Recent advances in the genetic regulation of the shape of simple leaves

Leaves are major photosynthetic organs, and their diverse shapes and sizes allow adaptation to the natural environment. The early control of leaf shape and size depends on the control of the rate and plane of cell division at the shoot apical meristem and the polarity-dependent cell differentiation in the leaf primordium. In this review, we first summarize knowledge regarding several genes that control the initial stages of leaf formation and leaf polarity (e.g. adaxial–abaxial polarity, symmetry, and flat morphology). Formation of the lateral leaf morphology involves co-ordination of the rates of division and enlargement of leaf cells. Thus, we also summarize information on a number of genes that control these stages of two-dimensional lateral leaf growth (e.g. polarized cell expansion, specific control of cell proliferation, and integration of cell proliferation and expansion). In addition, we discuss several recently identified microRNAs, which are important factors affecting the development of leaf shape via control of spatial and temporal expression of target gene families. We focus on the genetic regulation of leaf shape in the model plant Arabidopsis thaliana from the perspective of spatial and temporal balance among cell proliferation, enlargement, and differentiation, with special emphasis on the results of our own studies.     

Dissection of enhanced cell expansion processes in leaves triggered by a defect in cell proliferation, with reference to roles of endoreduplication

Leaf development relies on cell proliferation, post-mitotic cell expansion and the coordination of these processes. In several Arabidopsis thaliana mutants impaired in cell proliferation, such as angustifolia3 (an3), leaf cells are larger than normal at their maturity. This phenomenon, which we call compensated cell enlargement, suggests the presence of such coordination in leaf development. To dissect genetically the cell expansion system(s) underlying this compensation seen in the an3 mutant, we isolated and utilized 10 extra-small sisters (xs) mutant lines that show decreased cell size but normal cell numbers in leaves. In the xs single mutants, the palisade cell sizes in mature leaves are about 20-50% smaller than those of wild-type cells. Phenotypes of the palisade cell sizes in all combinations of xs an3 double mutants fall into three classes. In the first class, the compensated cell enlargement was significantly suppressed. Conversely, in the second class, the defective cell expansion conferred by the xs mutations was significantly suppressed by the an3 mutation. The residual xs mutations had effects additive to those of the an3 mutation on cell expansion. The endopolyploidy levels in the first class of mutants were decreased, unaffected or increased, as compared with those in wild-type, suggesting that the abnormally enhanced cell expansion observed in an3 could be mediated, at least in part, by ploidy-independent mechanisms. Altogether, these results clearly showed that a defect in cell proliferation in leaf primordia enhances a part of the network that regulates cell expansion, which is required for normal leaf expansion.     

Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

Organ shape and size: a lesson from studies of leaf morphogenesis

Control of the shape and size of indeterminate organs, such as roots and stems, is directly related to the control of the shape and size of the cells in these organs, as predicted by orthodox cell theory. For example, the polarity-dependent growth of leaf cells directly affects the polar expansion of leaves. Thus, the control of leaf shape is related to the control of the shape of cells within the leaf, as suggested by cell theory. By contrast, in determinate organs, such as leaves, the number of cells does not necessarily reflect organ shape or size. Genetic evidence shows that a compensatory system(s) is involved in leaf morphogenesis, and that an increase in cell volume can be triggered by a decrease in cell number and vice versa. Studies of chimeric leaves also suggest interaction between leaf cells that coordinates the behaviour of these cells at the organ level. Moreover, leaf size also appears to be coordinated at the whole-plant level. The recently hypothesised neo cell theory describes how leaf shape- and size-control mechanisms control leaf shape at the organ-level via cell-cell interaction.     

Development of Leaf Forms in Ipomoea reptans (Linn.) Poir

Development of leaves of two chromosomal biotypes of Ipomoeareptans, one with broad leaves and the other with narrow leaves,was studied. Early development of the ‘narrow’ typeinvolves a rapid multiplication of cells without expansion.This phase is followed by cell enlargement with a negligibleamount of multiplication. In the ‘broad’ type onthe other hand, multiplication and enlargement are simultaneousand proceed slowly, but steadily. The lamina shape of both thebiotypes is primarily related to a difference of cell multiplicationrate in the basal, median and apical parts. Some gradient influencingcell multiplication probably exists from the base to the tipof lamina, the optimum point being in the middle. Maturationand enlargement of cells is basipetal and continuous. The wavynature of the leaf margin is related to a very accurate geometricaladjustment of cell number variation in depressions and mounds,which may be controlled by a product of the lateral veins.     

All's well that ends well: arresting cell proliferation in leaves

The transition from cell proliferation to cell expansion is critical for determining leaf size. Andriankaja et al. (2012) demonstrate that in leaves of dicotyledonous plants, a basal proliferation zone is maintained for several days before abruptly disappearing, and that chloroplast differentiation is required to trigger the onset of cell expansion.     

THE DEVELOPMENTAL BASIS OF ANISOPHYLLY IN SELAGINELLA MARTENSII. I. INITIATION AND MORPHOLOGY OF GROWTH

The dorsiventral shoot system of Selaginella martensii is characterized by opposite pairs of ventral and dorsal leaves that are dimorphic in size and form. This study was undertaken to determine if the smaller dorsal leaf can be appropriately regarded as an arrested form of the larger leaf. Although the pattern of cell divisions and cell enlargement associated with leaf initiation is similar for both leaf types, the extent of localized growth results in distinctly larger primordia on the ventral side of the shoot. Ventral leaf primordia are also distinguished by the early formation of more extensive mesophyll tissue. Regression analysis of quantitative data on leaf length vs. position and leaf width vs. length indicates that the growth pattern of ventral and dorsal leaves is significantly different. These observations indicate that the developmental pathways of the dimorphic leaves of Selaginella martensii do not diverge at a relatively late developmental stage, but rather can be distinguished from inception.     

Exogenously applied jasmonic acid induces changes in apical meristem morphology of potato stolons

Hooked apex stolons and initial swelling stolons of potato plants were treated with 3 x 10-8 mol l-1 jasmonic acid (JA) to study the effect of this compound on histology, cell expansion and tissue differentiation. In hooked apex stolons, JA application increased the meristem thickness and reduced the length of the leaf primordia, whereas in initial swelling stolons narrowing of the apical region, absence of leaf primordia and swelling of the subapical meristem were evident. Early vascular tissue differentiation was observed in response to JA treatment, especially of xylem elements from regions proximal to the tunic. Protoxylem elements, such as tracheal elements, were present with thin primary cell walls. The cell area was measured in two zones: zone I, central mother cells situated immediately under the tunic; and zone II, rib meristem cells. JA caused a four- and six-fold increase in cell area in both zones in hooked apex stolons and initial swelling stolons, respectively. Thus, tuber formation is concluded to occur as a consequence of increased cell expansion, a reduction in the length of leaf primordia, enlargement of meristems, and early vascular tissue differentiation.     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Analysis of leaf development in fugu mutants of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs

In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1-fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways.     

Radial leaves of the maize mutant ragged seedling2 retain dorsiventral anatomy

ragged seedling2 (rgd2) is a novel, recessive mutation affecting lateral organ development in maize. The mutant phenotype of homozygous rgd2-R leaves is variable. Mild leaf phenotypes have a reduced midrib and may be moderately narrow and furcated; severe Rgd2-R(-) leaves are filamentous or even radial. Despite their radial morphology, severe Rgd2-R(-) mutant leaves develop distinct adaxial and abaxial anatomical features. Although Rgd2-R(-) mutants exhibit no reduction in adaxial or abaxial cell types, areas of epidermal cell swapping may occur that are associated with misaligned vascular bundles and outgrowths of ectopic margins. Scanning electron microscopy of young primordia and analyses of leaf developmental-marker gene expression in mutant apices reveal that RGD2 functions during recruitment of leaf founder cells and during expansive growth of leaf primordia. Overall, these phenotypes suggest that development is uncoordinated in Rgd2-R(-) mutant leaves, so that leaf components and tissues may develop quasi-independently. Models whereby RGD2 is required for developmental signaling during the initiation, anatomical patterning, and lateral expansion of maize leaves are discussed.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Observation of Apparently Unchanging Mesophyll Cell Diameters Throughout Leaf Ontogeny in Woody Species

The expansion of plant leaves usually lasts 3–6 weeks and it is widely believed that most cell types (epidermal and mesophyll) continue to expand in unison over a similar time period. The evidence supporting this account was derived from studies of herb leaves. We observed in woody species, however, that the diameter of mesophyll cells (spongy and palisade) changed little during leaf expansion from about 5 to 100 % maximum size. To keep pace with epidermal cell enlargement and leaf area expansion, mesophyll cells divided but palisade cell length expanded as leaves grew thicker. The prolonged division of mesophyll and apparently unchanging mesophyll cell diameters constitute a novel pattern of leaf cell development, different from that previously described for herbs. Possible mechanisms that attribute the varied expansion direction and speed to the different cellulose distributions in woody and herbaceous species are suggested. This finding could contribute to an enhanced understanding of the overall mechanism of leaf development.     

Patterns of asymmetry in the twining vine Dalechampia scandens (Euphorbiaceae): ontogenetic and hierarchical perspectives

We studied patterns of fluctuating asymmetry (FA) in leaves of four populations of the neotropical vine Dalechampia scandens to obtain insight into the origin of leaf FA and the level at which it is controlled. We analysed correlations in signed and unsigned asymmetry at different organizational levels. We also analysed the ontogeny of FA during leaf expansion to test whether asymmetry is regulated during cell expansion, and whether fast-expanding leaves are more or less asymmetrical. Signed asymmetry was negatively correlated between successive leaves, that is, when the right side of a leaf was larger than the left side, the next leaf on the shoot tended to show the opposite pattern. The magnitude of FA, however, was very weakly correlated among successive leaves or among leaves measured on different shoots. The direction of asymmetry did not change during leaf expansion, but the relative asymmetry, that is, asymmetry corrected for difference in trait size, decreased during expansion. We found a weak negative relationship between leaf expansion rate and relative asymmetry on the fully expanded leaves. These results suggest that leaf asymmetry in Dalechampia originates from perturbations in cell proliferation in the stem, creating asymmetries in opposite directions in successive leaves. These asymmetries persist during leaf expansion, but tend to be reduced by unknown mechanisms.     

Leaf growth of white mustard (Sinapis alba) in different environments

Summary The numbers of cells and area of fully expanded leaves were determined on successive leaves of Sinapis alba grown either in 8 hr. photoperiod (vegetative plants) or 16 hr. photoperiod (flowering plants) at a constant temperature of 20°C. In the 8 hr. photoperiod leaf 9 had the greatest area but leaf 12 had most cells. In 16 hr. photperiod leaf 5 had the greatest area but leaf 9 had most cells. The relationship between area and cell number of successive leaves on the main stem fell into 3 distinct phases: in phase (1), cell number increased at a greater rate than leaf area; in phase (2), leaf area decreased while cell number increased; in phase (3), cell number and leaf area decreased proportionally. For an increase in unit area, cell number increased more in 8 hr. than in 16 hr. photoperiod.Using final area and final cell number of successive leaves, by extrapolation the cell number of unit area of primordium has been deduced. Cell number per unit area increased in successive primordia up to a certain node after which it remained constant at succeeding nodes. It was found that in plants grown under different conditions the cell number per unit area in successive primordia increased at a constant logarithmic rate. That is, cells became progressively smaller. It is concluded that changes in cell size of successive primordia are not influenced by the environment but are under internal control.