Three non-allelic epistatically interacting methyltransferase mutations produce novel tocopherol (vitamin E) profiles in sunflower

Abstract

Wildtype sunflower (Helianthus annuus L.) seeds are a rich source of alpha-tocopherol (vitamin E). The g = Tph(2) mutation disrupts the synthesis of alpha-tocopherol, enhances the synthesis of gamma-tocopherol, and was predicted to knock out a gamma-tocopherol methyltransferase (gamma-TMT) necessary for the synthesis of alpha-tocopherol in sunflower seeds--wildtype (g(+) g(+)) lines accumulated > 90% alpha-tocopherol, whereas mutant (g g) lines accumulated > 90% gamma-tocopherol. We identified and isolated two gamma-TMT paralogs (gamma-TMT-1 and gamma-TMT-2). Both mapped to linkage group 8, cosegregated with the g locus, and were transcribed in developing seeds of wildtype lines. The g mutation greatly decreased gamma-TMT-1 transcription, caused alternative splicing of gamma-TMT-1, disrupted gamma-TMT-2 transcription, and knocked out one of two transcription initiation sites identified in the wildtype; gamma-TMT transcription was 36 to 51-fold greater in developing seeds of wildtype (g(+) g(+)) than mutant (g g) lines. F(2) populations (B109 x LG24 and R112 x LG24) developed for mapping the g locus segregated for a previously unidentified locus (d). B109, R112, and LG24 were homozygous for a null mutation (m = Tph(1)) in MT-1, one of two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs identified in sunflower. The d mutations segregating in B109 x LG24 and R112 x LG24 were allelic to a cryptic mutation identified in the other MPBQ/MSBQ-MT paralog (MT-2) and disrupted the synthesis of alpha- and gamma-tocopherol in F(2) progeny carrying m or g mutations--m m g(+) g(+) d d homozygotes accumulated 41.5% alpha- and 58.5% beta-T, whereas m m g g d d homozygotes accumulated 58.1% gamma- and 41.9% delta-T. MT-2 cosegregated with d and mapped to linkage group 4. Hence, novel tocopherol profiles are produced in sunflower seed oil by three non-allelic epistatically interacting methyltransferase mutations.

Genome-Wide Association Study and Pathway-Level Analysis of Tocochromanol Levels in Maize Grain

“…PCC6803 (reviewed in DellaPenna and Mène-Saffrané 2011). In concordance with transgenic and mutagenesis studies of the core tocochromanol pathway genes ( HPPD , VTE1 through 5 and HGGT ; reviewed in DellaPenna and Mène-Saffrané 2011), QTL data from several species show intervals containing these genes are important for determining seed tocopherol composition and content, but also that numerous other unknown loci are involved, often with contributions similar to that of presumed core pathway loci (Chander et al 2008; Dwiyanti et al 2011; Fritsche et al 2012; Gilliland et al 2006; Haddadi et al 2012; Hass et al 2006; Jackson et al 2008; Li et al 2012; Shutu et al 2012; Sookwong et al 2009; Tang et al 2006; Wang et al 2012; Wong et al 2003). Aside from ZmVTE4 (Li et al 2012), the degree to which additional pathway genes and other loci contribute to natural variation for tocochromanols in maize grain is still unclear.…”

“…Linkage analysis studies of natural variation for tocopherol levels in seed of Arabidopsis , soybean, rapeseed, and sunflower have identified quantitative trait loci (QTL), including some that contain within their support intervals tocochromanol biosynthetic pathway genes, primarily VTE3 , VTE4 , and HPPD (Dwiyanti et al 2011; Fritsche et al 2012; Gilliland et al 2006; Haddadi et al 2012; Hass et al 2006; Tang et al 2006; Wang et al 2012). In maize, VTE4 , HPPD , and VTE5 were found to reside within support intervals of QTL detected for variation of tocopherol levels in grain (Wong et al 2003; Chander et al 2008; Shutu et al 2012).…”

Vitamin Deficiencies in Humans: Can Plant Science Help?

“…It has been shown that tocopherols present in seed can be converted nearly completely to a-tocopherol by engineering increased expression of the two pathway methyl transferases, VTE3 and VTE4 (Van Eenennaam et al, 2003), and, in another study, manipulation of four VTE genes, singly and in combination, was shown to impact both tocopherol composition and total content (Karunanandaa et al, 2005). Several groups have identified QTL explaining the variance of seed vitamin E composition and content in biallelic populations of Arabidopsis (Gilliland et al, 2006), sunflower (Helianthus annuus; Hass et al, 2006), maize (Chander et al, 2008), and the fruit of tomato (Schauer et al, 2006;Almeida et al, 2011). For many of these studies, vitamin E biosynthetic genes colocalized to some of the QTL intervals, but the size of the QTL intervals had limited definitive demonstration of the molecular basis of the QTL and, thus, whether they are due to modifications of biosynthetic gene function or expression.…”

Specific Roles of α- and γ-Tocopherol in Abiotic Stress Responses of Transgenic Tobacco

“…To date, mutants with decreased g-TMT activity have been isolated from Arabidopsis (vte4), sunflower (Helianthus annuus), and Synechocystis (slr0089) and all of them accumulate g-tocopherol instead of a-tocopherol (Shintani and DellaPenna, 1998;Bergmü ller et al, 2003;Hass et al, 2006). Interestingly, the total tocopherol content in sunflower and Arabidopsis mutant leaves remained similar to the corresponding wild type (Bergmü ller et al, 2003;Hass et al, 2006), while the Synechocystis mutant lacked a-tocopherol without any compensation in g-tocopherol (Shintani and DellaPenna, 1998;Sakuragi et al, 2006). Among these g-TMT mutants, only the Arabidopsis vte4 mutant was tested for abiotic stress tolerance and did not exhibit an altered stress response toward heat, cold, and high light compared to the wild type (Bergmü ller et al, 2003).…”