3 Mutations and Trait Variation
Walter P. Suza; Philip W. Becraft; Faizo Kasule; and Madan K. Bhattacharyya
Introduction
Crop breeding and genetic research rely on genetic variation, which is synonymous with DNA variation. Mutations are the source of genetic variation that drives trait improvement. Although most mutations are rare and deleterious and are selected against during evolution, some create beneficial genetic variation. Therefore, it is often of interest for plant breeding or genetic studies to induce genetic mutations by exposing seed or plant tissue to certain agents, called mutagens. The chapter on mutations and variation in Crop Genetics extensively covered the genetic and molecular basis of mutations and variation. The practical utilization of genetic variability from induced mutations was covered in Plant Breeding Methods. Therefore, for more information, it may be helpful for you to review the texts.
Mutation and Epimutation
Since transcription is the first step in gene expression, it makes sense in terms of cellular economy to regulate expression at this point, which is one of the most important regulatory points. We described and discussed the involvement of RNA polymerase in the transcription process. However, it is important to note that other protein factors are required. Proteins involved in transcriptional regulation are known as transcription factors. The interaction of these transcription factors with specific DNA sequences regulates the process of gene transcription.
DNA Base Substitutions
The simplest type of mutation is a substitution of one base for another in the DNA sequence. Substitutions most often arise as errors during DNA replication or repair. The most common type is the transition, where one pyrimidine may be substituted by another or a purine by another purine. The less common type is the transversion, in which a purine may be substituted by a pyrimidine or vice versa. As described and discussed in the next section, these can have various effects on gene function depending on where they occur in a gene.
Insertions and Deletions
Insertions and deletions, collectively known as indels, are other types of DNA mutations that occur frequently. They vary in size from one to thousands or more nucleotide bases. Their effects on gene function depend on the size of the insertion or deletion and the location relative to the gene. As described below, insertions and deletions in multiples of 3 are referred to as in-frame insertions and deletions, resulting in the addition or deletion of amino acids from the protein sequence. When not in multiples of three, insertions and deletions lead to frameshift mutations, altering the amino acid sequence of the protein encoded by the mutated gene.
A common source of inserted DNA is transposons, which are DNA sequences that can move from one genomic site and insert into another. Transposons were discovered in maize by Barbara McClintock. She was awarded the Nobel Prize in Physiology and Medicine in 1983 for this discovery (Figure 1).
Some transposons copy themselves and multiply in number within a genome. Transposons can cause wide-scale chromosome rearrangements and gene mutations. A feature of mutations induced by many plant transposons is instability. Excision of a transposon from the mutated gene can often restore it to wild type or create a new stable mutant allele. Transposons are found in every living organism studied and are a major driving force in genome evolution.
Epimutations
As previously described, epigenetic mechanisms, including DNA methylation, histone modification, and RNA interference (RNAi), are essential to regulate gene expression for plant development and defend genomes against widespread mutagenic activity of transposons. Epimutations are heritable changes in gene expression that occur without any alteration in gene sequence (Daxinger and Whitelaw, 2010). Some epimutations may affect the function or regulation of a single gene. In contrast, others may interfere with the epigenetic mechanisms that control transposons, leading to increased transposon activity that can negatively impact growth and development. For example, the SUPERMAN gene of Arabidopsis has known epialleles that show heritable changes in flower morphology, producing extra anthers, even though the DNA base sequence is not altered. These alleles show heritable changes in the DNA methylation pattern (Jacobsen and Meyerowitz, 1997). The Arabidopsis decrease in DNA methylation1 (ddm1) mutant (Vongs et al., 1993) provides a good example of the impact of genome-wide epimutations on plant growth and development. The wild-type DDM gene controls DNA methylation, and when compared to the wild type, the ddm1 mutant shows progressive loss of DNA methylation, enhanced transposon activity, and developmental defects after several generations of inbreeding (Kakutani et al., 1996; Hirochika et al., 2000).
- Read: Transgenerational epigenetic inheritance
- Read: Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis
- Read: Arabidopsis thaliana methylation mutants
- Read: Developmental abnormalities and epimutations associated with DNA hypomethylation mutations
- Read: Silencing of retrotransposons in Arabidopsis and reactivation by the ddm1 mutation.
Effects of Mutations on Gene Function
Coding Region Mutations
Silent Mutation
A silent mutation is a mutation that results in the change of a codon without a change in the amino acid represented by the codon. Because of the redundancy of the genetic code, DNA base substitutions may not lead to the incorporation of an incorrect amino acid in the protein (Figure 2).
Missense Mutation (Amino Acid Substitution)
A missense mutation is a single-nucleotide base substitution that alters a codon such that it codes for a different amino acid that is incorporated into the encoded protein. The amino acid substitution may affect the function of the protein if it occurs in a critical portion of the protein (Figure 3).
Frameshift Mutation
Due to the triplet nature of the genetic code, an insertion or deletion can change the reading frame for the entire subsequent sequence. For example, if a particular sequence is read sequentially, such as in Figure 4.
Because the new sequence of codons is different from the original, the entire amino acid sequence is changed from the point of insertion (nucleotide sequence will be CCX CAA AGG and change in amino acid sequence accordingly if X is inserted; note that the change from A to X is a point mutation in the figure not an insertion since third C residue of the codon is not carried over to the next codon). A similar effect is seen for deletions. This effect is seen whenever the number of nucleotide bases inserted or deleted is not a multiple of 3, and it usually results in the loss of the function of the protein.
Amino Acid Additions or Deletions
Alternatively, if insertions or deletions are in multiples of 3, amino acid additions or deletions can arise. Consider the following 3-base insertion (Figure 5). If such insertions or deletions occur in important regions of the protein, they might impair function.
Nonsense Mutation
A nonsense mutation arises when a functional codon is changed to a stop codon. Nonsense mutations may cause premature termination of translation (Figure 6).
Regulatory Region Mutations
Promoters and enhancers control temporal, spatial, and quantitative aspects of gene expression. As such, mutations in these regulatory regions of a gene can alter the regulation of the gene’s expression. Genes can be expressed at inappropriate times, places, or levels, which can affect a plant’s phenotype.
Teosinte, the progenitor of modern maize, produces multiple branches (Figure 7A) due to reduced apical dominance. In teosinte and modern maize, apical dominance is controlled by a gene called teosinte branched1, tb1 ( Doebley et al. 1997). An insertion of a transposon in the regulatory region of the tb1 gene in modern maize causes the gene to be strongly expressed (Doebley et al. 1997; Studer et al. 2011), resulting in enhanced apical dominance and suppression of excessive branching (Figure 7B).
Introns
A mutation in an intron may lead to aberrant pre-mRNA splicing. For example, a change from GT to A-T in the splice site sequence of intron 3 of the maize brittle-2 gene involved in starch biosynthesis abolishes the use of the splice site. Consequently, the bt2 mutants accumulate multiple non-wild-type-sized transcripts that encode non-functional proteins and thus produce shrunken or brittle kernels with high sugar content due to low starch synthesis (Lal et al., 1999).
Recessive vs. Dominant Mutations
Recessive Mutations
Recessive mutations most commonly result from a loss of function. A loss-of-function mutation causes a decrease in the function of a gene. A complete loss of function is known as a null mutation, while partial loss of function mutants are often called hypomorphs or “leaky.” Loss-of-function mutations can affect gene expression or the function of the resultant protein. Most loss-of-function mutations are recessive because the normal allele can provide the necessary function in a heterozygote.
In corn, a recessive loss-of-function mutation in the y1 gene, which encodes the phytoene synthase enzyme required for yellow carotenoid pigment synthesis, causes a white kernel phenotype often seen segregating in sweet corn hybrids (Figure 8).
Dominant Mutations
Dominant mutations can affect genes in several different ways. One general class is gain-of-function mutations. These encompass several different types.
- Overexpression mutants. As the name implies, the affected gene is expressed at inappropriately high levels, causing too much of a gene product to be produced.
- Neomorphic mutations arise when a mutation causes a qualitatively new effect not seen with normal alleles. This can occur when a mutation affects the regulation of a gene such that it becomes expressed at a new time or place in the plant. It can also happen when a mutation alters the properties of the encoded protein. For example, a transcription factor could be mutated to recognize a different promoter sequence, or an enzyme could recognize a new substrate (Figure 9).
- Dominant negative mutations alter a gene product such that the mutant gene product interferes with the function of the normal one. This can occur at the protein level. For example, a protein might function as a multimeric complex, and the presence of one mutant subunit could compromise the function of the entire complex. Dominant negatives can also occur at the RNA level, for example, if an insertion, deletion, or rearrangement causes the wrong strand to be transcribed, creating an antisense or RNAi suppression effect. The subject of RNAi will be discussed in greater detail in Chapter 6.
Effects of Mutations on Traits
Example of a Loss of Function Mutation—Gene Function is Required for a Trait
Sweetness is an important quality trait in maize (corn). One of the loss-of-function mutants used to improve sweetness in corn is sugary1 (su1). The Su1 gene is expressed in the endosperm (Lertrat and Pulam, 2007), and it encodes an enzyme required for normal starch biosynthesis (Rahman et al., 1998). In the su1 mutant, with defective starch biosynthesis, sugar concentration is 3 times higher than wild type, making this mutant valuable for sweet corn (Lertrat and Pulam, 2007). In addition, su1 kernels accumulate a highly branched polysaccharide called phytoglycogen, which gives the kernels a creamy texture. New commercial sweet corn hybrids are developed using a combination of starch biosynthesis mutants (Nelson and Pan, 1995; Lertrat and Pulam, 2007).
Example of a Gain-of-Function Mutation—Gene Function is Sufficient for a Trait
The plant growth hormone, gibberellin (GA), regulates plant height; thus, a plant that fails to produce GA, or does not respond to GA, will have a dwarfed phenotype (Figure 10). At the molecular level, the cell response to GA involves several processes, including the destruction of repressor proteins, referred to as DELLAs.
The “green revolution gene” Reduced height-1 (Rht-1) from wheat encodes a DELLA repressor of GA signaling (Peng et al. 1999). A stretch of five amino acids referred to as the DELLA domain is required to destroy the repressor in response to GA. Mutations in the DELLA domain of Rht-1 genes result in a protein product resistant to degradation, leading to failure to signal a GA response. The inability to signal the presence of GA in the rht-1 mutants is responsible for their short stature. When the mutant rht-1 gene is transformed into rice, the result is also short plants, proving that the DELLA protein’s new function is sufficient to induce dwarfism (Peng et al. 1999).
Semidominant traits often imply that the level of gene function is proportional to the expression of a trait. A classic example of this occurs in flower pigmentation. As seen in Figure 11, the white flowers are homozygous for a recessive allele that carries a null mutation for a gene required for anthocyanin biosynthesis. In the homozygous dominant, red flowers are produced. In the heterozygote, with only one functional copy of the gene, only half as much pigment is produced, resulting in a pink flower color.
Read: Mutation Breeding in Plant Breeding Methods.
Generation of Mutations
Because mutations are the ultimate source of genetic variation for breeding and genetic studies, it is sometimes of interest to generate new mutations. New mutations can arise spontaneously, or they can be induced by experimental methods.
Spontaneous Mutations
Spontaneous mutations arise “naturally.” That is, the scientist makes no effort to increase the mutation rate. Spontaneous mutations generally occur at very low rates—approximately one mutation per gene per million gametes.
Mistakes in DNA Replication
Very rarely, incorrect bases are incorporated or omitted from a DNA strand during synthesis. Such mistakes can lead to spontaneous substitutions, insertions, or deletions—for example, strand slippage due to the formation of a loop.
Environmental Mutagens
Several chemical agents are present in the environment with the potential to damage DNA and create mutations. These include naturally occurring compounds as well as man-made pollutants. Some modify DNA nucleotide bases through deamination (e.g., nitrous acid), while others promote oxidative reactions that may damage DNA (e.g., ozone).
Radiation is another type of environmental mutagen. Ionizing radiation (e.g., X-rays) can shatter DNA sequences and promote chromosome rearrangements. The less powerful ultraviolet rays can penetrate the cell and promote the formation of pyrimidine dimers, which may inhibit replication and transcription. Of course, radiation can also be applied experimentally, and the use of radiation-induced mutations in plant breeding was discussed in Plant Breeding Methods. Several pathogens are also potentially mutagenic, including Agrobacterium tumefaciens (see Chapter 8).
Experimental Mutations
Experimental mutagenesis involves applying a mutagenic agent to a plant or plant part to generate a mutation. For the mutation to be useful, it must be heritable, which limits the type of tissues that can be treated. Following the mutagenic treatment, a subsequent breeding scheme must be implemented to generate pure breeding lines of appropriate genotypes to display mutant phenotypes. These will depend on the species and the design of the mutagenesis.
Chemical Mutagenesis
A commonly used chemical mutagen for experimental plant biology and mutation breeding is ethyl methanesulfonate (EMS). The compound is an alkylating agent and induces CG to TA transition.
Seed Mutagenesis
This is the most common method of chemical mutagenesis in most species. Seeds are soaked in EMS solution, planted, and allowed to flower and set seed. During EMS mutagenesis, every cell in the embryo will be independently mutagenized, and the resultant seed and subsequent plant will be chimeric. The pollen and egg cells arise from different progenitor cells that may or may not be derived from the same ancestral cell mutagenized in the seed, depending on the reproductive development of the specific species. Therefore, a breeding scheme must be devised to generate families that segregate 1:2:1 +/+: m*/+: m*/m*, where m* represents a new mutant allele generated by the mutagenesis. These seeds can be sown, and plants screened for traits of interest. The required breeding for self-pollinating species is very simple, whereas it would be much more elaborate and labor-intensive for dioecious species.
Pollen Mutagenesis
Pollen mutagenesis is the method of choice for a few species, such as maize. Pollen is treated with EMS and applied to the silks of an acceptor plant. The benefit is that the resultant seed is not chimeric; instead, all the embryo cells carry the same mutagenized paternal genome inherited from the pollen grain.
Insertional Mutagenesis
The idea behind insertional mutagenesis is that a mobile or introduced piece of DNA can sometimes insert into a gene, thereby inactivating or modifying the function of the gene. The approach is helpful in gene cloning because the inserted DNA serves as a “tag” that can be used to locate the gene of interest and subsequently isolate that gene. Various approaches to gene cloning will be discussed in more detail in later sections of the course.
Early transposon mutagenesis experiments in snapdragon (Antirrhinum majus) provided key information about genes that control floral development (Carpenter and Coen, 1990). From a mutagenesis experiment using the Tam3 transposon (Figure 12), Coen and co-workers (Coen et al. 1990) discovered a mutant, which they referred to as flo-613.
The flo-613 mutant produced inflorescence meristems in place of the floral meristem. Since they knew the sequence of the Tam3 transposon, these researchers were able to isolate the gene that was “tagged” by the transposon. The gene is called floricaula (flo) and belongs to a class of homeotic genes (Coen et al. 1990). Homeotic genes control organ morphogenesis, and mutations in homeotic genes transform one structure into another
Another important property of transposons that helped in the isolation of floricaula is their genomic instability, which results in unstable mutations. This occurs because the transposon can spontaneously excise from the gene it first mutated by insertion. Suppose excision of the transposon from the gene (e.g., floricaula) leads to reversion to wild wild-type phenotype. In that case, that is evidence that the cloned gene is responsible for the mutant phenotype observed.
Another strategy researchers use is mutagenesis using T-DNA from the Agrobacterium tumefaciens Ti plasmid. A larger number of plants (up to 10,000 for Arabidopsis) are transformed with T-DNA to generate mutants at random. The process of plant transformation will be discussed in detail in subsequent lessons.
Arabidopsis has been used for T-DNA mutagenesis for several reasons. One is that it has a small genome size, helping to limit the number of transformants needed to obtain a mutation in the gene of interest. However, other plant species, including crops such as rice and other grasses, are now subjected to T-DNA mutagenesis to study gene function. T-DNA mutagenized seed can be obtained from various organizations. A few examples of these organizations can be found in the web links below.
Like the example of transposon mutagenesis above, a gene can be “tagged” by a T-DNA, and the tag is used to isolate the gene. However, T-DNA insertions are stable and thus disrupt the gene permanently (Figure 13).
In recent years, insertional mutagens have been engineered in many innovative ways. For example, strong promoters have been engineered into transposons such that if the transposon inserts near a gene, it can cause strong transcriptional expression of that gene. Such “activation tagging” approaches have successfully identified many genes that were not amenable to other mutational approaches.
Chapter Summary
Mutations alter A-T and G-C base pairs in DNA. A mutation in a coding sequence may alter the sequence and function of the protein product. A frameshift mutation changes the reading frame through insertions or deletions to produce an entirely novel product. A point mutation, on the other hand, alters only the amino acid represented by the codon in which the mutation exists. Mutations in regulatory regions might alter the expression of a gene. Mutations have been powerful tools for genetic studies as well as to study biological functions, for example, growth and development. The natural occurrence of mutations can be enhanced experimentally by applying agents referred to as mutagens. Researchers have used certain mutagens to induce mutations to study gene function and apply new traits for crop improvement.
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