Validation of metabolites in resistant vs. susceptible maize
lines
Using 29 of the most resistant and 28 of the most susceptible maize
lines in this study, we sought correlations between the presence of
zeaxanthin, ß-carotene, lutein, chlorophyll a, chlorophyll b, and
pheophytin b, with 7- and 14-day damage rating. The carotenoids were
measured because of the multiple carotenoid-related pathways identified
by PAST, as were the chlorophylls and pheophytins. Pheophytins are
identical in structure to chlorophylls but lack magnesium(II).
Chlorophylls can be degraded to pheophytins under low pH or
enzymatically. Along with chlorophylls and ß-carotene pheophytins are
also involved in photosynthesis (photosystem II). We were unable to find
measurable levels of zeaxanthin in the leaf samples, which is
unsurprising as this compound tends to be produced in maize grains. How
the biosynthesis pathway is then associated with reduced leaf feeding by
FAW is thus unknown. The four other metabolites measured showed
phenotypic variation among the maize lines tested, although about 80%
of the lines showed low levels, with less than 300 µg/g of lutein, 2,000
µg/g of ß-carotene, 3E6 counts/g of chlorophyll a, 2E6 counts/g of
chlorophyll b and 2E6 counts/g of pheophytin b (Supplemental Table 5).
Levels of these four metabolites were correlated with each other, and
some with damage ratings (Fig. 2A). Lutein and pheophytin b were
correlated with an increase in FAW damage, and chlorophyll a was
correlated with a decrease in FAW damage, in agreement with pathway
PWY-6927 (chlorophyll a degradation II) being associated with increased
FAW damage ratings at 7 days (and less significantly so at 14 days,
along with pathway PWY-5098, chlorophyll a degradation I), (Supplemental
Table 4). Chlorophyll b is positively correlated to ß-carotene, lutein
and pheophytin but negatively correlated with chlorophyll a. The
chlorophyll cycle converts a to b forms of chlorophyll via a step
through the intermediary 3,8-divinyl-chlorophyllide a; thus, it makes
sense that a and b are negatively correlated and also that pathway
CHLOROPHYLL-SYN, which produces 3,8-divinyl-chlorophyllide a, is
associated with increased damage in the PAST analysis. ß-carotene was
positively correlated to pheophytin and lutein, which were correlated to
FAW damage, but ß-carotene itself was not significantly correlated with
FAW damage scores. These metabolites appear to agree with the metabolic
pathway analysis that found carotenoids to be important in FAW
resistance but are not conclusive.
The wide distribution of phenotype in relation to metabolite content
made straightforward conclusions difficult. The cluster analysis (Fig.
2B) and PCA (Fig. 2C) of metabolites and FAW ratings showed six groups,
formed based on FAW rankings and also metabolite content. For example,
two of the clusters showed high levels of lutein content, one of which
were FAW-resistant and the other FAW-susceptible. For a clearer
breakdown of metabolite and resistance phenotype, Supplemental Fig. 2
shows the levels of each metabolite within each of the six clusters; it
is apparent that some of the clusters are highly significantly different
than the others for levels of that metabolite. Although the numbers of
lines within each cluster reduce the statistical power of the analysis,
it is likely that within the resistant lines, there is more than one
mechanism leading to resistance, but that levels of lutein, pheophytin,
and chlorophyll a in particular are almost certainly contributing to
resistance (or susceptibility). Although the effect of each metabolite
on resistance may not be enough to create resistant lines, they do help
to validate the results of the PAST metabolic pathway analysis.
Figure 2 : Targeted metabolite analysis for lutein, β-carotene,
chlorophyll a and b and pheophytin b. A) Correlation matrix, B)
dendrogram, and C) principal component analysis (PCA) of metabolite and
Fall Armyworm rating analysis. In the dendrogram, maize lines are
colored salmon (BEST) and blue (WORST) representing resistance
phenotype. In the dendrogram and PCA, clusters are represented with the
following colors, 1 = black, 2 = blue, 3 = yellow, 4 = green, 5 = purple
and 6 = red. In the correlation matrix graph, blue represents positive
correlation, red negative correlation and black “X” indicates that the
comparison was not significantly correlated with a p-value cut-off of
0.05. In the PCA, shape of points represents resistant phenotype, circle
(BEST) and triangle (WORST).