Molecular Plant Genetics
Molecular Plant Genetics
The appearance and chemical composition of all life are determined by the action of genes functioning in the context of the conditions surrounding the organism. While both genes and environment are important in determining the characteristics of plants, it is becoming clearer that genes control many more characteristics, and to a higher degree, than we had previously imagined. Hence, the study of genes and their effects on organisms, genetics, has allowed us to combat a wide range of human diseases. The burgeoning plant biotechnology industry, which promises to produce revolutionary plants and plant products in the twenty-first century, has also arisen. An intriguing tenet of modern genetics is that the cellular molecules that carry the genetic information (deoxyribonucleic acid [DNA]) and transmit this data to cells (ribonucleic acid [RNA] and proteins) are the same in plants and animals, so that geneticists speak one universal language that can be interpreted and manipulated, through science, to beneficially alter any species.
DNA, Genes, and Chromosomes
DNA is the molecule that constitutes genes. The main component of each cell's DNA is found in its nucleus. The individual, very large DNA molecules of the nucleus are chromosomes, each of which consists of thousands of genes, and each cell of an individual plant species has the same DNA and chromosomal composition. Copies of all genes are transmitted from both parents to their offspring, accounting for inheritance, the principle wherein offspring resemble their parents.
The chemical structure of DNA allows it to store information and for that information to be incorporated into the design of developing cells and organs. DNA molecules are very long linear structures comprised of millions of repeating units. Segments, consisting typically of a few thousand of these units, constitute individual genes. Each gene carries the information that dictates the structure of a single protein. Proteins catalyze all of the chemical reactions in cells generating its components and forming the cells into recognizable tissues and organs.
The backbone of a DNA molecule consists of alternations of the 5-carbon sugar, 2-deoxyribose, and phosphate. Note that the sugars are linked at their number three position (3′, read as "three prime") to a phosphate and their number five position (5′) at the other end to another phosphate. Further, the sugars are all oriented by these links in the same direction so that the backbone has direction—that is, a 5′ and a 3′ end. Connected to each sugar, at its 1′ position, is one of four nitrogenous bases: adenine, cytosine, guanine, or thymine. Each DNA backbone is actually paired for its full length with a second DNA backbone, with the chemical linkage between the two occurring via weak hydrogen bonding between the bases of the two chains. Two aspects of this pairing should be noted: 1) the two sugar-phosphate backbones have opposite orientations (they are antiparallel); and 2) any adenine of either chain is bonded (paired) with a thymine, and each guanine is paired with a cytosine. Consequently, the sequence of bases of the two chains are complementary to one another so that one can be predicted from the other. It is the sequence of bases within a gene that determines the type of protein that the gene codes for, including the protein's function in plant cells. Within a gene for a particular protein, three successive bases determine one amino acid. For example, A (abbreviation for adenine), followed by T (thymine), and then G (guanine) code for the amino acid methione (ATG is the term for this code in DNA sequence terminology), and each of the twenty possible amino acids that are incorporated into proteins have their own three-base determinants, or codons. For most amino acids, there are several three-base sequences that will code for a particular amino acid.
For DNA to function as a hereditary molecule, it must be duplicated (replicated) so that the daughter cells produced by cell division can receive identical copies. Replication of DNA is accomplished by a large complex of enzymes , within which the main replication enzyme, DNA polymerase, carries out the main synthesizing reaction. In DNA replication, the following steps are accomplished by the synthesis complex:
- the two halves of the starting double-stranded DNA, which are wound together in a ropelike helix, are separated so that the bases are exposed;
- the replication complex reads each half of the unwound DNA so that molecules complementary to each of original halves of the helix are synthesized from new subunits; and
- these new chains are left bonded to old ones so that there are now two half-new, half-old identical DNAs. DNA replication must occur in each cell before the cell can divide and is also necessary in reproduction prior to the generation of pollen grains and ovules.
The process by which genes are read and the sequence used to form a polymer of amino acids in a protein consists of two steps. In the first transcription, a copy of the gene is made in the form of RNA. Then, via the process of translation, the RNA sequence is interpreted by the translation machinery to make the actual protein. RNA is a molecule that is similar to DNA in structure, with the following differences: 1) its sugar is ribose, also a 5-carbon molecule, but which has an OH group at the 2′ position, 2) it is usually single stranded, rather than consisting of two paired strands, and 3) it utilizes the base uracil in place of thymine, which does have similar base pairing characteristics. Hence, RNA has a similar, but not identical, sugar-phosphate backbone to DNA, and the sequences of bases in its structure can convey information in the same fashion.
In a biochemical sense, the events of transcription (DNA-dependent RNA synthesis) are similar to the steps of DNA replication. The paired halves of the DNA constituting one end of the gene are separated, and an enzyme complex is attached. Included in this complex is an enzyme called RNA polymerase that reads the DNA and builds an RNA molecule having a base sequence complementary to that of the template DNA.
Once the RNA copy, called messenger RNA (mRNA), is made in a plant nucleus, it undergoes several modifications and is then transmitted to the cytoplasm. Here, the mRNA is utilized by the process of translation that generates a protein having an amino sequence corresponding to the base sequence of the mRNA and its gene. The process of translation, or protein synthesis, takes place on ribosomes, which are composed of ribosomal RNA (rRNA) and more than one hundred proteins. The ribosome attaches to an mRNA and moves along its length, synthesizing a protein by adding the correct amino acids, in sequence, one at a time. The addition of the correct amino acid at each point is accomplished by the pairing of three bases of the mRNA with a transfer RNA, which has a three-base segment complementary to this set of bases and which was previously attached to the correct amino acid by an enzymatic reaction. Consequently, the correct functioning of transcription and translation allows the information of each gene to be interpreted and converted into a protein, which carries out a very specific metabolic reaction in the cell.
An interesting feature of plant chromosomes that is much less common in animals is polyploidy. Polyploidy occurs when the entire set of chromosomes is multiplied, relative to the normal two of each kind per cell. For example, the normal diploid number of corn chromosomes is twenty; that is, each cell of a normal plant contains two of each of ten different chromosomes. If this number were doubled so that there were a total of forty, with each of the ten different types being represented four times, the result would be tetraploid corn containing four of each chromosome. The common peanut is a natural tetraploid species. Polyploid strawberries have been created artificially to increase the desirable characteristics of the fruit.
Mutations and Polymorphisms
Any change in a DNA molecule of a plant or animal is called a mutation, whether occurring in nature or induced experimentally. Changes in DNA occur in nature as a result of either environmental agents or rare but inevitable mistakes in the DNA replication process. The resulting natural variations of DNA sequence among the individuals of a species, DNA polymorphisms, fuel evolution. These polymorphisms can be analyzed through molecular techniques and can be used to determine the relationship among plants and molecular plant improvement as well as identifying individual plants. Hence, we have seen the development of DNA fingerprinting for intellectual property protection of novel genetic improvements in plant breed-ing—which is similar to the fingerprinting techniques used in several human criminological contexts.
Specific mutational changes in DNA may affect the function of the resulting protein, usually by reducing its efficiency or rendering it completely nonfunctional. However, in rare cases, a mutation may make the enzyme more useful for metabolism in some way. The former type of change is widely used by plant scientists to discover the roles of genes in growth and development; the latter represents the goal of protein engineering and is the basis of plant biotechnology.
Uses of Mutants
The genetic dissection of plant growth and development is one of the outstanding uses of mutations for scientific analysis. For example, a number of mutants block aspects of flower development. One mutant was discovered whose flowers lack petals, another lacks both the male and female reproductive parts of the flower, and still another lacks sepals and petals. Detailed analysis of the effects of these mutants, along with the cloning and characterization of the genes themselves, has led to a partial understanding of how a plant makes flowers. It is likely that a complete picture will eventually result. Interestingly, the original flower development model was developed for the small dicot, Arabidopsis, which has both sexes in one flower, but the same regulators act in the crop plant corn, which has separate male and female flowers, and which are completely different in appearance from those of Arabidopsis.
Another experimental application of mutant analysis illustrates the use of genetics in biotechnology and the generation of transgenic plants. Plants are said to be transgenic when DNA from some external source is introduced by scientists through biotechnology. In this case, a mutation was discovered in Arabidopsis called "leafy." This mutation is a loss-of-function change, which results in the replacement of flowers and fruits by leaves. Hence, the normal version of this gene must promote the ability to produce flowers. Subsequently, the normal gene was cloned and inserted into different plant species by transgenic techniques. When poplar trees received the gene, the genetically modified tree seedlings germinated normally but flowered within months rather than several years later, as occurs in normal trees.
Transgenically modified plants used in agriculture are often referred to as GMOs or genetically modified organisms. An example of GMOs are Roundup-Ready soybeans, which have resistance to this effective, nonpolluting herbicide through a transgene. These beans are widely used but are somewhat controversial. The public debate over the use of GMOs in agriculture involves a number of complex political issues in addition to the public health and environmental concerns that may also be relevant for certain types of GMOs. The handling of this issue represents one of the important public policy issues of our era. Another example of a potentially beneficial GMO is rice that is altered to carry more iron in its seeds. This should dramatically improve its nutritional value and prove especially valuable in areas of the world where food is scarce and human diets are typically not well balanced.
Improvement of crop plants has been practiced by plant breeders for centuries. The molecular tools discussed above simply enhance the range of alterations that are possible for improving crops. Traditional crop breeding involves finding and evaluating potentially useful genetic variants of a species, intercrossing them so that the most optimal set of characteristics can be combined into one strain, and then evaluating a number of resulting strains for final use in actual production farming. This is a long and costly process. In addition, many of the traits, which are of interest from an agronomic perspective, are quantitative as opposed to qualitative in inheritance. That is, they are controlled by large numbers of genes, each of which has a relatively small effect on performance. When this is the case, the application of classical genetics and molecular biology is difficult, since individual genes affecting a quantitative trait are very difficult to identify or clone. However, molecular markers can be correlated with important quantitative traits of a segregating population and utilized to pinpoint the general chromosomal locations where greater-than-average effects on the quantitative traits are exerted. Loci found in this way are referred to as quantitative trait loci or QTLs. QTL approaches are being pursued in many crops as alternative means of developing improved crop varieties and understanding the genetic basis of quantitatively inherited traits.
see also Breeding; Cell Cycle; Chromosomes; Creighton, Harriet; Genetic Engineer; Genetic Engineering; Genetic Mechanisms and Development; McClintock, Barbara; Mendel, Gregor; Polyploidy; Quantitative Trait Loci; Transgenic Plants; Warming, Johannes.
Fletcher, C. "A Garden of Mutants." Discover 16 (1995): 54-69.
Klug, William R. and Michael R. Cummings. Essentials of Genetics, 2nd ed. Upper Saddle River, NJ: Prentice-Hall, 1996.
Arabidopsis thaliana is a small plant that has played a large part in unraveling the molecular genetics of plants. It has an approximately two-to four-inch-wide cluster of leaves and a several-inch-tall flowering structure and is capable of producing thousands of tiny seeds within four to six weeks after germination. Because of its small size and short generation time, it has long been used for genetic research. In the 1980s it was discovered that the deoxyribonucleic acid (DNA) content of its genome was very small, and it was therefore adopted as the favorite model for basic study of molecular control of plant development and metabolism. In the 1990s more research was published on Arabidopsis than on any other plant. Further, as biotechnology has developed, it was realized that a model organism could form the focus for initial evaluation of key systems, and several startup biotechnology firms that have substantial Arabidopsis research components have been established.
A new biological discipline called genomics has recently arisen. A genome is defined simply as the entire set of chromosomes (and thus DNA) of a species. Genomics is the analysis of the entire set (or at least a very large subset) of an organism's genes. Plant genomics is made possible by two circumstances: 1) the capability to clone and determine the base sequence of the entire length of all of the chromosomes of a plant, and 2) the development of new technologies to assay whether genes are being transcribed on a genome-wide scale. The Arabidopsis Genome Initiative (AGI), an international collaboration, was established in 1995 with the goal of determining the DNA base sequence of the entire Arabidopsis chromosome set (genome). By the time the project finished in 2000, all of the estimated twenty thousand plus genes of this plant were available for molecular and biological analyses.
Given the availability of the complete genomic sequence data, the next focus of international Arabidopsis cooperation is functional genomics. Functional genomics is simply the analysis of genes and their effects on the full scale of the entire genome, that is, all (or most) of the genes at once. In one of the most powerful full-genome approaches, microar-rays of copies of each of a large number of genes are bound to small glass slides so they can be used to quantify the amounts of their ribonucleic acids (RNA) produced under differing conditions. For example, RNA can be isolated from plants both infected and not infected with a pathogenic fungus so that the genes that are turned on in response to the fungus can be rapidly identified. These and many other similarly large-scale analyses will allow swift determination of how plants respond to their environment and the full set of changes that occur in different stages of development.