Control of stem-cell niche establishment in Arabidopsis flowers by REVOLUTA and the LEAFY-RAX1 module

Plants retain the ability to produce organs throughout their life by maintaining active stem cell niches called meristems. The shoot apical meristem (SAM) is responsible for the growth of aerial plant structures. In Arabidopsis thaliana, the SAM initially produces leaves during the vegetative phase and later flowers during reproductive development. In the early stages of floral initiation, a group of cells first emerges from the SAM to form a stereotypically organized meristematic structure on its flank. However, the molecular mechanisms underlying the acquisition of this specific meristematic organization remain elusive. We show here that the transcription factors LEAFY (LFY) and REVOLUTA (REV) control two partially redundant pathways controlling meristematic organization in early flower primordia. We found that LFY acts through the transcription factor REGULATOR OF AXILLARY MERISTEM1 (RAX1) and we provide mechanistic insights in how RAX1 allows meristem identity establishment in young flowers. Our work provides a molecular link between the processes of meristem formation and floral identity acquisition in the nascent flower.


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Plants retain the capacity to initiate new organs throughout their life. To this end, they 42 maintain self-sustaining pools of stem cells in organized niches called meristems. 43 The SAM gives rise to most post-embryonic aerial organs thanks to stem cells 118 Because REV appears as a possible candidate to act in parallel with the LFY/RAX1 119 pathway in flowers, we combined mutations in both pathways to study their potential 120 role in the acquisition of the meristematic structure of the flower primordium. We  (Otsuga et al., 2001). One representative line (thereafter named rev-c1) was further 138 characterized: its leaves were slightly over-curved downward, the number of axillary 139 stems was reduced, 20% of flowers lacked internal whorls, and some rare flowers 140 were replaced by filaments (Supplemental Figure 3 A,B). 141 We then crossed this line still containing the REV-targeting CRISPR construct into  Figure 1A). Co-segregation analysis after one back-153 cross to wild-type showed that the newly observed filamentous phenotype is specific 154 to lfy-12 rev-c4 double mutants (Supplemental Table 1).

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The growth of short determinate filaments instead of flowers suggests that the lfy-12   In silico prediction of putative direct RAX1 targets 307 We aimed at identifying putative direct targets of RAX1 amongst the differentially 308 expressed genes. For this, we determined RAX1 DNA-binding properties using  Table 2).

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Among the predicted RAX1 direct targets, we identified ABF2, a protein linked to  Table 2).

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RAX1 directly regulates CLV1 expression 338 CLV1, a known negative regulator of WUS, was identified as a putative direct RAX1 339 target and was repressed over two-fold in seedlings in response to RAX1 induction.   Table 3). Despite differences in the age and growth conditions of the 451 samples between these two datasets, it suggests that RAX1 and REV act in different  Since there is no evidence that LFY/RAX1 or REV act downstream of the L1 signals, 491 we can imagine that they are required to regulate WUS level in parallel of the L1

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Spacers with no predicted off-targets were selected (Supplemental Table 4). Spacers  Table   540 5). The sequence was subsequently amplified with oGD115 and oGD116, which 541 added the compatible GreenGate overhangs and flanking BsaI sites, and was cloned 542 in pGGC000, producing pGD41. The GR coding sequence was amplified from plants 543 carrying an APETALA1-GR construct (Wellmer et al., 2006) with oGD109 and 544 oGD110 to be cloned in pGGC000 to produce pGD38. For the cloning of GR in 545 pGGD000, a linker sequence was amplified from pGGD001 with oGD118 and 546 oGD119 and the GR sequence was amplified with oGD110 and oGD111. Both or REV carried a homozygous mutation (namely rax1-c1 and rev-c1), however the 581 double mutant line carried heteroallelic mutations at each locus (rax1-c2/c3 rev-582 c2/c3; see Figure S1). Progeny of these plants was used for further characterization.

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CRISPR lines targeting REV were crossed to lfy-12 and T2 was screened for double 584 mutant genotype. A line carrying homozygous mutation at the REV loci (rev-c4) and 585 heterozygous lfy-12 mutation was selected.  In vitro DNA-binding assay 650 Protein production was performed as previously described (Sayou et al., 2016).