Molecular, cellular, and population analysis of vegetative actin mutants in Arabidopsis thaliana

Actin is an abundant cytoskeletal protein found in all eukaryotes and is essential for a variety of cellular and developmental functions, but the purpose of encoding actin in a diverse family of genes in higher plants and animals is less clear. We hypothesize the ancient and divergent subclasses of plant actin genes have been preserved through evolution because they have distinct temporal and spatial patterns of expression and/or encode actin protein isovariants with specialized cytoskeletal functions. In Arabidopsis thaliana, there are eight expressed actin genes with different expression patterns and encoding distinct isovariants. ACT2 is strongly expressed in most vegetative tissues. The T-DNA insertion in the act2-1 allele fully disrupts expression of ACT2 RNA and lowers total actin protein levels. Plants homozygous for the act2-1 mutation have root hairs that are only 15% the length of wild-type that may bulge severely at the base, indicating a role for ACT2 in root hair tip growth. ACT7 is a vegetatively expressed actin showing the greatest response to developmental and physiological cues of any family member. A T-DNA insertion just after the stop codon in the act7-1 allele produced several species of abundant transcripts yet it significantly reduced protein expression. Plants homozygous for the act7-1 insertion are defective in callus formation, display exaggerated root waving with a reduction in root length, and show delayed and less efficient germination. The varied phenotypes of the act7-1 and recently isolated act7-4 mutants suggest a role for ACT7 in rapid growth in response to environmental stimulation. Both the act2-1 and act7-1 mutations are fully rescued by the addition of a complementing genomic fragment.; When grown as populations from a single heterozygous individual, the act2-1 and act7-1 mutant alleles were found at extremely low frequencies compared to wild-type in the F₂ generation, indicating they both were deleterious. In a multi-generation population study, the act2-1 mutant allele was found at normal frequencies in the F ₁, but at significantly lower frequencies than expected in the F ₂ and F₃ generations, suggesting the reduction in fitness occurs in the 2N sporophytic portion of the life cycle. This attests that each family member contributes to plant success. When the act2-1 mutation is combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibits extensive synergistic phenotypes in both shoot and root ranging from developmentally lethal to severe, depending on medium supplementation. We propose that enough redundancy exists in the actin gene family of Arabidopsis thaliana that single actin mutants have viable phenotypes restricted to only certain tissues, presumably either because those tissues have large proportions of a single sovereign due to expression patterns, or certain roles are performed there by a specific sovereign. Disruption of multiple actin genes with related expression patterns and similar potables prevents the plant from performing numerous vital tasks, presumably from a shortage of total actin protein.