Lignin

These three compounds differ from each other in the substitution pattern of the B-ring: apimaysin has one hydroxyl group on the 4' position of the Bring, maysin has a 3',4'-dihydroxy substitution pattern, and methoxymaysin has a 4'-hydroxy, 3'-methoxy substitution pattern. These compounds accumulate in the silks of maize (i.e. the styles attached to the ovules) and are thought to act in a manner similar to chlorogenic acid (see Figure 6-1) when insects damage the silks.

(6.17)

Maysin is generally the most abundant of these three compounds, and is typically present at concentrations of 0.3% fresh silk weight, which is very high for a single compound. As a consequence, the C-glycosyl flavones can be considered preformed defense compounds.

The concentration of C-glycosyl flavones varies considerably among different maize lines. Since the concentration of the C-glycosyl flavones does not have a discrete value, but rather varies along a continuum, it can be considered a quantitative trait (see Chapter 3, Section 3.4). In order to identify loci controlling maysin concentration in silks, Byrne et al. (1996) investigated the role of a number of structural and regulatory genes known to play a role in flavonoid biosynthesis. They generated an F2 population derived from the maize inbred lines GT119 and GT114, which had low (0.031%) and high (0.56%) maysin levels, respectively. They determined the genotype at a number of loci at or near flavonoid biosynthetic genes, and concluded that the P1 ('P-one'; see Chapter 3, Section 9.2) and a locus referred to as recessive enhancer of maysin (rem1) near the Brown pericarp1 (Bp1) locus accounted for 58 and 11% of the variance, respectively. In addition, a QTL near the centromere of chromosome 1 was uncovered, but there were no obvious candidate genes at this locus.

The effects of the P1 gene and the tightly linked homolog P2 on maysin biosynthesis were further investigated by Zhang et al. (2003). These researchers used introduced the P1 and P2 cDNA's under control of the ubiquitin promoter in cultured Black Mexican Sweet maize cells using microprojectile bombardment. This is a method in which small gold or tungsten particles coated with an expression construct are introduced into target cells using a burst of pressure. Based on gene expression studies, P1 and P2 activate the same genes, including phenylalanine ammonia lyase (Chapter 3, Section 7), chalcone synthase, chalcone isomerase, and dihydroflavonol 4-reductase (Chapter 3, Section 9), but not genes involved in the biosynthesis of flavonols and anthocyanins. Increased levels of flavones were detected in extracts obtained from the transformed cells. Further evidence for a role of both genes in maysin biosynthesis came from the observation that maize plants in which both P1 and P2 were deleted did not produce maysin, whereas plants in which P1 was deleted, but P2 was still present, still produced maysin, albeit at reduced levels.

Maysin is two times more effective in its ability to inhibit growth of the corn earworm, which is attributed to the fact that two neighboring hydroxyl groups (such as on the on the 3' and the 4' positions in maysin) will result in the efficient formation of a toxic quinone, whereas the quinone formation from apimaysin and methoxymaysin is less efficient (Elliger et al., 1980; Snook et al., 1994). The genetic basis of the substitution reactions of the Bring have been the subject of several studies. Using an F2 population derived from the maize inbred lines GT114 (moderately high levels of maysin, negligible levels of apimaysin) and NC7A (moderately high levels of apimaysin, maysin, and chlorogenic acid (6.4), Lee et al. (1998) determined that the reml locus identified by Byrne et al. (1996) explained 55% of the variance for maysin, whereas a QTL that mapped near the Prl gene, which is thought to encode flavonoid 3' hydroxylase (F3'H), explained 65% of the variance for apimaysin. Furthermore, the levels of maysin and apimaysin were independent of each other, suggesting these two compounds are synthesized via different pathways. Surprisingly, a functional Pr1 gene was not required for maysin production. Lee et al. (1998) speculated that the actual gene responsible for maysin biosynthesis may be near Prl, but does not have to be Prl, that a second F3 H gene is responsible for maysin production, or that the hydroxylation at C3' occurs at the level of the hydroxycinnamoyl CoA ester rather than at the level of the flavone.

The genetic control of the substitution of the C-glycosyl flavones was investigated in further detail by Cortes-Cruz et al. (2003). Two F2 populations were generated from maize inbred lines that differed from each other in the relative concentrations of maysin, apimaysin, and methoxymaysin. In both F2 populations the main QTL associated with levels of chlorogenic acid, maysin and methoxymaysin was located on the short arm of chromosome 4, whereas the main QTL associated with levels of apimaysin was located on the long arm of chromosome 5. Presence of a specific allele in the QTL on chromosome 4 resulted in higher levels of methoxymaysin and lower levels of maysin and chlorogenic acid. The fact that a single QTL affects the concentrations of three compounds (methoxymaysin, maysin and chlorogenic acid) suggests that there may be a regulatory gene underlying the QTL, or that there is a branched rather than a linear biosynthetic pathway leading to these different compounds. The QTL for apimaysin on chromosome 5 coincided with the Pr1 locus, consistent with the data reported by Lee et al. (1998).

The biosynthetic pathway leading to maysin starts with flavanone (6.18), which is hydroxylated by flavone 3' hydroxylase to yield di-hydroxyl flavanone (6.19) and is the reduced by flavone synthase to the flavone luteolin (6.20). The next steps were recently investigated in more detail by McMullen et al. (2004) using two salmon silk mutants, sm1 (Anderson, 1921) and a newly discovered mutant sm2. These mutants have salmon colored silks instead of green silks as a result of pigment accumulation throughout the shaft of the silks, as opposed to only in the silk hairs, but do require a functional P1 gene in order for the mutant phenotype to be apparent (see also Chapter 3, Section 9.2).

Detailed chemical analyses of flavone composition in the silks in wildtype, sm1, sm2 and sm1-sm2 plants revealed that isoorientin (6.21) is the only flavone accumulating in sm1-sm2 double mutants, indicating the synthesis of this compound precedes the action of the gene products of the functional Sm1 and Sm2 genes. Isoorientin (6.21) is present at high levels in sm2 but not sm1 mutants, so that a functional Sm2 gene is required for the formation of rhamnosyl-isoorientin (6.22) from isoorientin. The Sm2 gene may encode a rhamnosyl transferase, or control the expression of a rhamnosyl tranferase gene. Rhamnosyl-isoorientin (6.22) accumulates in sm1 mutants, suggesting that Sm1 encodes a protein that catalyzes the formation of maysin (6.17a), or otherwise controls the expression of gene(s) encoding the necessary enzymes.

Taking all of the abovementioned data into consideration, the most likely biosynthetic pathway leading to maysin is as shown in Figure 6-2, although further research is needed to fully elucidate the pathway and the regulatory genes.

Figure 6-2. Biosynthesis of maysin proposed by McMullen et al. (2004) based on the analysis of flavones in the silks of maize salmon silk mutants. a. flavone 3' hydroxylase (encoded by the maize Pr1 gene), b. flavone synthase, c. C-glucosyltransferase, d. putative rhamnosyl transferase (encoded by the Salmon silk2 gene), e. the step(s) controlled by the Salmon silk1 gene.

Figure 6-2. Biosynthesis of maysin proposed by McMullen et al. (2004) based on the analysis of flavones in the silks of maize salmon silk mutants. a. flavone 3' hydroxylase (encoded by the maize Pr1 gene), b. flavone synthase, c. C-glucosyltransferase, d. putative rhamnosyl transferase (encoded by the Salmon silk2 gene), e. the step(s) controlled by the Salmon silk1 gene.

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