4221 RESEARCH ARTICLE INTRODUCTION Organ size is determined by the number and size of constituent cells and is genetically regulated in a highly reproducible manner. Cell proliferation and postmitotic cell expansion are hence coordinated, yet the mechanisms behind these processes remain poorly understood. In plants, spatiotemporal regulation of cell proliferation and postmitotic cell expansion is seen during leaf development. Cell proliferation is active throughout the developing leaf primordium, but it ceases from the distal to proximal region (Donnelly et al., 1999; Ichihashi et al., 2010; Kazama et al., 2010; Nath et al., 2003; White, 2006). There is a proximal-distal gradient of cell proliferation activity, whereby proliferation in the proximal region and postmitotic expansion in the distal region occur simultaneously in the same leaf primordium. During leaf development, a defect in cell proliferation often triggers enhanced cell expansion, a phenomenon that was first described nearly half a century ago in -irradiated wheat (Haber, 1962). More recently, similar phenomena have been reported using various mutants and transgenic strains of Arabidopsis thaliana (hereafter Arabidopsis) (Micol, 2009; Tsukaya, 2008). These phenomena have been collectively termed ‘compensation’ (Beemster et al., 2003; Tsukaya, 2002; Tsukaya, 2008). Compensation seems to occur only in determinate organs, such as leaves (Ferjani et al., 2007). It suggests an interaction of cell proliferation and expansion, and provides clues that are relevant to an understanding of organ size control (Tsukaya, 2008). The loss-of-function mutant angustifolia3 (an3) and the overexpressor of the cyclin-dependent kinase inhibitor gene KIP- RELATED PROTEIN2 (KRP2 o/x) show features typical of compensation (De Veylder et al., 2001; Ferjani et al., 2007; Hemerly et al., 1995; Horiguchi et al., 2005). In the leaves of an3 and KRP2 o/x, cell number is decreased by more than 70% and 90% and cell size is increased by 50% and 100%, respectively, as compared with the wild type (WT) (De Veylder et al., 2001; Ferjani et al., 2007; Horiguchi et al., 2005). AN3 (which is also known as GRF-INTERACTING FACTOR1) encodes a transcriptional coactivator (Kim and Kende, 2004). KRP2, which is identical to Arabidopsis Cdc-2a-interacting protein ICK2, binds and inhibits A- type cyclin-dependent kinase, thereby restricting cell cycle progression (De Veylder et al., 2001; Lui et al., 2000; Verkest et al., 2005). Studies on an3-dependent compensation (Ferjani et al., 2007; Fujikura et al., 2007a; Fujikura et al., 2009) have indicated that the size of mitotic cells is normal but that enhanced expansion occurs in postmitotic cells in the an3 mutant; furthermore, there is a threshold decrease in cell number or cell proliferation activity that induces compensation. Moreover, an3-dependent compensation is completely suppressed in a group of extra-small sisters (xs) mutants that are specifically defective in postmitotic cell expansion. These data suggest that an3-dependent compensation is not simply the result of a defect in cell proliferation; it probably involves an interaction between cell proliferation and postmitotic cell expansion. We consider that compensation occurs in three successive steps. The induction step involves a defect in cell proliferation. The intermediary step links cell proliferation with postmitotic cell expansion through an unknown signal. This signal regulates the Development 137, 4221-4227 (2010) doi:10.1242/dev.057117 © 2010. Published by The Company of Biologists Ltd 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2 Department of Life Science, College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan. 3 National Institute for Basic Biology, 38 Nishigo-Naka, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan. *Author for correspondence (ghori@rikkyo.ac.jp) Accepted 15 October 2010 SUMMARY The 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-cell- autonomous 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 cell- autonomous compensation. Together, our results revealed two previously unknown pathways that coordinate cell proliferation and postmitotic cell expansion for organ size control in plants. KEY WORDS: AN3, Compensation, Leaf chimera, KRP2, Organ size, Arabidopsis thaliana Non-cell-autonomously coordinated organ size regulation in leaf development Kensuke Kawade 1 , Gorou Horiguchi 2, * and Hirokazu Tsukaya 1,3 DEVELOPMENT