Genetic analysis of amino acid accumulation in opaque-2 maize endosperm

Abstract

The opaque-2 mutation in maize (Zea mays) is associated with an increased level of free amino acids (FAA) in the mature endosperm. In particular, there is a high concentration of lysine, the most limiting essential amino acid. To investigate the basis for the high-FAA phenotype of opaque-2 maize, we characterized amino acid accumulation during endosperm development of several wild-type and opaque-2 inbreds. Oh545o2 was found to have an exceptionally high level of FAA, in particular those derived from aspartate (Asp) and intermediates of glycolysis. The FAA content in Oh545o2 is 12 times greater than its wild-type counterpart, and three and 10 times greater than in Oh51Ao2 and W64Ao2, respectively. We crossed Oh545o2 to Oh51Ao2 and analyzed the F(2:3) progeny to identify genetic loci linked with the high FAA level in these mutants. Quantitative trait locus mapping identified four significant loci that account for about 46% of the phenotypic variance. One locus on the long arm of chromosome 2 is coincident with genes encoding a monofunctional Asp kinase 2 and a bifunctional Asp kinase-homo-Ser dehydrogenase-2, whereas another locus on the short arm of chromosome 3 is linked with a cytosolic triose phosphate isomerase 4. The results suggest an alternation of amino acid and carbon metabolism leads to overproduction and accumulation of FAA in opaque-2 mutants.

Figure 1

FAA in developing endosperm of Oh545+/o2 and W64A+/o2. FAA analysis was performed with pooled kernel samples as described in “Materials and Methods.” The values are the means of three independent extractions and measurements.

Figure 2

Relative concentration of FAA in mature endosperm of Oh545o2, Oh51Ao2, their reciprocal F₁ crosses, and F_2:3 progeny. Flour was prepared from a pool of 20 kernels from the middle of well-filled ears and assayed with ninhydrin to determine the FAA content. The relative content of FAA in Oh51Ao2 (the low-FAA parent) was defined as “1.” The relative FAA content of each F_2:3 individual is the mean of two measurements from two independent extractions. Plants were grown in the spring (A) and fall (B) of 1996.

Figure 3

Interval mapping of QTLs influencing FAA content in maize endosperm. The plots are derived from the interval mapping of loci associated with FAA content in the F₂ population of Oh51Ao2 × Oh545o2. A free regression model was used to perform interval mapping. The solid curve illustrates the LRS; SSR DNA markers are indicated on the left. The significant threshold values of LRS for each chromosome were estimated with 1,000 permutations using Map Manager QTXb03. The LRS considered as a significant threshold value (95%) for chromosomes 2, 3, and 7 are 12.2, 11.5, and 12.1, respectively (shown with dashed lines).

Figure 4

Metabolic pathways showing the major points of regulation for amino acid biosynthesis and Lys degradation. The color scale shows the relative increase (blue, >10-fold; green, 6–10-fold; yellow, 3–6-fold; orange, 1–3-fold; red, <1-fold) in FAA concentration in mature endosperm of Oh545o2 compared with Oh51Ao2. AHAS, Acetohydroxyacid synthase; DAHP, 7-phospho-2-keto-3-deoxy-d-arabino-heptonate; DHDPS, dihydrodipicolinate synthase; FPK, Fru phosphate kinase; LKR, Lys ketoglutarate reductase; PK, pyruvate kinase; PPDK, pyruvate phosphate dikinase; SDH, sacchropine dehydrogenase; TPI, triose phosphate isomerase.