Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana.

Vitamin E is an essential nutrient for humans and is obtained primarily from food, especially oil, derived from the seed of plants. Genes encoding the committed steps in vitamin E synthesis in plants (VTE, loci 1-5) have been isolated and used for tocopherol pathway engineering with various degrees of success. As a complement to such approaches we have used quantitative trait loci analysis with two sets of Arabidopsis thaliana recombinant inbred lines and have identified 14 QVE (quantitative vitamin E) loci affecting tocopherol content and composition in seeds. Five QVE intervals contain VTE loci that are likely QVE gene candidates. Nine QVE intervals do not contain VTE loci and therefore identify novel loci affecting seed tocopherol content and composition. Several near-isogenic lines containing introgressions of the accession with increased vitamin E levels were shown to confer significantly elevated tocopherol levels compared with the recurrent parent. Fine-mapping has narrowed QVE7 (a gamma-tocopherol quantitative trait loci) to an 8.5-kb interval encompassing two genes. Understanding the basis of the QVE loci in Arabidopsis promises to provide insight into the regulation and/or metabolism of vitamin E in plants and has clear ramifications for improving the nutritional content of crops through marker-assisted selection and metabolic engineering.

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination.

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.

Detection of deleterious genotypes in multigenerational studies. III. Estimation of selection components in highly selfing populations.

New paradigms in genetics have increased the chance of finding genes that appear redundant but in fact may have been preserved due to a small level of positive selection potential acting during each generation. Monitoring changes in genotypic frequencies within and between generations allows the dissection of the fertility, viability and meiotic drive selection components acting on such genes in natural and experimental populations. Here, a formal maximum likelihood procedure is developed to identify and estimate these selection components in highly selfing populations by fitting the time-dependent solutions for genotypic frequencies to observed multigenerational counts. With adult census alone, we can not simultaneously estimate all three selection components considered. In such cases, we instead consider a hierarchy of 11 models with either fewer selection components, complete dominance, or multiplicative meiotic drive with a single parameter. We identify the best-fitting of these models by applying likelihood ratio tests to nested models and Akaike's Information Criterion (AIC) and the Bayesian Information Criterion (BIC) to non-nested models. With seed census, fertility and viability selection are not distinguishable and thus can only be estimated jointly. A combination of joint seed and adult census data allows us to estimate all three selection components simultaneously. Simulated data validate the estimation procedure and provide some practical guidelines for experimental design. An application to Arabidopsis data establishes that viability selection is the major selective force acting on the ACT2 actin gene in laboratory-grown Arabidopsis populations.

Arabidopsis actin gene ACT7 plays an essential role in germination and root growth.

Arabidopsis contains eight actin genes. Of these ACT7 is the most strongly expressed in young plant tissues and shows the greatest response to physiological cues. Adult plants homozygous for the act7 mutant alleles show no obvious above-ground phenotypes, which suggests a high degree of functional redundancy among plant actins. However, act7-1 mutant plants are at a strong selective disadvantage when grown in competition with wild-type plants and therefore must have undetected physical defects. The act7-1 and act7-4 alleles contain T-DNA insertions just after the stop codon and within the first intron, respectively. Homozygous mutant seedlings of both alleles showed less than 7% of normal ACT7 protein levels. Mutants displayed delayed and less efficient germination, increased root twisting and waving, and retarded root growth. The act7-4 mutant showed the most dramatic reduction in root growth. The act7-4 root apical cells were not in straight files and contained oblique junctions between cells suggesting a possible role for ACT7 in determining cell polarity. Wild-type root growth was fully restored to the act7-1 mutant by the addition of an exogenous copy of the ACT7 gene. T-DNA insertions just downstream of the major polyadenylation sites (act7-2, act7-3) appeared fully wild type. The act7 mutant phenotypes demonstrate a significant requirement for functional ACT7 protein during root development and explain the strong negative selection component seen for the act7-1 mutant.

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation.

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.