Developmental processes of leaf morphogenesis in<Emphasis Type="Italic">arabidopsis</Emphasis>

The leaf is a suitable subject with which to study plant morphogenesis because of its diversity of shape. Although mechanisms for leaf initiation and lateral morphogenesis have been suggested, the exact means for determining shape remain unclear. Many genes involved in those developmental processes have now been identified. Here, we summarize the early events in the genetic regulation ofArabidopsis leaf formation, including initiation, dorsoventrality, and the spatial and temporal control of cell proliferation and enlargement. We focus on recent progress within the model plantArabidopsis, placing special emphasis on our own findings.     

Leaf shape: genetic controls and environmental factors

In recent years, many genes have been identified that are involved in the developmental processes of leaf morphogenesis. Here, I review the mechanisms of leaf shape control in a model plant, Arabidopsis thaliana, focusing on genes that fulfill special roles in leaf development. The lateral, two-dimensional expansion of leaf blades is highly dependent on the determination of the dorsoventrality of the primordia, a defining characteristic of leaves. Having a determinate fate is also a characteristic feature of leaves and is controlled by many factors. Lateral expansion is not only controlled by general regulators of cell cycling, but also by the multi-level regulation of meristematic activities, e.g., specific control of cell proliferation in the leaf-length direction, in leaf margins and in parenchymatous cells. In collaboration with the polarized control of leaf cell elongation, these redundant and specialized regulating systems for cell cycling in leaf lamina may realize the elegantly smooth, flat structure of leaves. The unified, flat shape of leaves is also dependent on the fine integration of cell proliferation and cell enlargement. Interestingly, while a decrease in the number of cells in leaf primordia can trigger a cell volume increase, an increase in the number of cells does not trigger a cell volume decrease. This phenomenon is termed compensation and suggests the existence of some systems for integration between cell cycling and cell enlargement in leaf primordia via cell-cell communication. The environmental adjustment of leaf expansion to light conditions and gravity is also summarized.     

miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.     

The orientation of cell divisions determines the shape of Drosophila organs

Organ shape depends on the coordination between cell proliferation and the spatial arrangement of cells during development. Much is known about the mechanisms that regulate cell proliferation, but the processes by which the cells are orderly distributed remain unknown. This can be accomplished either by random division of cells that later migrate locally to new positions (cell allocation) or through polarized cell division (oriented cell division; OCD). Recent data suggest that the OCD is involved in some morphogenetic processes such as vertebrate gastrulation, neural tube closure, and growth of shoot apex in plants; however, little is known about the contribution of OCD during organogenesis. We have analyzed the orientation patterns of cell division throughout the development of wild-type and mutant imaginal discs of Drosophila. Our results show a causal relationship between the orientation of cell divisions in the imaginal disc and the adult morphology of the corresponding organs, indicating a key role of OCD in organ-shape definition. In addition, we find that a subset of planar cell polarity genes is required for the proper orientation of cell division during organ development.     

Cell cycling and cell enlargement in developing leaves of Arabidopsis.

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.     

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.     

The genetic control of plastid division in higher plants

The division of plastids is an important part of plastid differentiation and development and in distinct cell types, such as leaf mesophyll cells, results in large populations of chloroplasts. The morphology and population dynamics of plastid division have been well documented, but the molecular controls underlying plastid division are largely unknown. With the isolation of Arabidopsis mutants in which specific aspects of plastid and proplastid division have been disrupted, the potential exists for a detailed knowledge of how plastids divide and what factors control the rate of division in different cell types. It is likely that knowledge of plant homologues of bacterial cell division genes will be essential for understanding this process in full. The processes of plastid division and expansion appear to be mutually independent processes, which are compensatory when either division or expansion are disrupted genetically. The rate of cell expansion appears to be an important factor in initiating plastid division and several systems involving rapid cell expansion show high levels of plastid division activity. In addition, observation of plastids in different cell types in higher plants shows that cell-specific signals are also important in the overall process in determining not only the differentiation pathway of plastids but also the extent of plastid division. It appears likely that with the exploitation of molecular techniques and mutants, a detailed understanding of the molecular basis of plastid division may soon be a reality.     

Making leaves

Leaves are determinate organs that develop from the flanks of the shoot apical meristem through founder cell recruitment, establishment of proximodistal, dorsoventral and mediolateral axes, and subsequent growth, expansion and differentiation along these axes. Maintenance of the shoot apical meristem and production of leaves requires balanced partitioning of cells between pluripotent and differentiation fates. Hormones have a significant role in this balance but it is becoming apparent that additional intrinsic and extrinsic inputs influence hormone signalling to control meristem function and leaf initiation. As leaves develop, temporal and spatial regulation of growth and maturation determines leaf shape and complexity. Remarkably genes involved in leaf development in the context of the shoot apical meristem are also involved in elaboration of the leaf shape to generate subtle marginal serrations, more prominent lobes or a dissected compound leaf. Potentially these common regulatory modules represent a fundamental means of setting up boundaries separating discrete zones of growth. Defining gene networks involved in leaf shape variation and exploring interspecies differences between such networks is enabling exciting insight into changes that contribute to natural variation of leaf form.     

Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation

We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

Exit from proliferation during leaf development in Arabidopsis thaliana: a not-so-gradual process

Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.     

Whole organ, venation and epidermal cell morphological variations are correlated in the leaves of Arabidopsis mutants

Despite the large number of genes known to affect leaf shape or size, we still have a relatively poor understanding of how leaf morphology is established. For example, little is known about how cell division and cell expansion are controlled and coordinated within a growing leaf to eventually develop into a laminar organ of a definite size. To obtain a global perspective of the cellular basis of variations in leaf morphology at the organ, tissue and cell levels, we studied a collection of 111 non-allelic mutants with abnormally shaped and/or sized leaves, which broadly represent the mutational variations in Arabidopsis thaliana leaf morphology not associated with lethality. We used image-processing techniques on these mutants to quantify morphological parameters running the gamut from the palisade mesophyll and epidermal cells to the venation, whole leaf and rosette levels. We found positive correlations between epidermal cell size and leaf area, which is consistent with long-standing Avery's hypothesis that the epidermis drives leaf growth. In addition, venation parameters were positively correlated with leaf area, suggesting that leaf growth and vein patterning share some genetic controls. Positional cloning of the genes affected by the studied mutations will eventually establish functional links between genotypes, molecular functions, cellular parameters and leaf phenotypes.     

Manipulation of leaf shape by modulation of cell division

The role of cell division as a causal element in plant morphogenesis is debatable, with accumulating evidence supporting the action of cell division-independent mechanisms. To directly test the morphogenic function of cell division, we have utilised a microinduction technique to locally and transiently manipulate the expression in transgenic plants of two genes encoding putative effectors of the cell cycle, a tobacco A-type cyclin and a yeast cdc25. The results show that local expression of these genes leads to modulation of cell division patterns. Moreover, whereas altered cell division in the apical meristem had no influence on organogenesis, local induction of cell proliferation on the flanks of young leaf primordia led to a dramatic change in lamina development and, thus, leaf shape. These data indicate that the role of cell division in plant morphogenesis is context dependent and identify cell division in the leaf primordium as a potential target for factors regulating leaf shape.     

Characterization of the AE7 gene in Arabidopsis suggests that normal cell proliferation is essential for leaf polarity establishment

The formation of leaf polarity is critical for leaf morphogenesis. In this study, we characterized and cloned an Arabidopsis gene, AS1/2 ENHANCER7 (AE7), which is required for both leaf adaxial-abaxial polarity formation and normal cell proliferation. The ae7 mutant exhibited leaf adaxial-abaxial polarity defects and double mutants combining ae7 with the leaf polarity mutants as1 (asymmetric leaves1), as2, rdr6 (RNA-dependent RNA polymerase6) or ago7/zip (argonaute7/zippy) all resulted in plants with an apparently enhanced loss of adaxial leaf identity. In addition, ae7 also showed decreased cell proliferation in both leaves and roots, compensated by increased cell sizes in leaves. AE7 encodes a protein conserved in many eukaryotic organisms, ranging from unicellular yeasts to humans; however, the functions of AE7 family members from other species have not been reported. In situ hybridization revealed that AE7 is expressed in a spotted pattern in plant tissues, similar to cell-cycle marker genes such as HISTONE4. Moreover, the ae7 endoploidy and expression analysis of several cell-cycle marker genes in ae7 suggest that the AE7 gene is required for cell cycle progression. As the previously characterized 26S proteasome and ribosome mutants also affect both leaf adaxial-abaxial polarity and cell proliferation, similar to the defects in ae7, we propose that normal cell proliferation may be essential for leaf polarity establishment. Possible models for how cell proliferation influences leaf adaxial-abaxial polarity establishment are discussed.     

Shaping up: the genetic control of leaf shape

Leaf initiation at the shoot apical meristem involves a balance between cell proliferation and commitment to make primordia. Several genes, such as CLAVATA1, CLAVATA3, WUSCHEL, KNOTTED1, and PHANTASTICA, play key roles in these processes. When expressed in the leaf primordium, however, these 'meristem' genes can profoundly affect leaf shape and size, possibly by regulating hormone gradients and transport. The KNOTTED1-like genes are involved in regulating changes in hormonal levels. Recent studies have elaborated on the role that hormones, such as auxin, play in releasing biophysical constraints on leaf initiation and growth. Final leaf form is elaborated by a coordination of these hormonally regulated processes, cell division and cellular differentiation.