Characterization of the LATERAL SUPPRESSOR Promoter and the New Regulator of Axillary Meristem Formation ENHANCER OF LATERAL SUPPRESSOR 5

Field: Agricultural and Biological Sciences (Life Sciences)

Aerial architecture of flowering plants is largely based on the activities of the shoot apical meristem and axillary meristems (AM), which are initiated in the axils of leaves. The LATERAL SUPPRESSOR (LAS) loss-of-function mutant is characterized by a lack AM development during the vegetative growth phase, identifying LAS as a key regulator of this process. LAS is expressed in a very specific band-shaped domain at the adaxial side of leaf primordia, the site of later formation of AMs. In order to understand how this specific expression pattern is established, and to be able to subsequently address the question which factors control LAS, the LAS promoter was analyzed in detail in this work. Complementation of the las-4 phenotype with various promoter deletion constructs revealed that less than 117 bp 5' of the transcription start are necessary for gene function. However, the ability to complement is lost when constructs harbor less than 3547 bp of 3' sequences. The importance of the 3' region is emphasized by results showing that complementation is still achieved if the 5' promoter is replaced by a minimal 35S promoter or a PISTILLATA (PI) promoter fragment, which does not confer expression in the apex. In both cases visualization of expression profiles using promoter GUS constructs showed specific expression in axils of leaves and flowers, alike the endogenous LAS promoter activity. In summary an LAS 3' promoter element, extending from bp 3239 to 3547 behind the ORF, was found to be necessary for complementation. We speculate that it is this element, which causes the highly specific LAS expression pattern. A previous las-4 second site modifier screen led to the identification of the new regulator of AM development ENHANCER OF LATERAL SUPPRESSOR 5 (EOL5). The eol5 las-4 double mutant was identified owing to the lack of AM formation in cauline leaf axils. Additionally, eol5 las-4 plants could be shown to exhibit leaf fusions and defects in meristem maintenance and floral primordia development. The gene underlying the eol5 mutation could be identified by map based cloning as CZS, a putative histone methyl transferase, previously shown to be involved in the epigenetic regulation of FLOWERING LOCUS C (FLC) and displays a mild delay of flowering. Complementation with an endogenous pCZS::CZS construct led to a recovery of the ability to form axillary shoots in cauline leaf axils, likewise an allelism test showed that the T-DNA insertion alleles czs-1 and czs-2 are allelic to eol5. However, the single mutant phenotype of eol5 is more pronounced, leading to a lack of AM formation in most rosette leaf axils, whereas czs-1 and czs-2 plants only exhibited few barren axils in the lower rosette. Accordingly, the delay of flowering observed in long day conditions was most distinct in eol5 plants. To address the question which genes are regulated by CZS, the expression of various candidates was compared between mutants and wild-type by real-time PCR. FLC could be shown to be upregulated in czs mutants. Analysis of FRI FLC plants, which strongly express FLC, revealed side shoot development defects, suggesting that FLC is involved in the process of AM formation. This indicates that similar mechanisms regulate lateral meristem development and flowering, thus the AM initiation defects observed in czs mutants are likely to be caused by the upregulation of FLC.