Carol J. Rivin

The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development

Mutation at the TOUSLED locus of A. thaliana results in a complex phenotype, the most dramatic aspect of which being the abnormal flowers produced in mutant plants. tsl flowers show a random loss of floral organs, and organ development is impaired. The TSL gene appears to be required in the floral meristem for correct initiation of floral organ primordia and for proper development of organ primordia. Loss of TSL function also affects flowering time and leaf morphology. Using a mutation derived by T-DNA insertion mutagenesis, we have cloned the TSL gene and found that it encodes a protein kinase homolog with a novel N-terminal domain. This protein kinase gene identifies a novel signaling/regulatory pathway used during development in Arabidopsis.

Evaluation of genomic variability at the nucleic acid level

T he studv of variation is the basis of genetic analysis, evolutionary studies, and population t~iology, and of course, (s fundamental in the improvement of agricultural plants. In this context, the uncovering of new sources of variation and the ability to describe novel types of variation is of great importance to plant biologists. In maize, as well as other plants, variation has been widely studied at several levels: for phenotypic characters such as morphology and plant products (Goodman and Bird, 1977), for protein level changes as are revealed by isozyme analysis (Newton and Schwartz, 1980), and at the level of tile whole genome by analyzing the kinetics and stability of DNA:DNA reassociations in solution hybridization (Hake and Walbot, 1980). Recently, it has become possible to examine genome variation in much greater detail through the use of specific cloned DNA probes. The use of cloned probes makes direct nucleic acid analysis a fairly simple and extremely sensitive method of detecting types of variability that are inaccessible in phenotype or protein analysis. We will show belong, that we can easily detect extensive variation between modern inbred lines of maize, as well as between maize and its close relatives, the teosmtes. By using clones of repetitive DNA sequences, we have discovered considerable changes in the copy number of repeated DNA, and many restriction site polymorphisms. These types of variation cannot be detected at the phenotypic or protein level, although they may have important consequences in plant growth and adaptation. We also show that large numbers of restriction site polymorphisms can be found in the vicinity of genes defined by the use of cloned eDNA probes, and that these can be used to identify and map new genetic loci without having to find new phenotypes or new enzyme activities.

Characterization and Expression of a cDNA Encoding a Seed-Specific Metallothionein in Maize

Developing embryos of many higher plants undergo a maturation process in which a variety of storage proteins and putative dessication protectants are synthesized and stored. One of the proteins found to accumulate in maturing wheat embryos i s a Zn2+-associated class I1 metallothionein called E, (for early Cys) (Lane et al., 1987). Like many other maturation proteins, the promoter regions of wheat E, genes have been found to contain ABA-response elements, and their expression is ABA inducible (Kawashima et al., 1992). We have characterized a cDNA from maize (Zea mays L.), pMEC (maize EJ, whose sequence, regulation, and expression patterns indicate that it is the equivalent of wheat E, (Table I). Severa1 clones having strong homology with transcripts encoded by the wheat E, genes were isolated from a cDNA library that was differentially screened for messages expressed in maturing wild-type embryos but absent from vpl mutant embryos. These mutant embryos synthesize normal levels of ABA but are not ABA responsive (Neill et al., 1986; Robichaud et al., 1986). The product of the Viviparous-1 locus has been shown to act as a transcriptional activator of promoter targets carrying ABA-response elements (Hattori et al., 1992). We observed complete sequence identity among a11 pMEC isolates and single-band patterns on genomic Southern analysis, indicating that pMEC is probably encoded by a single gene. The approximately full-length clone, pMEC, contains a complete coding region for a polypeptide of 7.8 kD having 77% amino acid identity and 83% similarity with wheat E,. Maize embryos accumulate pMEC mRNA specifically in maturing seeds. On northem blots of RNA isolated from developing maize embryos, pMEC detects an mRNA species migrating at approximately 530 bases. The message leve1 is low in immature embryos and increases to a peak as embryos enter the mid-maturation phase. It remains high throughout later development. We have not assessed the relative contributions of transcription or stability to this persistence. Significant hybridization to pMEC was not found in RNA from seedling shoot, cob, tassle, or adult leaf.