Plant cell size: Links to cell cycle, differentiation and ploidy.

Cell size affects many processes, including exchange of nutrients and external signals, cell division and tissue mechanics. Across eukaryotes, cells have evolved mechanisms that assess their own size to inform processes such as cell cycle progression or gene expression. Here, we review recent progress in understanding plant cell size regulation and its implications, relating these findings to work in other eukaryotes. Highlights include use of DNA contents as reference point to control the cell cycle in shoot meristems, a size-dependent cell fate decision during stomatal development and insights into the interconnection between ploidy, cell size and cell wall mechanics.

Gibberellin and miRNA156-targeted SlSBP genes synergistically regulate tomato floral meristem determinacy and ovary patterning.

Many developmental processes associated with fruit development occur at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GAs) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, in tomato (Solanum lycopersicum), we show that GA and miR156-targeted SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL or SBP) genes interact in the tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low expression levels of miR156-silenced SBP genes, resulted in enlarged FMs, ovary indeterminacy and fruits with increased locule number. Conversely, low GA responses reduced indeterminacy and locule number, and overexpression of a S. lycopersicum (Sl)SBP15 allele that is miR156 resistant (rSBP15) reduced FM size and locule number. GA responses were partially required for the defects observed in 156OE and rSBP15 fruits. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBP genes, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to fine-tune aspects of fruit development that have been important for tomato domestication.

A Phloem-Expressed PECTATE LYASE-LIKE Gene Promotes Cambium and Xylem Development.

The plant vasculature plays essential roles in the transport of water and nutrients and is composed of xylem and phloem, both of which originate from undifferentiated cells found in the cambium. Development of the different vascular tissues is coordinated by hormonal and peptide signals and culminates in extensive cell wall modifications. Pectins are key cell wall components that are modified during cell growth and differentiation, and pectin fragments function as signals in defence and cell wall integrity pathways, although their role as developmental signals remains tentative. Here, we show that the pectin lyase-like gene PLL12 is required for growth of the vascular bundles in the Arabidopsis inflorescence stem. Although PLL12 was expressed primarily in the phloem, it also affected cambium and xylem growth. Surprisingly, PLL12 overexpression induced ectopic cambium and xylem differentiation in the inflorescence apex and inhibited development of the leaf vasculature. Our results raise the possibility that a cell wall-derived signal produced by PLL12 in the phloem regulates cambium and xylem development.

Cycling in a crowd: Coordination of plant cell division, growth, and cell fate.

The reiterative organogenesis that drives plant growth relies on the constant production of new cells, which remain encased by interconnected cell walls. For these reasons, plant morphogenesis strictly depends on the rate and orientation of both cell division and cell growth. Important progress has been made in recent years in understanding how cell cycle progression and the orientation of cell divisions are coordinated with cell and organ growth and with the acquisition of specialized cell fates. We review basic concepts and players in plant cell cycle and division, and then focus on their links to growth-related cues, such as metabolic state, cell size, cell geometry, and cell mechanics, and on how cell cycle progression and cell division are linked to specific cell fates. The retinoblastoma pathway has emerged as a major player in the coordination of the cell cycle with both growth and cell identity, while microtubule dynamics are central in the coordination of oriented cell divisions. Future challenges include clarifying feedbacks between growth and cell cycle progression, revealing the molecular basis of cell division orientation in response to mechanical and chemical signals, and probing the links between cell fate changes and chromatin dynamics during the cell cycle.

Cell size controlled in plants using DNA content as an internal scale.

How eukaryotic cells assess and maintain sizes specific for their species and cell type remains unclear. We show that in the Arabidopsis shoot stem cell niche, cell size variability caused by asymmetric divisions is corrected by adjusting the growth period before DNA synthesis. KIP-related protein 4 (KRP4) inhibits progression to DNA synthesis and associates with mitotic chromosomes. The F BOX-LIKE 17 (FBL17) protein removes excess KRP4. Consequently, daughter cells are born with comparable amounts of KRP4. Inhibitor dilution models predicted that KRP4 inherited through chromatin would robustly regulate size, whereas inheritance of excess free KRP4 would disrupt size homeostasis, as confirmed by mutant analyses. We propose that a cell cycle regulator, stabilized by association with mitotic chromosomes, reads DNA content as a cell size-independent scale.

ARABIDOPSIS THALIANA HOMEOBOX GENE 1 controls plant architecture by locally restricting environmental responses.

The diversity and environmental plasticity of plant growth results from variations of repetitive modules, such as the basic shoot units made of a leaf, axillary bud, and internode. Internode elongation is regulated both developmentally and in response to environmental conditions, such as light quality, but the integration of internal and environmental signals is poorly understood. Here, we show that the compressed rosette growth habit of Arabidopsis is maintained by the convergent activities of the organ boundary gene ARABIDOPSIS THALIANA HOMEOBOX GENE 1 (ATH1) and of the gibberellin-signaling DELLA genes. Combined loss of ATH1 and DELLA function activated stem development during the vegetative phase and changed the growth habit from rosette to caulescent. Chromatin immunoprecipitation high-throughput sequencing and genetic analysis indicated that ATH1 and the DELLA gene REPRESSOR OF GA1-3 (RGA) converge on the regulation of light responses, including the PHYTOCHROME INTERACTING FACTORS (PIF) pathway, and showed that the ATH1 input is mediated in part by direct activation of BLADE ON PETIOLE (BOP1 and BOP2) genes, whose products destabilize PIF proteins. We conclude that an organ-patterning gene converges with hormone signaling to spatially restrict environmental responses and establish a widespread type of plant architecture.

A Self-Activation Loop Maintains Meristematic Cell Fate for Branching.

In plants and animals, self-renewing stem cell populations play fundamental roles in many developmental contexts. Plants differ from most animals in their retained ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems. In seed plants, branching is achieved by stem-cell-containing axillary meristems, which are initiated from a leaf axil meristematic cell population originally detached from the shoot apical meristem. It remains unclear how the meristematic cell fate is maintained. Here, we show that ARABIDOPSISTHALIANAHOMEOBOXGENE1 (ATH1) maintains the meristem marker gene SHOOT MERISTEMLESS (STM) expression in the leaf axil to enable meristematic cell fate maintenance. Furthermore, ATH1 protein interacts with STM protein to form a STM self-activation loop. Genetic and biochemical data suggest that ATH1 anchors STM to activate STM as well as other axillary meristem regulatory genes. This auto-regulation allows the STM locus to remain epigenetically active. Taken together, our findings provide a striking example of a self-activation loop that maintains the flexibility required for stem cell niche re-establishment during organogenesis.

Cell Size Control in Plants.

The genetic control of the characteristic cell sizes of different species and tissues is a long-standing enigma. Plants are convenient for studying this question in a multicellular context, as their cells do not move and are easily tracked and measured from organ initiation in the meristems to subsequent morphogenesis and differentiation. In this article, we discuss cell size control in plants compared with other organisms. As seen from yeast cells to mammalian cells, size homeostasis is maintained cell autonomously in the shoot meristem. In developing organs, vacuolization contributes to cell size heterogeneity and may resolve conflicts between growth control at the cellular and organ levels. Molecular mechanisms for cell size control have implications for how cell size responds to changes in ploidy, which are particularly important in plant development and evolution. We also discuss comparatively the functional consequences of cell size and their potential repercussions at higher scales, including genome evolution.

Spatiotemporal coordination of cell division and growth during organ morphogenesis.

A developing plant organ exhibits complex spatiotemporal patterns of growth, cell division, cell size, cell shape, and organ shape. Explaining these patterns presents a challenge because of their dynamics and cross-correlations, which can make it difficult to disentangle causes from effects. To address these problems, we used live imaging to determine the spatiotemporal patterns of leaf growth and division in different genetic and tissue contexts. In the simplifying background of the speechless (spch) mutant, which lacks stomatal lineages, the epidermal cell layer exhibits defined patterns of division, cell size, cell shape, and growth along the proximodistal and mediolateral axes. The patterns and correlations are distinctive from those observed in the connected subepidermal layer and also different from the epidermal layer of wild type. Through computational modelling we show that the results can be accounted for by a dual control model in which spatiotemporal control operates on both growth and cell division, with cross-connections between them. The interactions between resulting growth and division patterns lead to a dynamic distributions of cell sizes and shapes within a deforming leaf. By modulating parameters of the model, we illustrate how phenotypes with correlated changes in cell size, cell number, and organ size may be generated. The model thus provides an integrated view of growth and division that can act as a framework for further experimental study.

The pillars of land plants: new insights into stem development.

In spite of its central importance in evolution, plant architecture and crop improvement, stem development remains poorly understood relative to other plant organs. Here, we summarise current knowledge of stem ontogenesis and its regulation, including insights from new image analysis and biophysical approaches. The stem initiates in the rib zone (RZ) of the shoot apical meristem, under transcriptional control by DELLA and BLH proteins. Links have emerged between these regulators and cell proliferation, patterning and oriented growth in the RZ. During subsequent internode elongation, cell wall properties and mechanics have been analysed in detail, revealing pectin modification as a prominent control point. Recent work has also highlighted signalling to coordinate growth of stem tissues with different mechanical properties.

DELLA genes restrict inflorescence meristem function independently of plant height.

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.

Coordination of plant cell growth and division: collective control or mutual agreement?

Plant tissue growth requires the interdependent cellular processes of cytoplasmic growth, cell wall extension and cell division, but the feedbacks that link these processes are poorly understood. Recent papers have revealed developmentally regulated coupling between plant cell growth and progression through both mitotic cycles and endocycles. Modeling has given insight into the effects of cell geometry and tissue mechanics on the orientation of cell divisions. Developmental inputs by auxin have been highlighted in the control of cell turgor, vacuole function and the microtubule dynamics that underlies oriented growth and division. Overall, recent work emphasizes growth and proliferation as processes that are negotiated within and between cells, rather than imposed on cells across tissues.

Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis.

The origin of the stem is a major but poorly understood aspect of plant development, partly because the stem initiates in a relatively inaccessible region of the shoot apical meristem called the rib zone (RZ). We developed quantitative 3D image analysis and clonal analysis tools, which revealed that the Arabidopsis homeodomain protein REPLUMLESS (RPL) establishes distinct patterns of oriented cell division and growth in the central and peripheral regions of the RZ. A genome-wide screen for target genes connected RPL directly to many of the key shoot development pathways, including the development of organ boundaries; accordingly, mutation of the organ boundary gene LIGHT-SENSITIVE HYPOCOTYL 4 restored RZ function and stem growth in the rpl mutant. Our work opens the way to study a developmental process of importance to crop improvement and highlights how apparently simple changes in 3D organ growth can reflect more complex internal changes in oriented cell activities.

Two-Step Regulation of a Meristematic Cell Population Acting in Shoot Branching in Arabidopsis.

Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.

Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis.

How cells regulate their dimensions is a long-standing question. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle, although there is evidence of cell-autonomous control in cell cultures. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures.

Control of patterning, growth, and differentiation by floral organ identity genes.

In spite of the different morphologies of sepals, petals, stamens, and carpels, all these floral organs are believed to be modified versions of a ground-state organ similar to the leaf. Modifications of the ground-state developmental programme are orchestrated by different combinations of MADS-domain transcription factors encoded by floral organ identity genes. In recent years, much has been revealed about the gene regulatory networks controlled by the floral organ identity genes and about the genetic pathways that control leaf development. This review examines how floral organ identity is connected with the control of morphogenesis and differentiation of shoot organs, focusing on the model species Arabidopsis thaliana. Direct links have emerged between floral organ identity genes and genes involved in abaxial-adaxial patterning, organ boundary formation, tissue growth, and cell differentiation. In parallel, predictive models have been developed to explain how the activity of regulatory genes can be coordinated by intercellular signalling and constrained by tissue mechanics. When combined, these advances provide a unique opportunity for revealing exactly how leaf-like organs have been 'metamorphosed' into floral organs during evolution and showing crucial regulatory points in the generation of plant form.

Arabidopsis JAGGED links floral organ patterning to tissue growth by repressing Kip-related cell cycle inhibitors.

Plant morphogenesis requires coordinated cytoplasmic growth, oriented cell wall extension, and cell cycle progression, but it is debated which of these processes are primary drivers for tissue growth and directly targeted by developmental genes. Here, we used ChIP high-throughput sequencing combined with transcriptome analysis to identify global target genes of the Arabidopsis transcription factor JAGGED (JAG), which promotes growth of the distal region of floral organs. Consistent with the roles of JAG during organ initiation and subsequent distal organ growth, we found that JAG directly repressed genes involved in meristem development, such as CLAVATA1 and HANABA TARANU, and genes involved in the development of the basal region of shoot organs, such as BLADE ON PETIOLE 2 and the GROWTH REGULATORY FACTOR pathway. At the same time, JAG regulated genes involved in tissue polarity, cell wall modification, and cell cycle progression. In particular, JAG directly repressed KIP RELATED PROTEIN 4 (KRP4) and KRP2, which control the transition to the DNA synthesis phase (S-phase) of the cell cycle. The krp2 and krp4 mutations suppressed jag defects in organ growth and in the morphology of petal epidermal cells, showing that the interaction between JAG and KRP genes is functionally relevant. Our work reveals that JAG is a direct mediator between genetic pathways involved in organ patterning and cellular functions required for tissue growth, and it shows that a regulatory gene shapes plant organs by releasing a constraint on S-phase entry.

Interplay between cell growth and cell cycle in plants.

The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.

JAGGED controls Arabidopsis petal growth and shape by interacting with a divergent polarity field.

A flowering plant generates many different organs such as leaves, petals, and stamens, each with a particular function and shape. These types of organ are thought to represent variations on a common underlying developmental program. However, it is unclear how this program is modulated under different selective constraints to generate the diversity of forms observed. Here we address this problem by analysing the development of Arabidopsis petals and comparing the results to models of leaf development. We show that petal development involves a divergent polarity field with growth rates perpendicular to local polarity increasing towards the distal end of the petal. The hypothesis is supported by the observed pattern of clones induced at various stages of development and by analysis of polarity markers, which show a divergent pattern. We also show that JAGGED (JAG) has a key role in promoting distal enhancement of growth rates and influences the extent of the divergent polarity field. Furthermore, we reveal links between the polarity field and auxin function: auxin-responsive markers such as DR5 have a broader distribution along the distal petal margin, consistent with the broad distal organiser of polarity, and PETAL LOSS (PTL), which has been implicated in the control of auxin dynamics during petal initiation, is directly repressed by JAG. By comparing these results with those from studies on leaf development, we show how simple modifications of an underlying developmental system may generate distinct forms, providing flexibility for the evolution of different organ functions.