The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G-->A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality.

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20-90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.

Arabidopsis map-based cloning in the post-genome era.

Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype. The major strength of this approach is the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes. One begins with an interesting mutant and allows plant biology to reveal what gene or genes are involved. Three major advances in the past 2 years have made map-based cloning in Arabidopsis fairly routine: sequencing of the Arabidopsis genome, the availability of more than 50,000 markers in the Cereon Arabidopsis Polymorphism Collection, and improvements in the methods used for detecting DNA polymorphisms. Here, we describe the Cereon Collection and show how it can be used in a generic approach to mutation mapping in Arabidopsis. We present the map-based cloning of the VTC2 gene as a specific example of this approach.

Engineering vitamin E content: from Arabidopsis mutant to soy oil.

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.