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

The development of the aerial plant body depends on the activity of the shoot apical meristem (SAM), whereby pluripotent stem cells transit from the central stem-cell zone towards the periphery and become specified as lateral organ founder cells (LOFCs) depending on their precise position. Coordinated cell divisions within small groups of LOFCs create an organ primordium that then acquires fate [1]. In Arabidopsis thaliana, leaves are initiated during the vegetative phase and axillary meristems remain dormant; in contrast, the floral transition consists of biphasic meristem identity, in which secondary inflorescences initiate in the axils of cauline leaves in a pre-floral inflorescence phase and following the complete acquisition of reproductive competence, floral primordia are initiated in the axils of subtending bracts [2], whose outgrowth in Arabidopsis is subsequently suppressed. Thus, consistent with phytomer theory, the floral meristem (FM) can be considered as an axillary meristem, whose initiation depends on that of the cryptic bract [3]. Bract growth is known to be linked with floral organ initiation [4] and a genetic determinant of bract identity and growth, LEAFY (LFY), also regulates floral primordium formation.

Groups of LOFCs in the IM are characterised by transcription of the DORNRÖSCHEN-LIKE (DRNL) AP2-type transcription factor gene in a spiral phyllotaxy from near the centre of the IM towards the morphologically apparent stage 1 floral buttress [5]. Here, the population of DRNL-expressing LOFCs bifurcates into two foci; one at the tip of the floral buttress where the abaxial sepal will develop [6] and the other basally at the cryptic bract position. Bract development in lfy and puchi mutants disrupts the unidirectional sequence of first-whorl floral organ initiation of wild type [6], which suggests a complex developmental dynamism of founder-cell specification and overlapping positional information for the abaxial sepal and bract in the wild type IM. LOFC specification in the outer floral whorl of sepals occurs in the absence of stem-cell markers such as CLAVATA3 (CLV3) or WUSCHEL (WUS) at the IM periphery, which regain activity after initation of the abaxial sepal, when a furrow separates the stage 2 primordium from the IM [7, 8].

A suitable genetic background in which to study the earliest stages of FM initiation is the apetala1 cauliflower (ap1 cal) double mutant, which overproliferates IMs before the delayed production of FMs [9]. The resulting inflorescence apices are massively enriched in synchronised IMs that specify LOFCs in a spiral phylotaxy at the IM periphery according to DRNL expression [6]. The ap1 cal genetic background has been combined with appropriate cell-type-specific fluorescent markers and used for fluorescence-activated cell sorting (FACS) coupled with microarray analysis to transcriptionally profile the meristem stem-cell niche [8, 10] or with chromatin immunoprecipitation analyses to identify the physical targets of MADS-box transcription factors [11]. The synchronisation of IMs in the ap1 cal apex restricts analyses to a short developmental window and the DRNL::GFP-expressing LOFCs can be separated via FACS from their non-expressing neighbours for comparative transcriptome analysis. This provides access to the earliest phase of cell-type specification in the IM peripheral zone.

The initiation of lateral organs involves the repression of the class I KNOX genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) by the ASYMMETRIC LEAVES1 (AS1) and AS2 transcription factors to promote cell differentiation [12]. In Arabidopsis, auxin is also a positional determinant, because polar auxin transport generates auxin response maxima at sites of incipient FM initiation [13] and mutation of the auxin polar transport and signalling components PIN-FORMED1 (PIN1) and MONOPTEROS (MP) completely blocks the formation of FMs [14, 15]. The downstream signalling cascade from MP in lateral organ initiation is partially known and includes the LFY, AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE6 (AIL6) and FILAMENTOUS FLOWER (FIL) transcription factors [16, 17]. However, auxin response is not the only phyllotactic signal, and it co-functions with cytokinin signalling via ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6) [18]. AHP6 is a target gene of DRNL [19] and the AHP6 and DRNL expression domains only partially overlap with that of the DR5 auxin response reporter and are more distal towards the IM periphery [6], indicating polarity with respect to auxin or cytokinin response. Polarity is an iterating scheme in lateral organ development in the IM, starting with progenitor cell divisions that occur near the central zone and defining an outward trajectory along an ad-/abaxial axis [20]. Furthermore, the polarity of the floral meristem is affected by several genes, including BLADE ON PETIOLE1 (BOP1) and BOP2 [21, 22], YABBY (YAB) [23] and ETTIN [24].

Despite the identification of some components of the gene regulatory networks (GRNs), including hormonal signals, which affect lateral organ initiation at the IM periphery, several problems remain: firstly, whether auxin or cytokinin signalling is causal or correlative with respect to LOFC specification; secondly, the relative timing of FM initiation in the axils of cryptic bracts within the IM, according to phytomer theory and thirdly, the basis of the interplay between founder-cell recruitment for the bract and FM, as is suggested by the altered series of sepal initiation in puchi and lfy mutants [6]. Resolving these issues is facilitated by detailed knowledge of the GRNs that are active in LOFCs compared to in the IM. Similar data are available at a single-cell resolution for specification of the hypophysis [25], lateral-root founder cells (reviewed in [26]), the endodermis/cortex initial [27] and the root phloem [28].

To optimise the resolution of studying the LOFC GRN, here, we have combined FACS using the DRNL::GFP founder-cell marker in the ap1 cal genetic background and RNA-seq, to capture the LOFC transcriptome at the earliest developmental time-point of lateral organ formation at the IM periphery. Next-generation sequencing, and especially RNA-seq [29], has become the method of choice for genome-wide transcriptional profiling, due to its ability to quantitate transcript expression over a large dynamic expression range and has to date been used in Arabidopsis to characterise the transcriptomes of pollen [30] and wild-type or homeotic mutant flowers [31]. We show here, that the combined use of FACS/RNA-seq is suitable to address cellular decisions in the SAM at a resolution not previously achieved. The LOFC transcriptome data represent a unique resource that allows the interrogation of aspects of transcriptional control and the molecular pathways that enable founder-cell specification, and that in comparison to the ap1 cal IM transcriptome, depicts the molecular repertoire that accompanies the cellular specification of bract, sepal or FM tissue at the IM periphery.