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.     

Patterns and symmetries in leaf development

The leaf is a coordinated mosaic of developmental domains, which are evident from leaf inception on the flanks of the apical meristem. The subdivision of the meristem into molecularly defined domains is regulated by the interactions of a number of gene products and by receptor kinase-mediated signals. The acquisition of symmetry axes in the emerging leaf is a process coordinated by hormones (such as auxin and cytokinins) and the expression of classes of genes (such as the knox and the ARP, as1/rs2/phan, genes). As with simple leaves, the architecture of compound leaves is defined by spatial/temporal gradients of regulatory gene functions: complexity results from the interplay between leaf differentiation processes and genes maintaining a partial level of indeterminacy in the developing primordium. Boundaries between regions with different molecular 'addresses' are considered, in plants as in Drosophila, as organizing centres for lateral organ development.     

Compound leaf development and evolution in the legumes

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.     

New Sphenophyllum plant from the Upper Devonian of Zhejiang Province,China

Sphenophyllum was an important and long-surviving sphenopsid genus in the Paleozoic floras, with a worldwide distribution. A new species, Sphenophyllum changxingense sp. nov., is described from the Upper Devonian Wutong Formation of Changxing County, Zhejiang Province, China. This plant is characterized by two orders of slender axes and wedge-shaped leaves borne in whorls. The axes bear short spines and show longitudinal ridges and furrows on surface. Three to eight isophyllous leaves, with one, two, or no second-order axes, are attached at each node of first-order axes. Leaves bear spines and show a bilobate morphology; the two leaf lobes divide distally to form several marginal segments, each segment with a leaf vein. Sphenophyllum changxingense represents an early and primitive species within the genus, in light of the absence of heterophylly and specialized hook-like leaves. Like some Carboniferous and Permian species, it appears to have formed dense mats with mutually supportive axes. This plant adds to the known diversity of early sphenopsids in the Late Devonian.     

Transforming compound leaf patterning by manipulating REVOLUTA in Medicago truncatula

Leaves are derived from the shoot apical meristem with three distinct axes: dorsoventral, proximodistal and mediolateral. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain‐leucine zipper (HD‐ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon‐tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliate leaves in most cases, in contrast to the trifoliate leaves of the wild type. Detailed characterization revealed that a lesion in a HD‐ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166‐insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the free auxin content was affected. Our results demonstrate that MtREV1 plays a key role in determination of leaf adaxial–abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis.     

SHOOT GROWTH AND LEAF DIMORPHISM IN BOSTON IVY (PARTHENOCISSUS TRICUSPIDATA)

Boston ivy, a common ornamental vine in the grape family, successively produces two kinds of leaves during the growing season. The two “early leaves” at the base of each shoot are preformed in the winter bud, and their expansion in the spring is accompanied by little stem elongation. At maturity they have large three-lobed blades and long petioles. Most short shoots produce no more leaves, but “late leaves” develop on all long shoots at intervals of less than 2 days. All but the first few undergo their entire development during the growing season. They are much smaller than early leaves, and the lateral lobes of their blades are reduced or eliminated. They are separated from the early leaves and from each other by long internodes. The early and late leaves differ in the circumstances and continuity of ontogeny, and diverge in form at an early stage. This vine and its relatives are unique in their three-node cyclical pattern of organ occurrence and internode length along the shoot. Lateral shoots and buds are present at every third node, with tendrils at intervening nodes. The long shoots branch freely and repeatedly, and the production of late leaves and new shoot axes by vigorous compound shoots is limited only by the growing season. Despite its specialized organization, Boston ivy resembles several tree species in its association between a seasonal type of leaf dimorphism and a shoot system constructed of long and short shoots.     

Reduced leaf complexity in tomato wiry mutants suggests a role for PHAN and KNOX genes in generating compound leaves

Recent work on species with simple leaves suggests that the juxtaposition of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf primordia is necessary for lamina outgrowth. However, how leaf dorsiventral symmetry affects leaflet formation in species with compound leaves is largely unknown. In four non-allelic dorsiventrality-defective mutants in tomato, wiry, wiry3, wiry4 and wiry6, partial or complete loss of ab-adaxiality was observed in leaves as well as in lateral organs in the flower, and the number of leaflets in leaves was reduced significantly. Morphological analyses and expression patterns of molecular markers for ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B (LeYAB B)] indicated that ab-adaxial cell fates were altered in mutant leaves. Reduction in expression of both LeT6 (a tomato KNOX gene) and LePHAN during post-primordial leaf development was correlated with a reduction in leaflet formation in the wiry mutants. LePHAN expression in LeT6 overexpression mutants suggests that LeT6 is a negative regulator of LePHAN. KNOX expression is known to be correlated with leaflet formation and we show that LeT6 requires LePHAN activity to form leaflets. These phenotypes and gene expression patterns suggest that the abaxial and adaxial domains of leaf primordia are important for leaflet primordia formation, and thus also important for compound leaf development. Furthermore, the regulatory relationship between LePHAN and KNOX genes is different from that proposed for simple-leafed species. We propose that this change in the regulatory relationship between KNOX genes and LePHAN plays a role in compound leaf development and is an important feature that distinguishes simple leaves from compound leaves.     

Functional Analysis and RNA Sequencing Indicate the Regulatory Role of Argonaute1 in Tomato Compound Leaf Development

Regardless of whether a leaf is simple or compound, the mechanism underlying its development will give rise to a full comprehension of plant morphogenesis. The role of Argonaute1 (AGO1) in the development of simple leaves has been established, but its role in the development of compound leaves remains to be characterized. In this paper, a virus-induced gene silencing (VIGS) strategy was used to dramatically down-regulate the expression of AGO1 ortholog in tomatoes, a model plant for research into compound leaves. AGO1-silenced tomato compound leaves exhibited morphological defects of leaf adaxial-abaxial and trichome development. Analysis of global gene expression profiles indicated that the silencing of AGO1 in tomato compound leaf caused significant changes in the expression of several critical genes, including Auxin Response Factor 4 (ARF4) and Non-expressor of PR5 (NPR5), which were involved in adaxial-abaxial formation and IAA15 that was found to contribute to growth of trichomes as well as Gibberellic Acid Insensitive (GAI) which participated in hormone regulation. Collectively, these results shed light on the complicated mechanism by which AGO1 regulates compound leaf development.     

Initiation of axillary and floral meristems in Arabidopsis

Shoot development is reiterative: shoot apical meristems (SAMs) give rise to branches made of repeating leaf and stem units with new SAMs in turn formed in the axils of the leaves. Thus, new axes of growth are established on preexisting axes. Here we describe the formation of axillary meristems and floral meristems in Arabidopsis by monitoring the expression of the SHOOT MERISTEMLESS and AINTEGUMENTA genes. Expression of these genes is associated with SAMs and organ primordia, respectively. Four stages of axillary meristem development and previously undefined substages of floral meristem development are described. We find parallels between the development of axillary meristems and the development of floral meristems. Although Arabidopsis flowers develop in the apparent absence of a subtending leaf, the expression patterns of AINTEGUMENTA and SHOOT MERISTEMLESS RNAs during flower development suggest the presence of a highly reduced, "cryptic" leaf subtending the flower in Arabidopsis. We hypothesize that the STM-negative region that develops on the flanks of the inflorescence meristem is a bract primordium and that the floral meristem proper develops in the "axil" of this bract primordium. The bract primordium, although initially specified, becomes repressed in its growth.     

LEAF TYPES IN THE ARACEAE

Leaf types in the Araceae are described and classified on the basis of their morphology and functional role. Four classes are recognized on the basis of their association with the initiation of new shoot axes, the continuation of axes, the resting of axes, or the termination and renewal of axes. The basic types are described with the terms leaf, prophyll, mesophyll, bracteole, mesobracteole, cataphyll, and blastophyll. These terms are modified with the terms monopodial, sympodial, proleptic, sylleptic, resting, flagellar, stolon, reduced, and foliage. This represents an unconventional terminology because some of the modifiers refer to the structure of the stem to which the leaves are attached, rather than to the form of the leaf itself. The intent is to draw attention to the impact of shoot organization on leaf form, and to develop a leaf terminology that will aid in describing shoot organization.     

Regulation of compound leaf development in Medicago truncatula by fused compound leaf1, a class M KNOX gene

Medicago truncatula is a legume species belonging to the inverted repeat lacking clade (IRLC) with trifoliolate compound leaves. However, the regulatory mechanisms underlying development of trifoliolate leaves in legumes remain largely unknown. Here, we report isolation and characterization of fused compound leaf1 (fcl1) mutants of M. truncatula. Phenotypic analysis suggests that FCL1 plays a positive role in boundary separation and proximal-distal axis development of compound leaves. Map-based cloning indicates that FCL1 encodes a class M KNOX protein that harbors the MEINOX domain but lacks the homeodomain. Yeast two-hybrid assays show that FCL1 interacts with a subset of Arabidopsis thaliana BEL1-like proteins with slightly different substrate specificities from the Arabidopsis homolog KNATM-B. Double mutant analyses with M. truncatula single leaflet1 (sgl1) and palmate-like pentafoliata1 (palm1) leaf mutants show that fcl1 is epistatic to palm1 and sgl1 is epistatic to fcl1 in terms of leaf complexity and that SGL1 and FCL1 act additively and are required for petiole development. Previous studies have shown that the canonical KNOX proteins are not involved in compound leaf development in IRLC legumes. The identification of FCL1 supports the role of a truncated KNOX protein in compound leaf development in M. truncatula.     

Native environment modulates leaf size and response to simulated foliar shade across wild tomato species

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits.     

CLAUSA restricts tomato leaf morphogenesis and GOBLET expression

Leaf morphogenesis and differentiation are highly flexible processes. The development of compound leaves is characterized by an extended morphogenesis stage compared with that of simple leaves. The tomato mutant clausa (clau) possesses extremely elaborate compound leaves. Here we show that this elaboration is generated by further extension of the morphogenetic window, partly via the activity of ectopic meristems present on clau leaves. Further, we propose that CLAU might negatively affect expression of the NAM/CUC gene GOBLET (GOB), an important modulator of compound‐leaf development, as GOB expression is elevated in clau mutants and reducing GOB expression suppresses the clau phenotype. Expression of GOB is also elevated in the compound leaf mutant lyrate (lyr), and the remarkable enhancement of the clau phenotype by lyr suggests that clau and lyr affect leaf development and GOB in different pathways.