Christopher Somerville

One of the main projects in the Somerville laboratory concerns a long-term attempt to understand the process by which a single cell gives rise to an embryo that contains the fundamental elements of the body plan of an adult plant. Considered in the abstract, realizing this goal devolves into answering a series of questions about the mechanisms that regulate whether a particular cell divides, how the division takes place, and what happens to the daughter cells after each division. Since there are many more cells in an adult plant than there are genes in the genome of a typical angiosperm, the genetic rules that govern development must be such that they can be interpreted generically by many different kinds of cells to result in different specific outcomes. In certain respects, the rules that govern plant development must be like the Constitution of the United States; the Constitution does not specify a precise answer to each question of civil governance but it does specify a process for deciding how to achieve an answer.

The researchers’ approach to identifying the basic tenets of the angiosperm constitution is to identify mutants that disrupt the normal process. During the past several years they isolated approximately 7,000 mutants of Arabidopsis with defects in embryogenesis. Then, using microscopy, they examined the morphology of the defective embryos in the mutant lines at a stage of development when the embryos contained only a few hundred to a few thousand cells. From this collection of mutants, the scientists selected about 350 lines in which the early-stage embryos exhibited readily apparent morphological differences when compared with wild-type embryos at the same stage. These mutants, which were maintained as heterozygotes, were then grouped into genetic complementation groups to determine how many genes were altered in the collection of the 350 lines. Although this process is not completely finished, it appears that the mutant collection represents mutations at only a few dozen loci. In other words, for each type of mutant recovered from this screen, the scientists identified a large number of mutant alleles at each locus. This finding indicates that the mutant screen was saturating--meaning that the researchers believe that because they isolated the same mutants repetitively, they observed all the genetic variation that is possible by this general approach.

Following the assignment of the various mutants into complementation groups, they began the process of identifying the genes that correspond to each of the mutations. This is accomplished by genetically mapping--with very high precision--each mutation onto a chromosome. The procedure is to test several thousand progeny from a cross between a mutant and a wild-type plant for the inheritance of the mutant phenotype, and also test for the inheritance of structural differences (polymorphisms) in each of the chromosomes. If a mutation is inseparable from a polymorphism, the investigators can conclude that the mutation is located very close to the polymorphism on the chromosome. Because the nucleotide sequence of all of the Arabidopsis chromosomes is now known, it has become relatively straightforward to proceed from knowing the precise location of a mutation on a chromosome to identifying the gene corresponding to that mutation. During the past year, the researchers have used this powerful approach to identify the genes corresponding to a number of embryo-defective mutations.

Two of the mutations that they characterized were found to have defects in enzymes that catalyze steps in the addition of sugars to proteins in a process called N-linked glycosylation. These were the first mutations that completely disrupted this process in plants, and the properties of the mutants demonstrated that this activity is an indispensable plant function. Interestingly, although hundreds of proteins are N-glycosylated, most of the phenotypic effects of the mutations seemed to be due to an almost complete absence of cellulose, the principal polysaccharide polymer in plant cell walls. Thus, it appears that some component of cellulose synthase requires N-glycosylation for normal activity (Lukowitz and Somerville, in press; Gillmore and Somerville, in preparation).

Mutations at another locus, called MONOPOLE, were characterized by a defect in the organization of the lower half of the embryo; the upper half retained a relatively normal organization. Furthermore, the expression of a gene that is normally only expressed in a specialized cell type in the root meristem of the embryo was altered in the mutant (Figure 1). Instead of being expressed in the quiescent center near the base of the wild-type root, this gene was expressed near the lower border of the upper tier of cells in the mutant embryo. These and other phenotypes of the mutant indicated that the monopole mutant carries a defect in a fundamental aspect of the mechanisms that normally specify the identity of each cell. The MONOPOLE gene was cloned by map-based methods and found to encode a transcription factor. During the coming year the researchers will attempt to determine when and where this gene is expressed and what genes are controlled by MONOPOLE. If these questions can be answered, we may move one small step closer to understanding the set of genetic instructions that specify the characteristic transition of the fertilized egg into a multicellular plant.Figure Legend

Figure 1. These are images of embryos from a wild type (left) and a monopole mutant (DIRECTION TK). The embryos are from transgenic Arabidopsis plants that contain a gene for b-glucuronidase fused to a promoter that causes expression of the gene in the cells of the root meristem. The embryos have been stained for b-glucuronidase enzyme activity, which produces a blue color in the presence of a substrate called X-gluc.