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

Super determinant1A, a RAWULdomain-containing protein, modulates axillary meristem formation and compound leaf development in tomato

Shoot branching and complex leaf development relies on the establishment of boundaries that precedes the formation of axillary meristems (AMs) and leaflets. The tomato (Solanum lycopersicum) super determinant mutant is compromised in both processes, due to a mutation in Sde1A. Sde1A encodes a protein with a RAWUL domain, which is also present in Polycomb Group Repressive Complex 1 (PRC1) RING finger proteins and WD Repeat Domain 48 proteins. Genetic analysis revealed that Sde1A and BmilA cooperate, whereas Bmi1C antagonizes both activities, indicating the existence of functionally opposing PRC1 complexes that interact with Sde1A. Sde1A is expressed at early stages of boundary development in a small group of cells in the center of the leaf-axil boundary, but its activity is required for meristem formation at later stages. This suggests that Sde1A and Bmi1A promote AM formation and complex leaf development by safeguarding a pool of cells in the developing boundary zones. Genetic and protein interaction analyses showed that Sde1A and Lateral suppressor (Ls) are components of the same genetic pathway. In contrast to Is, sdela mutants are not compromised in inflorescence branching, suggesting that Sde1A is a potential target for breeding tomato cultivars with reduced side-shoot formation during vegetative development

The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Background: Although the pattern of lateral organ formation from apical meristems establishes species-specific plant architecture, the positional information that confers cell fate to cells as they transit to the meristem flanks where they differentiate, remains largely unknown. We have combined fluorescence-activated cell sorting and RNA-seq to characterise the cell-type-specific transcriptome at the earliest developmental time-point of lateral organ formation using DORNROSCHEN-LIKE::GFP to mark founder-cell populations at the periphery of the inflorescence meristem (IM) in apetala1 cauliflower double mutants, which overproliferate IMs. Results: Within the lateral organ founder-cell population at the inflorescence meristem, floral primordium identity genes are upregulated and stem-cell identity markers are downregulated. Additional differentially expressed transcripts are involved in polarity generation and boundary formation, and in epigenetic and post-translational changes. However, only subtle transcriptional reprogramming within the global auxin network was observed. Conclusions: The transcriptional network of differentially expressed genes supports the hypothesis that lateral organ founder-cell specification involves the creation of polarity from the centre to the periphery of the IM and the establishment of a boundary from surrounding cells, consistent with bract initiation. However, contrary to the established paradigm that sites of auxin response maxima pre-pattern lateral organ initiation in the IM, auxin response might play a minor role in the earliest stages of lateral floral initiation

Additional file 1: of The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Comparison of log2-transformed expression from qRT-PCR for a subset of 18 up- or downregulated genes from the transcriptome dataset normalised to ACTIN2 expression and the log2 (relative expression) from RNA-seq. (DOC 150 kb

Additional file 2: of The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem

Primer sequences and list of genes used for real-time PCR expression analysis. (DOCX 147 kb