Leaf-size control beyond transcription factors: Compensatory mechanisms

Plant leaves display abundant morphological richness yet grow to characteristic sizes and shapes. Beginning with a small number of undifferentiated founder cells, leaves evolve via a complex interplay of regulatory factors that ultimately influence cell proliferation and subsequent post-mitotic cell enlargement. During their development, a sequence of key events that shape leaves is both robustly executed spatiotemporally following a genomic molecular network and flexibly tuned by a variety of environmental stimuli. Decades of work on Arabidopsis thaliana have revisited the compensatory phenomena that might reflect a general and primary size-regulatory mechanism in leaves. This review focuses on key molecular and cellular events behind the organ-wide scale regulation of compensatory mechanisms. Lastly, emerging novel mechanisms of metabolic and hormonal regulation are discussed, based on recent advances in the field that have provided insights into, among other phenomena, leaf-size regulation

ANGUSTIFOLIA3 Signaling Coordinates Proliferation between Clonally Distinct Cells in Leaves

SummaryCoordinated proliferation between clonally distinct cells via inter-cell-layer signaling largely determines the size and shape of plant organs [1–4]. Nonetheless, the signaling mechanism underlying this coordination in leaves remains elusive because of a lack of understanding of the signaling molecule (or molecules) involved. ANGUSTIFOLIA3 (AN3, also called GRF-INTERACTING FACTOR1) encodes a putative transcriptional coactivator with homology to human synovial sarcoma translocation protein [5–7]. AN3 transcripts accumulate in mesophyll cells but are not detectable in leaf epidermal cells [8]. However, we found here that in addition to mesophyll cells [5, 6], epidermal cells of an3 leaves show defective proliferation. This spatial difference between the accumulation pattern of AN3 transcripts and an3 leaf phenotype is explained by AN3 protein movement across cell layers. AN3 moves into epidermal cells after being synthesized within mesophyll cells and helps control epidermal cell proliferation. Interference with AN3 movement results in abnormal leaf size and shape, indicating that AN3 signaling is indispensable for normal leaf development. AN3 movement does not require type II chaperonin activity, which is needed for movement of some mobile proteins [9]. Taking these findings together, we present a novel model emphasizing the role of mesophyll cells as a signaling source coordinating proliferation between clonally independent leaf cells

Mobility of signaling molecules: the key to deciphering plant organogenesis

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Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue.

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 μm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity

Parameter combinations in endoreduplication-related Arabidopsis mutants.

<p>(A) Optimization of the combination of parameters <i>k</i> and <i>t</i> based on RSS to estimate the ploidy profiles of the <i>rpt2a</i>, <i>rpt5a-4</i>, and <i>cyca2;3</i> mutants. Higher <i>t</i> or <i>k</i> values were required to minimize RSS if we considered fixed <i>k</i> (filled circle) or fixed <i>t</i> (open circle) values, respectively, in these mutants. (B) Ploidy profiles determined by mathematical modeling with <i>k</i> = 0.5 and the optimized <i>t</i> value. Experimentally measured (Exp.) and mathematically modeled (mod.) probabilities of 2C (green), 4C (blue), 8C (orange), 16C (red), and 32C (black) are shown.</p

Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses

<i>In silico</i> test of the theoretical framework for EPC size distribution.

<p>(A) Conceptual scheme for determining EPC size distribution. The stochastic property determines the probability density of each ploidy, which makes a steady-state ploidy profile. A Gaussian mean ± standard deviation for cell size is assumed within each ploidy. Because the ploidy effect exponentially enhances cell size, EPC size distributes with a long tail toward larger cells. (B) Histogram of measured EPC size (red bars) and the estimated cell size distribution (black dots with dashed line). The theoretical simulation was performed with our mathematical model using <i>k</i> = 0.5 and <i>t</i> = 2.27 for predicting the ploidy profile.</p

Description of the mathematical model for ploidy profile dynamics.

<p>(A) Kinetic scheme describing the sequential reaction of endoreduplication. <i>k</i>₂^<i>i</i> and <i>γ</i>₂^<i>i</i> denote the rate constant of endoreduplication from 2^<i>i</i>C to 2^<i>i</i>+1C cells and the removal constant of cells from the 2^<i>i</i>C population, respectively (1 ≤ <i>i</i> ≤ 5). <i>b</i> indicates the rate constant of mitotic cell division of 2C cells. This scheme could be simplified in this case as given by a single constant rate of endoreduplication <i>k</i> in time. (B) Time evolution of the probability of each ploidy obtained by the analytical solution with <i>k</i> = 0.5. (C) Parameter dependency for optimizing a ploidy profile with the mathematical model. Color-coded RSS values with various parameter combinations are shown. The white dotted line indicates a minimum RSS in a combination of the parameters <i>k</i> and <i>t</i>. The color bar is scaled from minimum to maximum in this diagram. (D) Valley-like dynamics of the relationship between <i>t</i> and RSS with <i>k</i> = 0.5. The RSS reached the minimum when <i>t</i> = 2.27. (E) Based on the relationship between <i>t</i> and RSS with <i>k</i> = 0.5, an optimized ploidy profile was obtained using <i>t</i> = 2.27. The probability distribution of each ploidy level from the experimentally measured data (Exp.), the mathematically modeled data at <i>t</i> = 2.27 (mod.), and the Poisson distribution (Poi.) are indicated. The probabilities of 2C (green), 4C (blue), 8C (orange), 16C (red), and 32C (black) are shown.</p

Epidermal pavement and palisade mesophyll cells in Arabidopsis leaf tissue.

<p>(A and B) Traced images of adaxial epidermal pavement cells (EPCs) (A) and palisade mesophyll cells (PMCs) (B) in the mature Arabidopsis leaf. Each single cell is outlined in magenta to aid visualization. Arrows indicate stomata guard cells, which are quite different in size as compared with other pavement cells. Scale bars = 100 μm.</p

Absence of the ploidy effect in PMC size distribution.

<p>(A–C) Optimization of the parameter <i>t</i> in our model with <i>k</i> = 0.5 based on the RSS value to estimate the ploidy profile of PMCs in WT (A) and the ploidy profile thus determined (B). Experimentally determined (Exp.) and mathematically modeled (mod.) probabilities of 2C (green), 4C (blue), 8C (orange), 16C (red), and 32C (black) are shown. (C) Histogram of the measured size of the PMCs (blue bars) and the estimated cell size distribution with the ploidy effect (+PE, black dots with solid line) or without (-PE, magenta dots with solid line).</p