A gain-of-function polymorphism controlling complex traits and fitness in nature

Abstract

Identification of the causal genes that control complex trait variation remains challenging, limiting our appreciation of the evolutionary processes that influence polymorphisms in nature. We cloned a quantitative trait locus that controls plant defensive chemistry, damage by insect herbivores, survival, and reproduction in the natural environments where this polymorphism evolved. These ecological effects are driven by duplications in the BCMA (branched-chain methionine allocation) loci controlling this variation and by two selectively favored amino acid changes in the glucosinolate-biosynthetic cytochrome P450 proteins that they encode. These changes cause a gain of novel enzyme function, modulated by allelic differences in catalytic rate and gene copy number. Ecological interactions in diverse environments likely contribute to the widespread polymorphism of this biochemical function.

Fig. 1

(A) Histograms show percent leaf area removed by the generalist herbivore Trichoplusia ni, total quantity of glucosinolates, and proportion of aliphatic glucosinolates from branched chain amino acid precursors. Greenhouse-grown plants descended from nine B. stricta populations. (B) Map showing the proportion of genotypes in each population that produce predominantly branched-chain derived glucosinolates (white) or methionine-derived glucosinolates (black). Parental populations of the crossing experiment are boxed.

Fig. 2

Fitness reductions under field conditions associated with 1% loss of leaf area by herbivory. Bars indicate reduction in components of fitness due to fecundity (gray) and survival (black) in 2008, 2009, and 2010 in Colorado and Montana. NIL plants in the clipping experiment were randomly assigned to artificial herbivory or control treatments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, NS = not significant.

Fig. 3

(A) Glucosinolate production in transgenic Arabidopsis expressing B. stricta BCMA genes, encoding CYP79F enzymes which catalyze amino acids in the first step of the glucosinolate pathway. Bars show amounts of aliphatic glucosinolates from MET, VAL, and ILE precursors catalyzed by each of the BCMA gene products. Gene phylogeny includes wildtype (WT) Arabidopsis with empty vector controls, the black triangle identifies the gene duplication in Boechera, red circle shows origin of branched chain amino acid catalysis, and blue circle indicates elevated ILE activity. Abbreviations: B1 = BCMA1, B2 = BCMA2, B3 = BCMA3, with alleles from CO or MT. N = 130 independent transgenic lines. (B) In vitro enzyme activity levels relative to controls, in nmol product/nmol enzyme/min, with standard error. Labels indicate CYP79F enzymes from Arabidopsis, and BCMA1, BCMA2, and BCMA3 from Boechera, with alleles from CO or MT. BCMA2 alleles encode identical proteins, so one allele was assayed. BCMA2 (green) retains the ancestral MET activity, and was engineered to change G134L, P536K, or both (pink). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Fig. 4

(A) Homology model of BCMA2 with the substrate binding cleft above the heme group (magenta) with putative substrate recognition regions in purple. Amino acid changes G134L and P536K (green) show statistical evidence for accelerated protein evolution, and alter catalytic function when changed by site-directed mutagenesis. Other mutations with statistical evidence of accelerated evolution (in blue) are not addressed in this study. The location of amino acid 529, which aligns with the last resolved residue in the CYP1A2 crystal structure, is colored since subsequent amino acids 530–540 cannot be accurately modeled. (B) Close-up view of substrate binding cleft with mutation G134L residing just above the heme.