Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity.

The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.

Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern.

The causal relationship between cell division and growth in plants is complex. Although altered expression of cell-cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under-explored, is that altered division patterns resulting from manipulation of cell-cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma-related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high-resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell-cycle gene expression.

A shift toward smaller cell size via manipulation of cell cycle gene expression acts to smoothen Arabidopsis leaf shape.

Understanding the relationship of the size and shape of an organism to the size, shape, and number of its constituent cells is a basic problem in biology; however, numerous studies indicate that the relationship is complex and often nonintuitive. To investigate this problem, we used a system for the inducible expression of genes involved in the G1/S transition of the plant cell cycle and analyzed the outcome on leaf shape. By combining a careful developmental staging with a quantitative analysis of the temporal and spatial response of cell division pattern and leaf shape to these manipulations, we found that changes in cell division frequency occurred much later than the observed changes in leaf shape. These data indicate that altered cell division frequency cannot be causally involved in the observed change of shape. Rather, a shift to a smaller cell size as a result of the genetic manipulations performed correlated with the formation of a smoother leaf perimeter, i.e. appeared to be the primary cellular driver influencing form. These data are discussed in the context of the relationship of cell division, growth, and leaf size and shape.

LEAFPROCESSOR: a new leaf phenotyping tool using contour bending energy and shape cluster analysis.

*Significant progress has been made in the identification of the genetic factors controlling leaf shape. However, no integrated solution for the quantification and categorization of leaf form has been developed. In particular, the analysis of local changes in margin growth, which define many of the differences in shape, remains problematical. *Here, we report on a software package (LEAFPROCESSOR) which provides a semi-automatic and landmark-free method for the analysis of a range of leaf-shape parameters, combining both single metrics and principal component analysis. In particular, we explore the use of bending energy as a tool for the analysis of global and local leaf perimeter deformation. *As a test case for the implementation of the LEAFPROCESSOR program, we show that this integrated analysis leads to deeper insights into the morphogenic changes underpinning a series of previously identified Arabidopsis leaf-shape mutants. Our analysis reveals that many of these mutants which, at first sight, show similar leaf morphology, can be distinguished via our shape analysis. *The LEAFPROCESSOR program provides a novel integrated tool for the analysis of leaf shape.

The NAC domain transcription factors FEZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells.

Because plant cells do not migrate, cell division planes are crucial determinants of plant cellular architecture. In Arabidopsis roots, stringent control of cell divisions leads to a virtually invariant division pattern, including those that create new tissue layers. However, the mechanisms that control oriented cell divisions are hitherto poorly understood. Here, we reveal one such mechanism in which FEZ and SOMBRERO (SMB), two plant-specific NAC-domain transcription factors, control the delicately tuned reorientation and timing of cell division in a subset of stem cells. FEZ is expressed in root cap stem cells, where it promotes periclinal, root cap-forming cell divisions. In contrast, SMB negatively regulates FEZ activity, repressing stem cell-like divisions in the root cap daughter cells. FEZ becomes expressed in predivision stem cells, induces oriented cell division, and activates expression of its negative regulator, SMB, thus generating a feedback loop for controlled switches in cell division plane.

Restoration of DWF4 expression to the leaf margin of a dwf4 mutant is sufficient to restore leaf shape but not size: the role of the margin in leaf development.

The role of the margin in leaf development has been debated over a number of years. To investigate the molecular basis of events in the margin, we performed an enhancer trap screen to identify genes specifically expressed in this tissue. Analysis of one of these lines revealed abnormal differentiation in the margin, accompanied by an abnormal leaf size and shape. Further analysis revealed that this phenotype was due to insertion of the trap into DWF4, which encodes a key enzyme in brassinolide biosynthesis. Transcripts for this gene accumulated in a specific and dynamic pattern in the epidermis of young leaf primordia. Targeted expression of DWF4 to a subset of these cells (the leaf margin) in a dwf4 mutant background led to both restoration of differentiation of a specific group of leaf cells (margin cells) and restoration of wild-type leaf shape (but not leaf size). Ablation of these cells led to abrogation of leaf development and the formation of small round leaves. These data support the hypothesis that events in the margin play an essential role in leaf morphogenesis, and implicate brassinolide in the margin as a key mediator in the control of leaf shape, separable from a general function of this growth factor in the control of organ size.