single-effect QTLs, rem1 and pr1. The interaction was only significant for “total flavones,” not for the individual chemicals themselves. This interaction indicates the presence of a mechanism governing total flavone levels. The synthesis of maysin and apimaysin was independent only as long as total flavone synthesis is under this ceiling level. The phenylpropanoid pathway, flavone synthesis, and corn earworm antibiosis continue to serve as excellent models for investigating and interpreting quantitative genetic theory in relation to a known biological system, demonstrating the interconnected dynamic nature of the pathway and the phenotypic consequences.

Note added in proof: Chemical analysis of silks from the test cross of NC7A × pr1 tester revealed that recessive pr1 is not sufficient for apimaysin synthesis.