Session 1: Cell Growth (Cell cycle/Cell death)
Chair: P. Doerner, The Salk Institute, La Jolla, CA, USA
email: peter_doerner@qm.salk.edu
This session covered several aspects of growth control in plants. Cell cycle regulation was discussed in relation to quantitative growth control as well as in developmental processes. These talks illustrated the distinct relationship of cell division and growth in plants when compared to other biological model systems. Regulated cell death plays a crucial role in plant defense responses and the recent isolation of genes implicated in these programs will accelerate the mechanistic analysis of the death program in plants. This session included three short communications highlighting posters presented at the meeting.
Peter Doerner, La Jolla, discussed the role of cell division in controlling root growth. In Arabidopsis, root growth accelerates two-fold when nitrogen availability is low. This leads to an increase of meristem size, measured by examining cell size and number in the root apex. However, this increase in the cycling population is not sufficient to quantitatively account for the enhanced overall growth rate. By counting cells that express a cyclin::GUS fusion, Doerner argued that a discrepancy between observed and predicted numbers indicated that the G2 phase of the cell cycle was truncated under these conditions. One prediction of this hypothesis was confirmed by the observation that proliferating cells in the rapidly growing root divided at a smaller size than equivalent cells in slower growing roots. Doerner proposed that control over the rate of cell cycle progression in G2 is a mechanism by which nutritional signals function in growth regulation.
Dirk Inze, Ghent, discussed the role of cyclin-dependent kinase genes in plant cell cycle regulation. Two CDK genes, cdc2a and cdc2b have been identified in Arabidopsis, with the former functioning throughout the cell cycle, the latter primarily at the G2/M transition. Cdc2b RNA levels as well as p35cdc2bAt-dependent histone H1 kinase activity peaks at the G2/M transition. In two-hybrid experiments, B-type cyclins were found to interact with the protein encoded by cdc2b while the G1-type D cyclins interacted with p34cdc2aAt, encoded by cdc2a. Two-hybrid screens also resulted in the isolation of an Arabidopsis CKS1 homolog, proteins which are thought to mediate interactions of the CDK complex with other proteins. Expression of the CKS1 homolog was found to be high in all dividing cells but also in cells undergoing endoreduplication. It was proposed that CKS1 functions in this process.
Bob Dietrich, Chapel Hill, presented recent work on the lsd1 mutant. LSD1 appears to be required to maintain cell viability, in the lsd1 mutant lesions are produced affected leaves in which spread unchecked after induction and kill the entire affected leaf. The LSD1 gene has no overall homology to any known genes. However, the corresponding protein contains three tandem zinc finger domains, raising the possibility that LSD1 may be a transcription factor. Current experiments focus on the functional analysis of LSD1, taking advantage of a null background for structure-function experiments with various deletion and point mutations of LSD1. A screen is underway to identify differences in gene expression between the LSD1 and lsd1 lines, based on the rationale that LSD1 might be a transcription factor. Concurrently, a yeast two hybrid screen is being used to identify proteins that interact with LSD1. In addition, a genetic screen to identify second-site suppressors is in progress to identify other genes which function in the LSD1 pathway.
Bill Crosby, Saskatoon, discussed how protein interactions involving the floral homeotic gene UFO suggest a role for selective cell cycle control during floral morphogenesis. Loss-of-function mutations in the UFO (unidentified floral organs) gene result in variably aberrant second and third whorl organs as well as defects in the transition from vegetative to floral development such that additional flower-like structures are generated in the place of individual inflorescences. Two-hybrid screens with UFO as bait resulted in the isolation of a class of interacting proteins (UIP for UFO-interacting protein) which show significant homology to the yeast cell cycle regulator SKP1. In-situ hybridization results show that UFO and UIPs are co-expressed in the same cells early during floral development. Crosby proposed that the role of UFO is to negatively regulate cell cycle progression mediated by an inhibition of UIP (SKP1) function.
Chun-Ming Liu, Stillwater, discussed three titan mutants. These mutants are defective in karyokinesis and form endosperm nuclei up to 100 times larger than the wild-type. Chromosome condensation has been observed in all titan mutants, while mitotic figures have only been seen in titan3. Development of titan1 and titan2 embryos is arrested at the 1-2 cell stage, whereas titan3 can produce fertile homozygous mutant plants. The TITAN1, TITAN2 and TITAN3 genes have been mapped to chromosomal 2, 1 and 5, respectively and progress towards their cloning is being made.
Raymond DiDonato, Boston, discussed the alf4-1 mutant which fails to develop lateral roots even when treated with exogenous IAA. These plants appear to be defective in the early stages of pericycle cell division. To examine cell division patterns in these mutants, we constructed transgenic lines of WT and alf4-1 plants containing a B cyclin (cyc1At) promoter fused to the GUS gene. Compared to WT, alf4-1 plants exhibit greatly decreased GUS staining in the root tip and pericycle cells, even when treated with IAA. These observations are consistent with the notion that alf4-1 mutants are defective in the early stages of pericycle cell division. We also mutagenized a WT line containing the cyc1At-GUS fusion construct to find plants displaying altered staining patterns. Two recessive, xcl- (extra cyclin levels) mutants were isolated which display a marked increase in staining levels in the roots, through the elongation zone and root shoot junction, and throughout all leaves.