Academic journal article Genetics

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

Academic journal article Genetics

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

Article excerpt

ABSTRACT

Plants synthesize an array of natural products that play diverse roles in growth, development, and defense. The plant-specific phenylpropanoid metabolic pathway produces as some of its major products flavonoids, monolignols, and hydroxycinnamic- acid conjugates. The reduced epidermal fluorescence 4 (ref4) mutant is partially dwarfed and accumulates reduced quantities of all phenylpropanoid-pathway end products. Further, plants heterozygous for ref4 exhibit intermediate growth and phenylpropanoid-related phenotypes, suggesting that these mutations are semidominant. The REF4 locus (At2g48110) was cloned by a combined map- and sequencing-based approach and was found to encode a large integral membrane protein that is unique to plants. The mutations in all ref4 alleles cause substitutions in conserved amino acids that are located adjacent to predicted transmembrane regions. Expression of the ref4-3 allele in wild-type and null REF4 plants caused reductions in sinapoylmalate content, lignin content, and growth, demonstrating that the mutant alleles are truly semidominant. Further, a suppressor mutant was isolated that abolishes a WW protein-protein interaction domain that may be important for REF4 function.

MANY mutations have been identified in structural genes that are required for the accumulation of phenylpropanoid-pathway end products, particularly in Arabidopsis and maize. The majority of mutant phenotypes identifiedin these forwardgenetic screens segregate as recessive traits. A theoretical basis for this observation has been provided bymetabolic control analysis (Kacser and Burns 1981), which suggests that the contribution of a single enzyme's activity within a metabolic pathway is generally small incomparisontothesummedactivityofall the enzymes in the entire pathway. Thus, this analysis predicts that only in very short metabolic pathways can a null or hypomorphicmutation in one of the biosynthetic enzyme-encoding genes be semidominant.

Although oftenmore difficult to interpret than simple loss-of-function alleles, dominant mutations often lead to interesting insights into the pathways within which the mutated gene operates. In humans, dominant and semidominant diseases are often caused by mutations in transcription factors, transporters, and components of signaling cascades ( Jimenez-Sanchez et al. 2001; Kondrashov and Koonin 2004). Mutations in such genesmay lead to dominant phenotypes through a number of different mechanisms. The simplest of these is haplo-insufficiency where the wild-type allele of a gene in a+/-heterozygote does not produce enough protein to generate a wild-type phenotype. Although few examples of haplo-insufficiency are known in plants (Weijers et al. 2001), in Antirrhinum flowers, haplo-insufficiency with regard to anthocyanin accumulation is revealed in the presence of null alleles of F3H (Coen et al. 1986;Martin et al. 1991). Further, because these null alleles operate in a biosynthetic pathway, they represent an exception to the predictions of metabolic control analysis and demonstrate that F3H is truly the rate-limiting enzyme in anthocyanin biosynthesis.

Another class of dominant mutations either increases the abundance of or stabilizes mRNA transcripts or their encoded proteins, preventing the normal turnover of these molecules essential for the wild-type phenotype. Although these gain-of-function mutations are now commonly generated synthetically by approaches such as activation tagging (e.g., Sundaresan et al. 1995), examples of such mutations generated via point mutations have been described. One such example is the Arabidopsis (Arabidopsis thaliana L. Heynh) atr1D (altered tryptophan regulation-dominant)mutant that exhibits upregulated transcription of tryptophan biosynthetic genes due to the stabilization of the ATR1 Myb transcription factor mRNA (Bender and Fink 1998; Smolen and Bender 2002). Further, mutations in the miRNA target sequences of REVOLUTA (REV), PHABULOSA (PHB), and PHAVOLUTA (PHV) cause these genes to escape miRNA-mediated transcript degradation, effectively resulting in dominant gain-of-function alleles (Emery et al. …

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