Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Abstract

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.

Novel Loci Underlie Natural Variation in Vitamin E Levels in Maize Grain

“…The chlorophyll degradation pathway has recently been elucidated in Arabidopsis (Schelbert et al, 2009;Hörtensteiner and Kräutler, 2011;Hörtensteiner, 2013) (Figure 4C), and the phytol released from pheophytin a by pheophytinase could be esterified to fatty acids to yield fatty acid phytyl esters or phosphorylated by VTE5 and VTE6 to yield phytyl-DP (Valentin et al, 2006;Tanaka et al, 2010;DellaPenna and Mène-Saffrané, 2011;Lippold et al, 2012;Zhang et al, 2014;Vom Dorp et al, 2015) (Figure 4C). This latter route clearly provides phytyl-DP for the large amounts of tocopherol synthesized by senescing Arabidopsis leaves, as mutation of the pheophytinase gene eliminates both chlorophyll degradation and the senescence-associated increases in tocopherol and fatty acid phytyl ester levels (Schelbert et al, 2009;Zhang et al, 2014;Vom Dorp et al, 2015). If flux through this chlorophyll degradation pathway provided the majority of phytol for tocopherol biosynthesis in other tissues and leaf development stages, one would expect a similarly severe impact on tocopherol levels in these tissues.…”

“…Correlations with chlorophyllide a and chlorophyll b with total tocopherols gradually decreased to r = 0.48 and r = 0.36, respectively, at 30 DAP, after which chlorophyll b correlation increased. Pheophytin a is a key intermediate and metabolite marker for the chlorophyll degradation pathway in senescing leaves (Schelbert et al, 2009;Hörtensteiner and Kräutler, 2011;Hörtensteiner, 2013), where it provides phytol for senescence-associated tocopherol biosynthesis (Schelbert et al, 2009;Zhang et al, 2014;Vom Dorp et al, 2015), but pheophytin a only showed weak correlations with total tocopherols at 16 and 20 DAP (r = 0.23 and 0.31, respectively) and was negatively correlated at later developmental stages. Tocopherols accumulated in endosperm to levels <2% that in embryos, and endosperm chlorophyll metabolite levels were similarly reduced, with only chlorophyll a being consistently above the limits of detection (Supplemental Data Set 9; Figure 4B).…”

“…In contrast, engineering total tocopherol content is more difficult, and even with coordinate overexpression of multiple pathway steps, the increases achieved were modest (Savidge et al, 2002;Collakova and DellaPenna, 2003;Karunanandaa et al, 2005;Raclaru et al, 2006;Lu et al, 2013). Identification of the tocopherol-deficient Arabidopsis vte5 and vte6 mutants (Valentin et al, 2006;Vom Dorp et al, 2015), encoding kinases that sequentially phosphorylate phytol to generate phytyl-DP, suggested a mechanism underlying the divergent engineering results for tocotrienols and tocopherols: While tocotrienol biosynthesis can directly utilize GGDP, tocopherol biosynthesis requires phytol to be produced from GGDP and then phosphorylated.…”

Current strategies for vitamin E biofortification of crops

“…Recently, the light-harvesting-like proteins LIL3:1 and LIL3:2 were shown to be involved in tocochromanol metabolism through their interaction and stabilization of GGR proteins in the chloroplast membrane [19]. In Arabidopsis seeds and leaves, tocopherol synthesis mostly depends on the phytol kinase VTE5 and the phytyl phosphate kinase VTE6 that sequentially phosphorylate phytol into PPP [20,21 ]. In senescent leaves, the phytol used for tocopherol synthesis originates mostly from chlorophyll hydrolysis [21 ].…”

“…In Arabidopsis seeds and leaves, tocopherol synthesis mostly depends on the phytol kinase VTE5 and the phytyl phosphate kinase VTE6 that sequentially phosphorylate phytol into PPP [20,21 ]. In senescent leaves, the phytol used for tocopherol synthesis originates mostly from chlorophyll hydrolysis [21 ]. In Arabidopsis seeds, chlorophyll degradation and recycling contributes at least 60% to tocopherol synthesis [20].…”