The establishment of the cambium-specific stem cell niche, which is being addressed by characterizing CBI1, a novel repressor of cambium identity in Arabidopsis thaliana
Final Report Abstract
In the forefront of the project, CBI1 was identified in a mutagenesis screen as a repressor of WOX4 promoter activity, therefore negatively acting upstream of cambium regulation. The cbi1-1 mutation could now be successfully mapped to the CTL1 locus, which encodes a factor involved in primary cell wall assembly. Even though indications on cell wall loosening being upstream of cell division initiation were available from studies on shoot apical meristems, the connection between cambium regulation and call wall dynamics is completely new. Though we could not detect cell ectopic cell proliferation in cbi1 stems after toluidine staining, several indications suggest that at least single events of “cambium-like” cell divisions are induced: 1) In addition to vascular bundles and the established cambium, DR5rev:GFP in the wild type is detected in starch sheath cells in the transition zone, preceding the first IC cell division. In cbi1 stems, in contrast, single DR5rev:GFP positive starch sheath cells were additionally detected in internode regions, where the wild type does not establish an IC. 2) In addition to the WOX4:GUS signal that originally lead to the isolation of cbi1 from the mutagenesis screen, we could also detect pith cells that were positive for the cell division marker H4:mCherry, as well as for the (upstream of WOX4) cambium marker PXY:YFP. The identification of CBI1 however redirected our focus from understanding ectopic induction of cell division to the regulation of the cambium where it is normally being induced. Indeed, changes in the regulation of cell wall genes occur at the onset of cambium initiation, and precede the induction of known cambium regulators. According to the new working hypothesis, the weakening of cell walls should facilitate cambium induction, and in line with this, cbi1 stems indeed show an enhancement of cambium activity and acropetal IC progression. Clonings were done and several plant lines produced which carry constructs for the local expression of cell wall weakening or strengthening genes in the cambium area. Even though the first round of experiments did not always deliver the expected results, the material produced in the course of the project will allow further understanding in the course of follow-up studies, and will therefore be highly informative and important for the lab as a whole. Various mutants were tested for their involvement in a potential signaling mechanism that communicates between cell wall loosening and induction of cell division, and among them, we could observe a phenotype with reduced cambium activity for fer in line with the idea of cell wall integrity sensing. In addition, the rlp44 mutant showed enhanced cambium activity, and therefore represents a novel cambium repressor. However, at this step no conclusion can be made with regard to the connection between RLP44 and cell wall dynamics. Taken together, the identification of a cell wall assembly factor acting upstream of cambium regulation lead to the idea of cell wall weakening inducing cambium cell division. In addition to promising first results which are in line with our new hypothesis, a tool box was generated that will allow further understanding in the course of follow-up studies.
Publications
- International Conference on Arabidopsis Research, Vienna 2012: “CAMBIUM ISLANDS 1 – A Novel Repressor of Cambium Initiation”
- Plant Vascular Development Conference, Vienna 2012: “CAMBIUM ISLANDS 1 – A Novel Repressor of Cambium Initiation”
