In plants, the shoot apical meristem (SAM) serves as a reservoir of pluripotent stem cells from which all above ground organs originate. To sustain proper growth, the SAM must maintain homeostasis between the self-renewal of pluripotent stem cells and cell recruitment for lateral organ formation. At the core of the network that regulates this homeostasis in Arabidopsis are the WUSCHEL (WUS) transcription factor specifying stem cell fate and the CLAVATA (CLV) ligand-receptor system limiting WUS expression. In this study, we identified the ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection-microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

Growth and development of the Ceratopteris hermaphroditic gametophytes are dependent on cell proliferation in the marginal meristem, which when destroyed will regenerate at a new location on the body margin. We established a laser ablation method to destroy a single initial cell in the meristem. Ablation caused the cessation of cell proliferation accompanied by the disappearance of the expression of an auxin synthesis gene (CrTAA2) and a cell proliferation marker gene (CrWOXB). New meristem regeneration occurred within a predictable distance from the original two days post-ablation, signified by cell proliferation and the expression of CrTAA2. Treatment with the naturally occurring auxin indole-3-acetic acid (IAA), synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), or the transport inhibitor naphthylphthalamic acid (NPA) altered positioning of the original marginal meristem toward the apex of the gametophyte. IAA altered positioning of the regenerated meristem after damaging the original meristem. A model of auxin involvement in the positioning of the marginal meristem in Ceratopteris is presented to encompass these results.

Phyllotaxis and vein formation are among the most conspicuous patterning processes in plants. The expression and polarization of the auxin efflux carrier PIN1 is the earliest marker for both processes, with mathematical models indicating that PIN1 can respond to auxin gradients and/or auxin flux. Here, we use cell-layer-specific PIN1 knockouts and partial complementation of auxin transport mutants to examine the interaction between phyllotactic patterning, which occurs primarily in the L1 surface layer of the meristem, and midvein specification in the inner tissues. We show that PIN1 expression in the L1 is sufficient for correct organ positioning, as long as the L1-specific influx carriers are present. Thus, differentiation of inner tissues can proceed without PIN1 or any of the known polar transporters. On theoretical grounds, we suggest that canalization of auxin flux between an auxin source and an auxin sink may involve facilitated diffusion rather than polar transport.

The stem cell niche and the size of the root meristem in plants are maintained by intercellular interactions and signalling networks involving a peptide hormone, root meristem growth factor 1 (RGF1)1. Understanding how RGF1 regulates the development of the root meristem is essential for understanding stem cell function. Although five receptors for RGF1 have been identified2-4, the downstream signalling mechanism remains unknown. Here we report a series of signalling events that follow RGF1 activity. We find that the RGF1-receptor pathway controls the distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a previously uncharacterized transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), that has a central role in mediating RGF1 signalling. Manipulating RITF1 expression leads to the redistribution of ROS along the root developmental zones. Changes in ROS distribution in turn enhance the stability of the PLETHORA2 protein, a master regulator of root stem cells. Our results thus clearly depict a signalling cascade that is initiated by RGF1, linking this peptide to mechanisms that regulate ROS.

We isolated flattened shoot meristem (fsm) mutants in rice that showed defective seedling growth and died in the vegetative phase. Since most fsm plants had flat and small shoot apical meristems (SAMs), we suggest that FSM is required for proper SAM maintenance. FSM encodes a putative ortholog of Arabidopsis FASCIATA1 (FAS1) that corresponds to the p150 subunit of chromatin assembly factor-1 (CAF-1). FSM is expressed patchily in tissues with actively dividing cells, suggesting a tight association of FSM with specific cell-cycle phases. Double-target in situ hybridization counterstained with cell-cycle marker genes revealed that FSM is expressed mainly in the G(1) phase. In fsm, expressions of the two marker genes representing S- and G(2)- to M-phases were enhanced in SAM, despite a reduced number of cells in SAM, suggesting that S- and G(2)-phases are prolonged in fsm. In addition, developmental events in fsm leaves took place at the proper time, indicating that the temporal regulation of development occurs independently of the cell-cycle period. In contrast to the fasciated phenotype of Arabidopsis fas1, fsm showed size reduction of SAM. The opposite phenotypes between fsm and fas1 indicate that the SAM maintenance is regulated differently between rice and Arabidopsis.

Roots and shoots of plant bodies develop from meristems-cell populations that self-renew and produce cells that undergo differentiation-located at the apices of axes [1].The oldest preserved root apices in which cellular anatomy can be imaged are found in nodules of permineralized fossil soils called coal balls [2], which formed in the Carboniferous coal swamp forests over 300 million years ago [3-9]. However, no fossil root apices described to date were actively growing at the time of preservation [3-10]. Because the cellular organization of meristems changes when root growth stops, it has been impossible to compare cellular dynamics as stem cells transition to differentiated cells in extinct and extant taxa [11]. We predicted that meristems of actively growing roots would be preserved in coal balls. Here we report the discovery of the first fossilized remains of an actively growing root meristem from permineralized Carboniferous soil with detail of the stem cells and differentiating cells preserved. The cellular organization of the meristem is unique. The position of the Körper-Kappe boundary, discrete root cap, and presence of many anticlinal cell divisions within a broad promeristem distinguish it from all other known root meristems. This discovery is important because it demonstrates that the same general cellular dynamics are conserved between the oldest extinct and extant root meristems. However, its unique cellular organization demonstrates that extant root meristem organization and development represents only a subset of the diversity that has existed since roots first evolved.

Chromatin structure requires proper modulation in face of transcriptional reprogramming in the context of organism growth and development. Chromatin-remodeling factors and histone chaperones are considered to intrinsically possess abilities to remodel chromatin structure in single or in combination. Our previous study revealed the functional synergy between the Arabidopsis chromatin-remodeling factor INOSITOL AUXOTROPHY 80 (AtINO80) and the histone chaperone NAP1-RELATED PROTEIN 1 (NRP1) and NRP2 in somatic homologous recombination, one crucial pathway involved in repairing DNA double strand breaks. Here, we report genetic interplay between AtINO80 and NRP1/2 in regulating inflorescence meristem (IM) and root apical meristem (RAM) activities. The triple mutant atino80-5 m56-1 depleting of both AtINO80 (atino80-5) and NRP1/2 (m56-1) showed abnormal positioning pattern of floral primordia and enlargement of IM size. Higher mRNA levels of several genes involved in auxin pathway (e.g., PIN1, FIL) were found in the inflorescences of the triple mutant but barely in those of the single mutant atino80-5 or the double mutant m56-1. In particular, the depletion of AtINO80 and NRP1/2 decreased histone H3 levels within the chromatin regions of PIN1, which encodes an important auxin efflux carrier. Moreover, the triple mutant displayed a severe short-root phenotype with higher sensitivity to auxin transport inhibitor NPA. Unusual high level of cell death was also found in triple mutant root tips, accompanied by double-strand break damages revealed by γ-H2A.X loci and cortex cell enlargement. Collectively, our study provides novel insight into the functional coordination of the two epigenetic factors AtINO80 and NRP1/2 in apical meristems during plant growth and development.

The shoot apical meristem (SAM) is responsible for the development of all the above-ground parts of a plant. Our understanding of the SAM at the molecular level is incomplete. This study investigates the gene expression repertoire of SAMs in the garden pea (Pisum sativum). To this end, 10 346 EST sequences representing 7610 unique genes were generated from SAM cDNA libraries. These sequences, together with previously reported pea ESTs, were used to construct a 12K oligonucleotide array to identify genes with differential SAM expression, as compared to axillary meristems, root apical meristems, or non-meristematic tissues. A number of genes were identified, predominantly expressed in specific cell layers or domains of the SAM and thus are likely components of the gene networks involved in stem cell maintenance or the initiation of lateral organs. Further in situ hybridization analysis confirmed the spatial localization of some of these genes within the SAM. Our data also indicate the diversification of some gene expression patterns and hence functions in legume crop plants. A number of transcripts highly expressed in all three meristems have also been uncovered and these candidates may provide valuable insight into molecular networks that underpin the maintenance of meristematic functionality.

A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.

Plant root growth is enabled by root meristems that harbor the stem cell niches as a source of progenitors for the different root tissues. Understanding the root development of diverse plant species is important to be able to control root growth in order to gain better performances of crop plants. In this study, we analyzed the root meristem of the fourth most abundant crop plant, barley (Hordeum vulgare). Cell division studies revealed that the barley stem cell niche comprises a Quiescent Center (QC) of around 30 cells with low mitotic activity. The surrounding stem cells contribute to root growth through the production of new cells that are displaced from the meristem, elongate and differentiate into specialized root tissues. The distal stem cells produce the root cap and lateral root cap cells, while cells lateral to the QC generate the epidermis, as it is typical for monocots. Endodermis and inner cortex are derived from one common initial lateral to the QC, while the outer cortex cell layers are derived from a distinct stem cell. In rice and Arabidopsis, meristem homeostasis is achieved through feedback signaling from differentiated cells involving peptides of the CLE family. Application of synthetic CLE40 orthologous peptide from barley promotes meristem cell differentiation, similar to rice and Arabidopsis. However, in contrast to Arabidopsis, the columella stem cells do not respond to the CLE40 peptide, indicating that distinct mechanisms control columella cell fate in monocot and dicot plants.

Co-ordination of auxin and cytokinin activities determines root meristem size during post-embryonic development. Calcineurin B-like proteins (CBLs) and their interacting protein kinases (CIPKs) constitute signaling modules that relay calcium signals. Here we report that CIPK25 is involved in regulating the root meristem size. Arabidopsis plants lacking CIPK25 expression displayed a short root phenotype and a slower root growth rate with fewer meristem cells. This phenotype was rescued by restoration of CIPK25 expression. CIPK25 interacted with CBL4 and -5, and displayed strong gene expression in the flower and root, except in the cell proliferation domain in the root apical meristem. Its expression in the root was positively and negatively regulated by auxin and cytokinin, respectively. The cipk25 T-DNA insertion line was compromised in auxin transport and auxin-responsive promoter activity. The cipk25 mutant line showed altered expression of auxin efflux carriers (PIN1 and PIN2) and an Aux/IAA family gene SHY2. Decreased PIN1 and PIN2 expression in the cipk25 mutant line was completely restored when combined with a SHY2 loss-of-function mutation, resulting in recovery of root growth. SHY2 and PIN1 expression was partially regulated by cytokinin even in the absence of CIPK25, suggesting a CIPK25-independent cytokinin signaling pathway(s). Our results revealed that CIPK25 plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development.

DELLA proteins associate with transcription factors to control plant growth in response to gibberellin 1 . Semi-dwarf DELLA mutants with improved harvest index and decreased lodging greatly improved global food security during the 'green revolution' in the 1960-1970s 2 . However, DELLA mutants are pleiotropic and the developmental basis for their effects on plant architecture remains poorly understood. Here, we show that DELLA proteins have genetically separable roles in controlling stem growth and the size of the inflorescence meristem, where flowers initiate. Quantitative three-dimensional image analysis, combined with a genome-wide screen for DELLA-bound loci in the inflorescence tip, revealed that DELLAs limit meristem size in Arabidopsis by directly upregulating the cell-cycle inhibitor KRP2 in the underlying rib meristem, without affecting the canonical WUSCHEL-CLAVATA meristem size regulators 3 . Mutation of KRP2 in a DELLA semi-dwarf background restored meristem size, but not stem growth, and accelerated flower production. In barley, secondary mutations in the DELLA gain-of-function mutant Sln1d 4 also uncoupled meristem and inflorescence size from plant height. Our work reveals an unexpected and conserved role for DELLA genes in controlling shoot meristem function and suggests how dissection of pleiotropic DELLA functions could unlock further yield gains in semi-dwarf mutants.

Plant growth is driven by cell proliferation and elongation. The hormone gibberellin (GA) regulates Arabidopsis root growth by controlling cell elongation, but it is currently unknown whether GA also controls root cell proliferation. Here we show that GA biosynthetic mutants are unable to increase their cell production rate and meristem size after germination. GA signals the degradation of the DELLA growth repressor proteins GAI and RGA, promoting root cell production. Targeting the expression of gai (a non-GA-degradable mutant form of GAI) in the root meristem disrupts cell proliferation. Moreover, expressing gai in dividing endodermal cells was sufficient to block root meristem enlargement. We report a novel function for GA regulating cell proliferation where this signal acts by removing DELLA in a subset of, rather than all, meristem cells. We suggest that the GA-regulated rate of expansion of dividing endodermal cells dictates the equivalent rate in other root tissues. Cells must double in size prior to dividing but cannot do so independently, because they are physically restrained by adjacent tissues with which they share cell walls. Our study highlights the importance of probing regulatory mechanisms linking molecular- and cellular-scale processes with tissue and organ growth responses.

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.

The shoot apical meristem (SAM) ensures continuous plant growth and organogenesis. In LD 30 °C, plants lacking AtFTSH4, an ATP-dependent mitochondrial protease that counteracts accumulation of internal oxidative stress, exhibit a puzzling phenotype of premature SAM termination. We aimed to elucidate the underlying cellular and molecular processes that link AtFTSH4 with SAM arrest. We studied AtFTSH4 expression, internal oxidative stress accumulation, and SAM morphology. Directly in the SAM we analysed H2O2 accumulation, mitochondria behaviour, and identity of stem cells using WUS/CLV3 expression. AtFTSH4 was expressed in proliferating tissues, particularly during the reproductive phase. In the mutant, SAM, in which internal oxidative stress accumulates predominantly at 30 °C, lost its meristematic fate. This process was progressive and stage-specific. Premature meristem termination was associated with an expansion in SAM area, where mitochondria lost their functionality. All these effects destabilised the identity of the stem cells. SAM termination in ftsh4 mutants is caused both by internal oxidative stress accumulation with time/age and by the tissue-specific role of AtFTSH4 around the flowering transition. Maintaining mitochondria functionality within the SAM, dependent on AtFTSH4, is vital to preserving stem cell activity throughout development.

The Asteraceae plant family is characterized by inflorescences, called flower heads or capitula that may combine hundreds of individual florets into a single flower-like structure. The florets are arranged in a regular phyllotactic pattern with Fibonacci numbers of left- and right-winding spirals. Such a pattern may be disrupted due to physical constraints or by wounding occurring during the early meristem development. Recovery from wounding re-establishes patterning although the mechanisms have remained elusive. In this study, we applied Gerbera hybrida as a model system and established methods to conduct wounding experiments either with syringe needles or using laser ablation combined with live imaging of head meristems. By revisiting the historical experiments in sunflower, we conducted wounding to transgenic auxin reporter lines of gerbera and followed the recovery of cellular growth and meristem patterning. We show that wounding disrupted the expression of the gerbera CLAVATA3 (GhCLV3) gene that marks the undifferentiated meristematic region and led to de novo re-initiation of patterning at the wound margin. During the recovery growth, three to five layers of elongated cells showing periclinal cell division planes and lacking auxin signal were formed at the wound rim. DR5 auxin signal was shown to localize and form regularly spaced maxima in a distance from the wound rim. Consequently, spiral pattern of contact parastichies was re-established by stacking of new auxin maxima on top of the previous ones. The developed methods facilitate future studies on understanding the molecular mechanisms of de novo patterning of meristems.

The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labeling, and transcriptome profiling of the SAM to provide a spatiotemporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways.

Leaves are flat determinate organs derived from indeterminate shoot apical meristems. The presence of a specific leaf meristem is debated, as anatomical features typical of meristems are not present in leaves. Here we demonstrate that multiple NGATHA (NGA) and CINCINNATA-class-TCP (CIN-TCP) transcription factors act redundantly, shortly after leaf initiation, to gradually restrict the activity of a leaf meristem in Arabidopsis thaliana to marginal and basal domains, and that their absence confers persistent marginal growth to leaves, cotyledons and floral organs. Following primordia initiation, the restriction of the broadly acting leaf meristem to the margins is mediated by the juxtaposition of adaxial and abaxial domains and maintained by WOX homeobox transcription factors, whereas other marginal elaboration genes are dispensable for its maintenance. This genetic framework parallels the morphogenetic program of shoot apical meristems and may represent a relic of an ancestral shoot system from which seed plant leaves evolved.