Soybean seeds contain approx. 20 to 30% 8-tocopherol, 2 to 5% P-tocopherol, 60 to 70% y-tocopherol, and 10 to 20% a-tocopherol. The overexpression of At-VTE3 (2-methyl-6-phytylbenzoquinol methyltransferase) in soybean seeds resulted in nearly a complete conversion of 5- and P-tocopherols to y- and a-tocopherols, which indicated that VTE3 controls the flux from MPBQ to y- and a-tocopherol . When At-VTE3 was co-overexpressed with At-VTE4 (Y-tocopherol methyltransferase) in soybean, almost all P-, y-, and 5-tocopherols were converted to a-tocopherol, with a seed accumulation of > 95% a-tocopherol and up to a fivefold increase in vitamin E activity (Table 18.3) [74, 103]. These combined experiments could be extended to other agronomically important crops.
It has been demonstrated recently that overexpression of VTE3 in soybean seeds is sufficient to funnel nearly all tocopherols into the pool of a- and Y-tocopherols [74, 98]. A nontransgenic soybean contains on average 30 to 35% P- and 5-tocopherol.
Overexpression of VTE1 in Arabidopsis leaves resulted in up to a sevenfold increase in tocopherols and a dramatic shift in tocopherol composition from a-tocopherol (16.5%) to Y-tocopherol (80.5%), which indicated that both VTE1 and Y-TMT are limiting factors for tocopherol biosynthesis . This differs from previous work, in which overexpression of y-TMT and HPT, singly or in combination, clearly demonstrated that y-TMT was not a limiting factor for tocopherol biosynthesis in Arabidopsis leaves in the absence of stress but became a limiting factor under abiotic stress [29, 93, 104]. Accumulation of tocopherol in VTE1 overexpressing plants led to a 60% and 40% decrease in ascorbate and glutathione, two key water-soluble antioxidants, respectively . It is unclear why increased VTE1 activity would lead to a reduction in ascorbate and glutathione, indicating that there is still much to be learned on the regulation of tocochromanol synthesis in plants.
Arabidopsis, Perilla, and canola seeds contain predominantly Y-tocopherol. Overexpression of the gene y-TMT led to the efficient conversion of Y-tocopherol to a-tocopherol and resulted in dramatic increase in seed a-tocopherol content [29, 81, 98, 105]. The gene encoding Arabidopsis y-TMT was overexpressed in lettuce (Latuca sativa), which resulted in an increase in the ratio of a-/y-tocopherol content (TR) by up to 0.8 to 320 from 0.6 to 1.2 in nontransformed plants (Table 18.3) . Among the transformed plants, the total tocopherol content did not change. Therefore, y-TMT may play an important role in determining the composition of toco-pherols, but not in the total tocopherol content in transgenic plants.
Y-Tocopherol methyl transferase cDNA from Arabidopsis thaliana, coding for the enzyme catalyzing the conversion of the large Y-tocopherol pool to a-tocopherol, was overexpressed in Brassica juncea plants. T1 transgenic lines showed a shift in tocopherol profile, having a-tocopherol levels as high as sixfold over the nontrans-genic controls .
In a recent report, designer transcription factors were used to regulate vitamin E synthesis . Five three-finger zinc finger proteins (ZFPs) were designed to bind to a target 9-bp-long sequence in the promoter or coding regions of the Arabidopsis
GMT gene (Y-tocopherol methyltransferase). These ZFPs were separately fused to the maize opaque-2 nuclear localization signal and the maize C1 activation ED to make synthetic zinc finger transcription factors (ZFP-TFs). Overexpression of these ZFP-TFs in Arabidopsis seeds under the control of an embryo-specific promoter resulted in a heritable 20-fold increase in a-tocopherol compared to the control seed (Table 18.3) . The dramatic increase of a-tocopherol was derived from the increased expression of y-TMT modulated by ZFP-TFs. This provides a graphic example of designer transcription factor regulating the expression of vitamin E pathway enzymes.
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