Functional Analysis of the GRAS Gene LATERAL SUPPRESSOR Arabidopsis Development of in Root and tomato

The genetic regulation of axillary meristem (AM) initiation is highly conserved throughout the majority of higher plants. Especially the GRAS gene LATERAL SUPPRESSOR (LAS), and its orthologues in various species constitute a nice example of this mechanistic conservation. LAS and its orthologues act as branching regulators, which promote the initiation of axillary meristems during vegetative development. Yet, the molecular mechanisms of LAS function are unknown. This study explored new perspectives, to gain a deeper understanding of the molecular basis of LAS function. Instead of focusing on AM initiation, the root was chosen as a promising new developmental context to analyze LAS function. Root-specific expression of LAS appears to be similarly conserved as LAS contribution to AM initiation, hence the root provides a new approach to study LAS function. Here, the previously identified cis-regulatory element B, located 2 kb downstream of LAS, is shown to be indispensable for LAS expression in the Arabidopsis root. LAS expression domains in the root mirror the cone-shaped domain of auxin response maxima in the tips of primary and lateral roots, showing transcript accumulation in the quiescent center, the columella stem cells and the mature columella cells. Monitoring of protein localization revealed that LAS protein accumulates in the nucleus and the cytoplasm. Also, LAS is a cell-to-cell mobile protein, traficking from the columella in the LRC. Additionally, the LAS locus encodes for a long noncoding antisense transcript, which is expressed in exactly the same domains as LAS. One notable difference between LAS expression domains in the tips of primary and lateral roots is the significantly elevated expression level in young lateral roots. This might correlate with the increased vertical growth trajectories of LRs in las-4 mutants, which is the only phenotypic deviation found in roots. LAS is thought to act as an inhibitor of cell differentiation. This hypothesis was tested by ectopic expression of LAS in the root apical meristem (RAM), which constitutes a powerful system to analyze cell differentiation. Ectopic expression reveals, that LAS promotes cell differentiation, and surprisingly cell differentiation also occurs in cells that express LAS endogenously. This indicated that LAS function might be dosage dependent. To test the applicability of root-derived knowledge about LAS protein characteristics, nuclear-targeted versions of LAS (LAS-NLS) were employed to analyze the relevance of protein movement during AM initiation. Interestingly, LAS-NLS versions were not able to complement the las-4 mutant branching phenotype. Addition of the nuclear targeting domain seems not to affect movement of the LAS protein, because in the root LAS-NLS versions are still cell-to-cell mobile. Interestingly, there is a positive correlation between dosage and function, based on the analysis of transgene copy-number in relation to las-4 complementation. Higher copy-number correlates with the loss of las-4 complementation. To account for the reoccurring theme of dosage-dependent function a spillover-model of LAS function is proposed. In this model, LAS is kept in check by a second interacting protein. If the buffering capacity of the interacting protein is compromised or exceeded, LAS might extend its functional scope, probably counteracting its own endogenous function