Coordinated proliferation between clonally distinct cells via inter-cell-layer signaling largely determines the size and shape of plant organs. 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. AN3 transcripts accumulate in mesophyll cells but are not detectable in leaf epidermal cells. However, we found here that in addition to mesophyll cells, 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. 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.

SUMMARYThe way in which the number and size of cells in an organ are determined poses a central challenge in our understanding of organ size control. Compensation is an unresolved phenomenon, whereby a decrease in cell proliferation below some threshold level triggers enhanced postmitotic cell expansion in leaf primordia. It suggests an interaction between these cellular processes during organogenesis and provides clues relevant to an understanding of organ size regulation. Although much attention has been given to compensation, it remains unclear how the cellular processes are coordinated. Here, we used a loss-of-function mutation in the transcriptional coactivator gene ANGUSTIFOLIA3 (AN3), which causes typical compensation in Arabidopsis thaliana. We established Cre/lox systems to generate leaves chimeric for AN3 expression and investigated whether compensation occurs in a cell-autonomous or non-cell-autonomous manner. We found that an3-dependent compensation is a non-cellautonomous process, and that an3 cells seem to generate and transmit an intercellular signal that enhances postmitotic cell expansion. The range of signalling was restricted to within one-half of a leaf partitioned by the midrib. Additionally, we also demonstrated that overexpression of the cyclin-dependent kinase inhibitor gene KIP-RELATED PROTEIN2 resulted in cellautonomous compensation. Together, our results revealed two previously unknown pathways that coordinate cell proliferation and postmitotic cell expansion for organ size control in plants.

Leaves are the most important, fundamental units of organogenesis in plants. Although the basic form of a leaf is clearly divided into the leaf blade and leaf petiole, no study has yet revealed how these are differentiated from a leaf primordium. We analyzed the spatiotemporal pattern of mitotic activity in leaf primordia of Arabidopsis (Arabidopsis thaliana) in detail using molecular markers in combination with clonal analysis. We found that the proliferative zone is established after a short interval following the occurrence of a rod-shaped early leaf primordium; it is separated spatially from the shoot apical meristem and seen at the junction region between the leaf blade and leaf petiole and produces both leaf-blade and leaf-petiole cells. This proliferative region in leaf primordia is marked by activity of the ANGUSTIFOLIA3 (AN3) promoter as a whole and seems to be differentiated into several spatial compartments: activities of the CYCLIN D4;2 promoter and SPATULA enhancer mark parts of it specifically. Detailed analyses of the an3 and blade-on-petiole mutations further support the idea that organogenesis of the leaf blade and leaf petiole is critically dependent on the correct spatial regulation of the proliferative region of leaf primordia. Thus, the proliferative zone of leaf primordia is spatially differentiated and supplies both the leaf-blade and leaf-petiole cells.

Leaves are ideal model systems to study the organ size regulation of multicellular plants. Leaf cell number and cell size are determinant factors of leaf size which is controlled through cell proliferation and post-mitotic cell expansion, respectively. To achieve a proper leaf size, cell proliferation and post-mitotic cell expansion should be co-ordinated during leaf morphogenesis. Compensation, which is enhanced post-mitotic cell expansion associated with a decrease in cell number during lateral organ development, is suggestive of such co-ordination. Genetic and kinematic studies revealed at least three classes of modes of compensation, indicating that compensation is a heterogeneous phenomenon. Recent studies have increased our understanding about the molecular basis of compensation by identifying the causal genes of each compensation-exhibiting mutant. Furthermore, analyses using chimeric leaves revealed that a type of compensated cell expansion requires cell-to-cell communication. Information from recent advances in molecular and genetic studies on compensation has been integrated here and its role in organ size regulation is discussed.

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.