Spatiotemporal control of axillary meristem formation by interacting transcriptional regulators.

Branching is a common feature of plant development. In seed plants, axillary meristems (AMs) initiate in leaf axils to enable lateral shoot branching. AM initiation requires a high level of expression of the meristem marker SHOOT MERISTEMLESS (STM) in the leaf axil. Here, we show that modules of interacting transcriptional regulators control STM expression and AM initiation. Two redundant AP2-type transcription factors, DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL), control AM initiation by regulating STM expression. DRN and DRNL directly upregulate STM expression in leaf axil meristematic cells, as does another transcription factor, REVOLUTA (REV). The activation of STM expression by DRN/DRNL depends on REV, and vice versa. DRN/DRNL and REV have overlapping expression patterns and protein interactions in the leaf axil, which are required for the upregulation of STM expression. Furthermore, LITTLE ZIPPER3, another REV-interacting protein, is expressed in the leaf axil and interferes with the DRN/DRNL-REV interaction to negatively modulate STM expression. Our results support a model in which interacting transcriptional regulators fine-tune the expression of STM to precisely regulate AM initiation. Thus, shoot branching recruits the same conserved protein complexes used in embryogenesis and leaf polarity patterning.

Clonal sector analysis and cell ablation confirm a function for DORNROESCHEN-LIKE in founder cells and the vasculature in Arabidopsis.

MAIN CONCLUSION: Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre-Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL, to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.

Functional dissection of the DORNRÖSCHEN-LIKE enhancer 2 during embryonic and phyllotactic patterning.

MAIN CONCLUSION: The Arabidopsis DORNRÖSCHEN-LIKE enhancer 2 comprises a high-occupancy target region in the IM periphery that integrates signals for the spiral phyllotactic pattern and cruciferous arrangement of sepals. Transcription of the DORNRÖSCHEN-LIKE (DRNL) gene marks lateral organ founder cells (LOFCs) in the peripheral zone of the inflorescence meristem (IM) and enhancer 2 (En2) in the DRNL promoter upstream region essentially contributes to this phyllotactic transcription pattern. Further analysis focused on the phylogenetically highly conserved 100-bp En2core element, which was sufficient to promote the phyllotactic pattern, but was recalcitrant to further shortening. Here, we show that En2core functions independent of orientation and create a series of mutations to study consequences on the transcription pattern. Their analysis shows that, first, in addition to in the inflorescence apex, En2core acts in the embryo; second, cis-regulatory target sequences are distributed throughout the 100-bp element, although substantial differences exist in their function between embryo and IM. Third, putative core auxin response elements (AuxREs) spatially activate or restrict DRNL expression, and fourth, according to chromatin configuration data, En2core enhancer activity in LOFCs correlates with an open chromatin structure at the DRNL transcription start. In combination, mutational and chromatin analyses imply that En2core comprises a high-occupancy target (HOT) region for transcription factors, which implements phyllotactic information for the spiral LOFC pattern in the IM periphery and coordinates the cruciferous array of floral sepals. Our data disfavor a contribution of activating auxin response factors (ARFs) but do not exclude auxin as a morphogenetic signal.

Specific chromatin changes mark lateral organ founder cells in the Arabidopsis inflorescence meristem.

Fluorescence-activated cell sorting (FACS) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were combined to analyse the chromatin state of lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis apetala1-1 cauliflower-1 double mutant inflorescence meristem. On a genome-wide level, we observed a striking correlation between transposase hypersensitive sites (THSs) detected by ATAC-seq and DNase I hypersensitive sites (DHSs). The mostly expanded DHSs were often substructured into several individual THSs, which correlated with phylogenetically conserved DNA sequences or enhancer elements. Comparing chromatin accessibility with available RNA-seq data, THS change configuration was reflected by gene activation or repression and chromatin regions acquired or lost transposase accessibility in direct correlation with gene expression levels in LOFCs. This was most pronounced immediately upstream of the transcription start, where genome-wide THSs were abundant in a complementary pattern to established H3K4me3 activation or H3K27me3 repression marks. At this resolution, the combined application of FACS/ATAC-seq is widely applicable to detect chromatin changes during cell-type specification and facilitates the detection of regulatory elements in plant promoters.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

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 DORNRÖSCHEN-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.

The AP2-type transcription factors DORNRÖSCHEN and DORNRÖSCHEN-LIKE promote G1/S transition.

The paralogous genes DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) encode AP2-type transcription factors that are expressed and act cell-autonomously in the central stem-cell zone or lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis shoot meristem (SAM), but their molecular contribution is unknown. Here, we show using the Arabidopsis thaliana MERISTEM LAYER 1 promoter that DRN and DRNL share a common function in cell cycle progression and potentially provide local competence for G1-S transitions in the SAM. Analysis of double transgenic DRN::erGFP and DRNL::erCERULEAN promoter fusion lines suggests that the trajectory of this cellular competence starts with DRN activity in the central stem-cell zone and extends locally via DRNL activity into groups of founder cells at the IM or FM periphery. Our data support the scenario that after gene duplication, DRN and DRNL acquired different transcription domains within the shoot meristem, but retained protein function that affects cell cycle progression, either centrally in stem cells or peripherally in primordial founder cells, a finding that is of general relevance for meristem function.

Founder-cell-specific transcription of the DORNRÖSCHEN-LIKE promoter and integration of the auxin response.

Transcription of the DORNRÖSCHEN (DRNL) promoter marks lateral-organ founder cells throughout Arabidopsis development, from cotyledons to flowers or floral organs. In the inflorescence apex, DRNL::GFP depicts incipient floral phyllotaxy, and organs in the four floral whorls are differentially prepatterned: the sepals unidirectionally along an abaxial-adaxial axis, the four petals and two lateral stamens in two putative morphogenetic fields, and the medial stamens subsequently in a ring-shaped domain, before two groups of carpel founder cells are specified. The dynamic DRNL transcription pattern is controlled by three enhancer elements, which redundantly and synergistically control qualitative or quantitative aspects of expression, and differentially integrate the auxin response in Arabidopsis inflorescence and floral meristems. The high sequence conservation of all three enhancer elements among the Brassicaceae is striking, which suggests that densely packed cis-regulatory elements are conserved to recruit multiple transcription factors, including auxin response factors, into higher-order enhanceosome complexes. The spatial organization of the enhancers is also conserved, by a microsynteny that extends beyond the Brassicaceae, which relates to enhancer sharing, as the distal element En1 bidirectionally serves DRNL and the upstream At1g24600 gene; the genes are transcribed in opposite directions and possibly comprise a conserved functional chromatin domain.

Symplesiomorphies in the WUSCHEL clade suggest that the last common ancestor of seed plants contained at least four independent stem cell niches.

Evolutionary studies addressing plant architecture have uncovered several significant dichotomies between lower and higher land plant radiations, which are based on differences in meristem histology and function. Here, we assess the establishment of different stem cell niches during land plant evolution based on genes of the stem cell-promoting WUSCHEL (WUS) clade of the WOX (WUSCHEL-related homeobox) gene family. WOX gene orthology was addressed by phylogenetic analyses of full-length WOX protein sequences and cellular expression pattern studies indicate process homology. Gene amplifications in the WUS clade were present in the last common ancestor (LCA) of extant gymnosperms and angiosperms. Whereas the evolution of complex multicellular shoot and root meristems relates to members in the WUS/WOX5 sub-branch, the evolution of marginal and plate meristems or the vascular cambium is associated with gene duplications that gave rise to WOX3 and WOX4, respectively. A fourth WUS clade member, WOX2, was apparently recruited for apical cell fate specification during early embryogenesis. The evolution and functional interplay of WOX3 and WOX4 possibly promoted a novel mode of leaf development, and evolutionary adaptations in their activities have contributed to the great diversity in shape and architecture of leaves in seed plants.

The invention of WUS-like stem cell-promoting functions in plants predates leptosporangiate ferns.

The growth of land plants depends on stem cell-containing meristems which show major differences in their architecture from basal to higher plant species. In Arabidopsis, the stem cell niches in the shoot and root meristems are promoted by WUSCHEL (WUS) and WOX5, respectively. Both genes are members of a non-ancestral clade of the WUS-related homeobox (WOX) gene family, which is absent in extant bryophytes and lycophytes. Our analyses of five fern species suggest that a single WUS orthologue was present in the last common ancestor (LCA) of leptosporangiate ferns and seed plants. In the extant fern Ceratopteris richardii, the WUS pro-orthologue marks the pluripotent cell fate of immediate descendants of the root apical initial, so-called merophytes, which undergo a series of stereotypic cell divisions and give rise to all cell types of the root except the root cap. The invention of a WUS-like function within the WOX gene family in an ancestor of leptosporangiate ferns and seed plants and its amplification and sub-functionalisation to different stem cell niches might relate to the success of seed plants, especially angiosperms.

Genetic integration of DORNRÖSCHEN and DORNRÖSCHEN-LIKE reveals hierarchical interactions in auxin signalling and patterning of the Arabidopsis apical embryo.

DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) encode AP2-domain transcription factors, which act redundantly in cotyledon organogenesis. A more detailed genetic study now integrates DRN and DRNL into the CUP-SHAPED COTYLEDON (CUC) regulatory network and places DRN and DRNL differentially within the auxin signalling network: DRNL function overlaps with that of PIN-FORMED1, and DRN with PINOID. DRN and DRNL act cell-autonomously and are co-expressed in the early globular embryo, whereas expression patterns diverge during later stages of embryogeny. Both genes synergize to provide essential patterning information in the apical embryo domain, to establish correct CUC, SHOOTMERISTEMLESS and WUSCHEL expression domains, which relates to the patterning of SAM anlagen to a central apical position to create two planes of bilateral symmetry in wild type Arabidopsis thaliana embryos.

DORNRÖSCHEN-LIKE expression marks Arabidopsis floral organ founder cells and precedes auxin response maxima.

Live imaging during floral development revealed that expression of the DORNRÖSCHEN-LIKE (DRNL) gene encoding an AP2-like transcription factor, marks all organ founder cells. Transcription precedes the perception of auxin response maxima as measured by the DR5 reporter and is unaffected in early organogenesis, by mutation of four canonical auxin response elements (AuxREs) in the DRNL promoter. DRNL expression identifies discrete modes of organ initiation in the four floral whorls, from individual or pairs of organ anlagen in the outer whorl of sepals to two morphogenetic fields pre-patterning petals and lateral stamens, or a ring-shaped field giving rise to the medial stamens before carpel primordia are specified. DRNL function only overlaps in the central stem cell zone with that of its paralogue, DORNRÖSCHEN (DRN). drnl mutants are affected in floral organ outgrowth, which functionally interplays with boundary specification as organ fusions are sensitized by loss of CUP-SHAPED COTYLEDON (CUC) gene activity, and synergistic interactions exist with mutants in local auxin biosynthesis and polar transport. DRNL apparently monitors and contributes to cellular decisions in the SAM and thus provides a novel molecular access to the interplay of founder cell specification, organ anlage and organogenesis in the SAM peripheral zone.

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress.

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

Discrete shoot and root stem cell-promoting WUS/WOX5 functions are an evolutionary innovation of angiosperms.

The morphologically diverse bodies of seed plants comprising gymnosperms and angiosperms, which separated some 350 Ma, grow by the activity of meristems containing stem cell niches. In the dicot model Arabidopsis thaliana, these are maintained by the stem cell-promoting functions of WUS and WUSCHEL-related homeobox 5 (WOX5) in the shoot and the root, respectively. Both genes are members of the WOX gene family, which has a monophyletic origin in green algae. The establishment of the WOX gene phylogeny from basal land plants through gymnosperms to basal and higher angiosperms reveals three major branches: a basal clade consisting of WOX13-related genes present in some green algae and throughout all land plant genomes, a second clade containing WOX8/9/11/12 homologues, and a modern clade restricted to seed plants. The analysis of the origin of the modern branch in two basal angiosperms (Amborella trichopoda and Nymphaea jamesoniana) and three gymnosperms (Pinus sylvestris, Ginkgo biloba, and Gnetum gnemon) shows that all members of the modern clade consistently found in monocots and dicots exist at the base of the angiosperm lineage, including WUS and WOX5 orthologues. In contrast, our analyses identify a single WUS/WOX5 homologue in all three gymnosperm genomes, consistent with a monophyletic origin in the last common ancestor of gymnosperms and angiosperms. Phylogenetic data, WUS- and WOX5-specific evolutionary signatures, as well as the expression pattern and stem cell-promoting function of the single gymnosperm WUS/WOX5 pro-orthologue in Arabidopsis indicate a gene duplication event followed by subfunctionalization at the base of angiosperms.

Plant development revolves around axes.

Arabidopsis thaliana has become a paradigm for dicot embryo development, despite its embryology being non-representative of dicots in general. The recent cloning of heterologous genes involved in embryonic development from maize and construction of robust phylogenies has shed light on the conservation of transcription factor function and now facilitates a comparison of maize and Arabidopsis embryogenesis orthology. In this review, we focus on a comparison of expression domains of WUSCHEL HOMEOBOX LIKE (WOX), SHOOTMERISTEMLESS (STM), DORNROESCHEN (DRN) and CUP-SHAPED COTYLEDON (CUC) genes and their role in axialization in both species, showing that despite significantly divergent modes of embryogenesis, most notably in terms of axes and planes of symmetry, there is considerable conservation of function as well as notable differences between maize and Arabidopsis.

Transcription of the WUSCHEL-RELATED HOMEOBOX 4 gene in Arabidopsis thaliana.

Phylogenetic shadowing and chromatin accessibility data suggested that essential regulatory elements are absent in the 2.9 kb immediate upstream region of the published WOX4pro::YFP cambium marker. Inclusion of an additional 6.3 kb of upstream promoter sequence and confocal imaging with different fluorophores in transgenic Arabidopsis lines revealed a much wider cell-type-specific expression pattern in parenchymous cells of the aerial plant body. The previously demonstrated activity of the WOX4pro::YFP marker in the cambium of vascular strands in the young Arabidopsis inflorescence stem depicts only sectors of a circular subcortical layer of parenchymous AtWOX4-positive cells. Transcription starts in subepidermal cells within the inflorescence apex in a phyllotactic pattern and extends into successively branching lateral organs, which are connected via small tube-like domains of AtWOX4-expressing cells with the circular subcortical parenchymal layer that extends basipetally down the stem. AtWOX4 expression is most dynamic in leaves, where promoter activity is observed transiently at the adaxial side of the lamina and remains detectable later in the palisade parenchyma, although at a weaker level than in the vasculature. In the root the extended AtWOX4 promoter is active through the proximal root meristem, i.e. in the quiescent centre (QC) and its surrounding initials, a pattern that is broader than transcription of its stem cell promoting relative AtWOX5 in the QC. Outside the proximal meristem AtWOX4 transcription is observed in upper cell layers of the columella root cap beneath or above within the stele in proto- and metaxylem cells, in a ribbon-type pattern which divides the central cylinder in two equal halves. This xylem-specific expression it the root stele relates to established AtWOX4 activity in xylem parenchyma specificity within vascular bundles of the stem.

The evolution of plant regulatory networks: what Arabidopsis cannot say for itself.

Genetic and molecular analyses in the dicot model plant Arabidopsis thaliana have begun to shed some light on regulatory networks in plants. However, comparisons with other species are necessary to validate networks identified in model species on the evolutionary scale. Many key regulatory proteins are encoded by members of transcription factor gene families. Orthologous genes can be identified by phylogenetic reconstructions based on conserved protein domains and functionally substantiated by gene expression patterns and mutant analyses. Recent comparative analyses of different pathways involved in shoot meristem development reveal not only conservation from basal land plants to angiosperms but also evolutionary freedom for significant adaptations in the course of plant speciation.

Histology versus phylogeny: Viewing plant embryogenesis from an evo-devo perspective.

The goal of evolutionary developmental (evo-devo) biology compares inter-organism developmental processes to infer ancestral relationships and evolutionary adaptations. Frameworks to address macroevolutionary traits such as plant embryogenesis commonly involve two complementary approaches. Historically, focus has been placed on comparative morphology and histology, but more recently, accumulating genome data from diverse taxa have elicited the construction of molecular phylogenies, which aid the identification of gene homologies and orthologies that have been adaptive and that underlie differences in form. Distinguishing between ancestral or derived traits in phyletic or cladistic-driven approaches is challenging, but relates to the broader applicability of existing developmental models such as Arabidopsis thaliana.

Nuclear import of the transcription factor SHOOT MERISTEMLESS depends on heterodimerization with BLH proteins expressed in discrete sub-domains of the shoot apical meristem of Arabidopsis thaliana.

The gene SHOOT MERISTEMLESS (STM) is required for the initiation and the maintenance of the shoot apical meristem (SAM) in Arabidopsis and encodes a MEINOX/three amino acid loop extension (TALE)-HD-type transcription factor. Translational fusions with the green fluorescent protein showed that STM is not nuclear by default. In a yeast two-hybrid screen performed with a meristem-enriched cDNA library, three interacting BLH (Bel1-like homeodomain) transcription factors were identified. According to bimolecular fluorescence complementation, STM is targeted into the nuclear compartment through heterodimerization with BLH partner proteins, which are expressed in distinct SAM domains from the center to the periphery. On a functional level, overexpression experiments in transgenic Arabidopsis plants suggest that individual heterodimers provide distinct contributions. These results contribute to our understanding of the STM transcription factor function in the SAM and also shed new light on the evolution of the TALE-HD super gene family in animal and plant lineages.