Genetics of Shoot Meristem and Shoot Regeneration.

Plants exhibit remarkable lineage plasticity, allowing them to regenerate organs that differ from their respective origins. Such developmental plasticity is dependent on the activity of pluripotent founder cells or stem cells residing in meristems. At the shoot apical meristem (SAM), the constant flow of cells requires continuing cell specification governed by a complex genetic network, with the WUSCHEL transcription factor and phytohormone cytokinin at its core. In this review, I discuss some intriguing recent discoveries that expose new principles and mechanisms of patterning and cell specification acting both at the SAM and prior to meristem organogenesis during shoot regeneration. I also highlight unanswered questions and future challenges in the study of SAM and meristem regeneration. Finally, I put forward a model describing stochastic events mediated by epigenetic factors to explain how the gene regulatory network might be initiated at the onset of shoot regeneration.

An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

PLETHORA transcription factors promote early embryo development through induction of meristematic potential.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential.

An updated model of shoot apical meristem regulation by ERECTA family and CLAVATA3 signaling pathways in Arabidopsis.

The shoot apical meristem (SAM) gives rise to the aboveground organs of plants. The size of the SAM is relatively constant due to the balance between stem cell replenishment and cell recruitment into new organs. In angiosperms, the transcription factor WUSCHEL (WUS) promotes stem cell proliferation in the central zone of the SAM. WUS forms a negative feedback loop with a signaling pathway activated by CLAVATA3 (CLV3). In the periphery of the SAM, the ERECTA family receptors (ERfs) constrain WUS and CLV3 expression. Here, we show that four ligands of ERfs redundantly inhibit the expression of these two genes. Transcriptome analysis confirmed that WUS and CLV3 are the main targets of ERf signaling and uncovered new ones. Analysis of promoter reporters indicated that the WUS expression domain mostly overlaps with the CLV3 domain and does not shift along the apical-basal axis in clv3 mutants. Our three-dimensional mathematical model captured gene expression distributions at the single-cell level under various perturbed conditions. Based on our findings, CLV3 regulates cellular levels of WUS mostly through autocrine signaling, and ERfs regulate the spatial expression of WUS, preventing its encroachment into the peripheral zone.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

The landscape of transcription factor promoter activity during vegetative development in Marchantia.

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterizing the transcriptional activity of TF genes in space and time is a critical step toward understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analyzed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem-cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics.

Antagonistic regulation of target genes by the SISTER OF TM3-JOINTLESS2 complex in tomato inflorescence branching.

Inflorescence branch number is a yield-related trait controlled by cell fate determination in meristems. Two MADS-box transcription factors (TFs)-SISTER OF TM3 (STM3) and JOINTLESS 2 (J2)-have opposing regulatory roles in inflorescence branching. However, the mechanisms underlying their regulatory functions in inflorescence determinacy remain unclear. Here, we characterized the functions of these TFs in tomato (Solanum lycopersicum) floral meristem and inflorescence meristem (IM) through chromatin immunoprecipitation and sequencing analysis of their genome-wide occupancy. STM3 and J2 activate or repress the transcription of a set of common putative target genes, respectively, through recognition and binding to CArG box motifs. FRUITFULL1 (FUL1) is a shared putative target of STM3 and J2 and these TFs antagonistically regulate FUL1 in inflorescence branching. Moreover, STM3 physically interacts with J2 to mediate its cytosolic redistribution and restricts J2 repressor activity by reducing its binding to target genes. Conversely, J2 limits STM3 regulation of target genes by transcriptional repression of the STM3 promoter and reducing STM3-binding activity. Our study thus reveals an antagonistic regulatory relationship in which STM3 and J2 control tomato IM determinacy and branch number.

Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Anisotropic cell growth at the leaf base promotes age-related changes in leaf shape in Arabidopsis thaliana.

Plants undergo extended morphogenesis. The shoot apical meristem (SAM) allows for reiterative development and the formation of new structures throughout the life of the plant. Intriguingly, the SAM produces morphologically different leaves in an age-dependent manner, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the SAM produces small orbicular leaves in the juvenile phase, but gives rise to large elliptical leaves in the adult phase. Previous studies have established that a developmental decline of microRNA156 (miR156) is necessary and sufficient to trigger this leaf shape switch, although the underlying mechanism is poorly understood. Here we show that the gradual increase in miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE transcription factors with age promotes cell growth anisotropy in the abaxial epidermis at the base of the leaf blade, evident by the formation of elongated giant cells. Time-lapse imaging and developmental genetics further revealed that the establishment of adult leaf shape is tightly associated with the longitudinal cell expansion of giant cells, accompanied by a prolonged cell proliferation phase in their vicinity. Our results thus provide a plausible cellular mechanism for heteroblasty in Arabidopsis, and contribute to our understanding of anisotropic growth in plants.

Plant and animal stem cells: similar yet different.

The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into new tissues. Plant stem cell niches are located within the meristems, which are organized structures that are responsible for most post-embryonic development. The continuous organ production that is characteristic of plant growth requires a robust regulatory network to keep the balance between pluripotent stem cells and differentiating progeny. Components of this network have now been elucidated and provide a unique opportunity for comparing strategies that were developed in the animal and plant kingdoms, which underlie the logic of stem cell behaviour.

RGF1 controls root meristem size through ROS signalling.

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.

The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM.

OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice.

Rice axillary meristems (AMs) are essential to the formation of tillers and panicle branches in rice, and therefore play a determining role in rice yield. However, the regulation of inflorescence AM development in rice remains elusive. In this study, we identified no spikelet 1-Dominant (nsp1-D), a sparse spikelet mutant, with obvious reduction of panicle branches and spikelets. Inflorescence AM deficiency in nsp1-D could be ascribed to the overexpression of OsbHLH069. OsbHLH069 functions redundantly with OsbHLH067 and OsbHLH068 in panicle AM formation. The Osbhlh067 Osbhlh068 Osbhlh069 triple mutant had smaller panicles and fewer branches and spikelets. OsbHLH067, OsbHLH068, and OsbHLH069 were preferentially expressed in the developing inflorescence AMs and their proteins could physically interact with LAX1. Both nsp1-D and lax1 showed sparse panicles. Transcriptomic data indicated that OsbHLH067/068/069 may be involved in the metabolic pathway during panicle AM formation. Quantitative RT-PCR results demonstrated that the expression of genes involved in meristem development and starch/sucrose metabolism was down-regulated in the triple mutant. Collectively, our study demonstrates that OsbHLH067, OsbHLH068, and OsbHLH069 have redundant functions in regulating the formation of inflorescence AMs during panicle development in rice.

Single-cell transcriptomic analysis of pea shoot development and cell-type-specific responses to boron deficiency.

Understanding how nutrient stress impacts plant growth is fundamentally important to the development of approaches to improve crop production under nutrient limitation. Here we applied single-cell RNA sequencing to shoot apices of Pisum sativum grown under boron (B) deficiency. We identified up to 15 cell clusters based on the clustering of gene expression profiles and verified cell identity with cell-type-specific marker gene expression. Different cell types responded differently to B deficiency. Specifically, the expression of photosynthetic genes in mesophyll cells (MCs) was down-regulated by B deficiency, consistent with impaired photosynthetic rate. Furthermore, the down-regulation of stomatal development genes in guard cells, including homologs of MUTE and TOO MANY MOUTHS, correlated with a decrease in stomatal density under B deficiency. We also constructed the developmental trajectory of the shoot apical meristem (SAM) cells and a transcription factor interaction network. The developmental progression of SAM to MC was characterized by up-regulation of genes encoding histones and chromatin assembly and remodeling proteins including homologs of FASCIATA1 (FAS1) and SWITCH DEFECTIVE/SUCROSE NON-FERMENTABLE (SWI/SNF) complex. However, B deficiency suppressed their expression, which helps to explain impaired SAM development under B deficiency. These results represent a major advance over bulk-tissue RNA-seq analysis in which cell-type-specific responses are lost and hence important physiological responses to B deficiency are missed. The reported findings reveal strategies by which plants adapt to B deficiency thus offering breeders a set of specific targets for genetic improvement. The reported approach and resources have potential applications well beyond P. sativum species and could be applied to various legumes to improve their adaptability to multiple nutrient or abiotic stresses.

STAMENLESS1 activates SUPERWOMAN 1 and FLORAL ORGAN NUMBER 1 to control floral organ identities and meristem fate in rice.

Floral patterns are unique to rice and contribute significantly to its reproductive success. SL1 encodes a C2H2 transcription factor that plays a critical role in flower development in rice, but the molecular mechanism regulated by it remains poorly understood. Here, we describe interactions of the SL1 with floral homeotic genes, SPW1, and DL in specifying floral organ identities and floral meristem fate. First, the sl1 spw1 double mutant exhibited a stamen-to-pistil transition similar to that of sl1, spw1, suggesting that SL1 and SPW1 may located in the same pathway regulating stamen development. Expression analysis revealed that SL1 is located upstream of SPW1 to maintain its high level of expression and that SPW1, in turn, activates the B-class genes OsMADS2 and OsMADS4 to suppress DL expression indirectly. Secondly, sl1 dl displayed a severe loss of floral meristem determinacy and produced amorphous tissues in the third/fourth whorl. Expression analysis revealed that the meristem identity gene OSH1 was ectopically expressed in sl1 dl in the fourth whorl, suggesting that SL1 and DL synergistically terminate the floral meristem fate. Another meristem identity gene, FON1, was significantly decreased in expression in sl1 background mutants, suggesting that SL1 may directly activate its expression to regulate floral meristem fate. Finally, molecular evidence supported the direct genomic binding of SL1 to SPW1 and FON1 and the subsequent activation of their expression. In conclusion, we present a model to illustrate the roles of SL1, SPW1, and DL in floral organ specification and regulation of floral meristem fate in rice.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

Single-cell genomics revolutionizes plant development studies across scales.

Understanding the development of tissues, organs and entire organisms through the lens of single-cell genomics has revolutionized developmental biology. Although single-cell transcriptomics has been pioneered in animal systems, from an experimental perspective, plant development holds some distinct advantages: cells do not migrate in relation to one another, and new organ formation (of leaves, roots, flowers, etc.) continues post-embryonically from persistent stem cell populations known as meristems. For a time, plant studies lagged behind animal or cell culture-based, single-cell approaches, largely owing to the difficulty in dissociating plant cells from their rigid cell walls. Recent intensive development of single-cell and single-nucleus isolation techniques across plant species has opened up a wide range of experimental approaches. This has produced a rapidly expanding diversity of information across tissue types and species, concomitant with the creative development of methods. In this brief Spotlight, we highlight some of the technical developments and how they have led to profiling single-cell genomics in various plant organs. We also emphasize the contribution of single-cell genomics in revealing developmental trajectories among different cell types within plant organs. Furthermore, we present efforts toward comparative analysis of tissues and organs at a single-cell level. Single-cell genomics is beginning to generate comprehensive information relating to how plant organs emerge from stem cell populations.

A mutation in THREONINE SYNTHASE 1 uncouples proliferation and transition domains of the root apical meristem: experimental evidence and in silico proposed mechanism.

A continuum from stem to transit-amplifying to a differentiated cell state is a common theme in multicellular organisms. In the plant root apical meristem (RAM), transit-amplifying cells are organized into two domains: cells from the proliferation domain (PD) are displaced to the transition domain (TD), suggesting that both domains are necessarily coupled. Here, we show that in the Arabidopsis thaliana mto2-2 mutant, in which threonine (Thr) synthesis is affected, the RAM lacks the PD. Through a combination of cell length profile analysis, mathematical modeling and molecular markers, we establish that the PD and TD can be uncoupled. Remarkably, although the RAM of mto2-2 is represented solely by the TD, the known factors of RAM maintenance and auxin signaling are expressed in the mutant. Mathematical modeling predicts that the stem cell niche depends on Thr metabolism and that, when disturbed, the normal continuum of cell states becomes aborted.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.