Ty3/gypsy-like retrotransposon knockout of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase is non-lethal, uncovers a cryptic paralogous mutation, and produces novel tocopherol (vitamin E) profiles in sunflower.

The m (Tph(1)) mutation partially disrupts the synthesis of alpha-tocopherol (vitamin E) in sunflower (Helianthus annuus L.) seeds and was predicted to disrupt a methyltransferase activity necessary for the synthesis of alpha- and gamma-tocopherol. We identified and isolated two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs from sunflower (MT-1 and MT-2), resequenced MT-1 and MT-2 alleles from wildtype (m(+) m(+)) and mutant (m m) inbred lines, identified m as a non-lethal knockout mutation of MT-1 caused by the insertion of a 5.2 kb Ty3/gypsy-like retrotransposon in exon 1, and uncovered a cryptic codominant mutation (d) in a wildtype x mutant F(2) population predicted to be segregating for the m mutation only. MT-1 and m cosegregated and mapped to linkage group 1 and MT-1 was not transcribed in mutant homozygotes (m m). The m locus was epistatic to the d locus--the d locus had no effect in m(+) m(+) and m(+) m individuals, but significantly increased beta-tocopherol percentages in m m individuals. MT-2 and d cosegregated, MT-2 alleles isolated from mutant homozygotes (d d) carried a 30 bp insertion at the start of the 5'-UTR, and MT-2 was more strongly transcribed in seeds and leaves of wildtype (d(+) d(+)) than mutant (d d) homozygotes (transcripts were 2.2- to 5.0-fold more abundant in the former than the latter). The double mutant (m m d d) was non-lethal and produced 24-45% alpha- and 55-74% beta-tocopherol (the wildtype produced 96% alpha- and 4% beta-tocopherol). MT-2 compensated for the loss of the MT-1 function, and the MT-2 mutation profoundly affected the synthesis of tocopherols without adversely affecting the synthesis of plastoquinone crucial for normal plant growth and development.