Auxin‐mediated lamina growth in tomato leaves is restricted by two parallel mechanisms

In the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild‐type leaf primordia, the miR160‐targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160‐targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160‐targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.     

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.     

PROCERA encodes a DELLA protein that mediates control of dissected leaf form in tomato

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Mechanisms that define leaflet number and position are poorly understood and their elucidation presents an attractive opportunity to understand mechanisms controlling organ shape in plants. In tomato (Solanum lycopersicum), a plant with dissected leaves, KNOTTED1-like homeodomain proteins (KNOX) are positive regulators of leaflet formation. Conversely, the hormone gibberellin (GA) can antagonise the effects of KNOX overexpression and reduce leaflet number, suggesting that GA may be a negative regulator of leaflet formation. However, when and how GA acts on leaf development is unknown. The reduced leaflet number phenotype of the tomato mutant procera (pro) mimics that of plants to which GA has been applied during leaf development, suggesting that PRO may define a GA signalling component required to promote leaflet formation. Here we show that PRO encodes a DELLA-type growth repressor that probably mediates GA-reversible growth restraint. We demonstrate that PRO is required to promote leaflet initiation during early stages of growth of leaf primordia and conversely that reduced GA biosynthesis increases the capability of the tomato leaf to produce leaflets in response to elevated KNOX activity. We propose that, in tomato, DELLA activity regulates leaflet number by defining the correct timing for leaflet initiation.     

Asymmetric auxin response precedes asymmetric growth and differentiation of asymmetric leaf1 and asymmetric leaf2 Arabidopsis leaves

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.     

SIMPLE LEAF3 encodes a ribosome‐associated protein required for leaflet development in Cardamine hirsuta

Leaves show considerable variation in shape, and may be described as simple, when the leaf is entire, or dissected, when the leaf is divided into individual leaflets. Here, we report that the SIMPLE LEAF3 (SIL3) gene is a novel determinant of leaf shape in Cardamine hirsuta – a dissected‐leaved relative of the simple‐leaved model species Arabidopsis thaliana. We show that SIL3 is required for leaf growth and leaflet formation but leaf initiation is less sensitive to perturbation of SIL3 activity. SIL3 is further required for KNOX (knotted1‐like homeobox) gene expression and localized auxin activity maxima, both of which are known to promote leaflet formation. We cloned SIL3 and showed that it encodes RLI2 (RNase L inhibitor 2), an ATP binding cassette‐type ATPase with important roles in ribosome recycling and translation termination that are conserved in eukaryotes and archaea. RLI mutants have not been described in plants to date, and this paper highlights the potential of genetic studies in C. hirsuta to uncover novel gene functions. Our data indicate that leaflet development is sensitive to perturbation of RLI2‐dependent aspects of cellular growth, and link ribosome function with dissected‐leaf development.     

LEAF DEVELOPMENT IN THE NORMAL AND SOLANIFOLIA MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM)

Leaf development in the normal (lobed margin) and the solanifolia (sf/sf) mutant (entire margin) of tomato (Lycopersicon esculentum) was compared at the light and scanning electron microscope levels. The shoot apices of the mutant plants contained microbodies near the axil of the youngest leaf, which were absent in the normal plants. The structural and morphological events in the initiation of leaf primordia were similar in the two genotypes. The pattern of leaflet emergence was also similar in the two types of plants, but the timing of leaflet production was different. The first pair of leaflet primordia in the normal plants was produced on P₃, whereas in the mutant it was not produced until P₅. The adult leaves of sf/sf plants were larger than those of normal, and the greater leaf area in the mutant was associated with a greater adaxial epidermal cell and areole area. A continuous marginal fimbriate vein (MFV) was present along the margin of each of the normal leaflets. However, a continuous MFV was absent in the mutant leaflets. It is suggested that the absence of a continuous MFV in the mutant might alter the nutritional and hormonal supply to the leaf margin, which ultimately leads to a modified leaf, i.e., with an entire margin.     

Auxin–cytokinin and auxin–gibberellin interactions during morphogenesis of the compound leaves of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression.     

Different expression patterns of duplicated PHANTASTICA-like genes in Lotus japonicus suggest their divergent functions during compound leaf development

Recent studies on leaf development demonstrate that the mechanism on the adaxial-abaxial polarity pattern formation could be well conserved among the far-related species, in which PHANTASTICA （PAHN）-Iike genes play important roles. In this study, we explored the conservation and diversity on functions of PHAN-Iike genes during the compound leaf development in Lotusjaponicus, a papilionoid legume. Two PHAN-Iike genes in L. japonicus, LjPHANa and LjPHANb, were found to originate from a gene duplication event and displayed different expression patterns during compound leaf development. Two mutants, reduced leafletsl （rell） and reduced leaflets3 （rel3）, which exhibited decreased adaxial identity of leaflets and reduced leaflet initiation, were identified and investigated. The expression patterns of both LjPHANs in rel mutants were altered and correlated with abnormalities of compound leaves. Our data suggest that LjPHANa and LjPHANb play important but divergent roles in regulating adaxial-abaxial polarity of compound leaves in L. japonicus.     

Different expression patterns of duplicated PHANTASTICA-like genes in Lotus japonicus suggest their divergent functions during compound leaf development

INTRODUCTION The leaf organs of higher plants can be classified as simple or compound leaves. Compound leaves are found in distantly related groups, and differ from simple leaves in that each petiole bears multiple leaflets lacking auxiliary buds [1, 2]. …     

Acropetal leaflet initiation of Eschscholzia californica is achieved by constant spacing of leaflets and differential growth of leaf

In compound leaves, leaflet primordia are initiated directionally along the lateral sides. Our understanding of the molecular basis of leaflet initiation has improved, but the regulatory mechanisms underlying spatio-temporal patterns remain unclear. In this study, we investigated the mechanisms of acropetal (from the base to the tip) progression of leaflet initiation in Eschscholzia californica. We established an ultraviolet-laser ablation system to manipulate compound-leaf development. Local ablation at the leaflet incipient site generated leaves with asymmetric morphology. In the majority of cases, leaflets that were initiated on the ablated sides shifted apically. Finite time-course observation revealed that the timing of leaflet initiation was delayed, but the distance from the leaf tip did not decrease. These results were suggestive of the local spacing mechanism in leaflet initiation, whereby the distance from the leaf tip and adjacent pre-existing leaflet determines the position of leaflet initiation. To understand how such a local patterning mechanism generates a global pattern of successive leaflet initiation, we assessed the growth rate gradient along the apical–basal axis. Our time-course analysis revealed differential growth rates along the apical–basal axis of the leaf, which can explain the acropetal progression of leaflet initiation. We propose that a leaflet is initiated at a site where the distances from pre-existing leaflets and the leaf tip are sufficient. Furthermore, the differential growth rate may be a developmental factor underlying the directionality of leaflet initiation.     

Regulation of leaf morphology by microRNA394 and its target LEAF CURLING RESPONSIVENESS

The present study identified Arabidopsis miR394 and its target, an F-box (SKP1-Cullin/CDC53-F-box) gene At1g27340 (here referred to as LEAF CURLING RESPONSIVENESS, LCR), for regulation of leaf curling-related morphology. The loss-of-function lcr mutants exhibit pleiotropic defects with semi-dwarfism, altered leaf shape and a shorter stem. Overexpression of an miR394-resistant version of LCR under the 35S promoter (35S:m5LCR) and target mimicry MIM394 resulted in a curled-down leaf defect. Conversely, transgenic plants overexpressing 35S:MIR394a/b display a curled-up leaf phenotype. Detailed analyses show that there is a certain level of LCR that is optimal for leaf morphology, but lower or higher levels lead to abnormal leaf development, indicating that expression of miR394 in the leaf lamina is necessary for proper leaf morphology. Because the phytohormone auxin plays a crucial role in leaf morphogenesis and patterning, the DR5-GUS reporter gene was used to monitor the auxin response. We show that DR5 expression patterns in lcr and 35S::m5LCR plants were significantly different from those in the wild type. Also, overexpression of LCR in 35S::m5LCR plants drastically decreased the expression of the auxin-responsive genes IAA3, AXR3 and IAMT1, whereas increased expression of the genes was found in 35S::MIR394a plants. These results indicate that miR394 and its target LCR are involved in the regulation of leaf development.     

The mutant crispa reveals multiple roles for PHANTASTICA in pea compound leaf development

Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple- and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.     

Auxin efflux transporter MtPIN10 regulates compound leaf and flower development in Medicago truncatula

Plant diversity in nature is to a large extent reflected by morphological diversity of their leaves. Both simple and dissected (with multiple blades or leaflets) leaves are initiated from shoot apical meristem (SAM) in a highly ordered fashion. Similarly, development of leaflets from leaf marginal meristem (marginal blastozone) is also highly ordered. How morphological diversity of plant leaves is regulated remains an important topic of studies on plant form evolution. Here, we describe isolation and characterization of loss-of-function mutants of auxin efflux transporter MtPIN10 of a legume species, Medicago truncatula. Mtpin10 mutants exhibit defects in diverse developmental processes including leaf and leaflet development. Cross species genetic complementation demonstrates that MtPIN10 and Arabidopsis PIN1 are functional orthologs. Double mutant analyses reveal complex genetic interactions between MtPIN10 and Medicago SINGLE LEAFLET1 (SGL1) and CUP-SHAPED COTYLEDON2 (MtCUC2), three regulatory genes involved in developmental processes including dissected leaf and flower development.Key words: auxin, auxin transport, compound leaf development, MtPIN10, SGL1, MtCUC2, Medicago truncatula     

Developmental analysis of a Medicago truncatula smooth leaf margin1 mutant reveals context-dependent effects on compound leaf development

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula, a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula.     

Control of Leaf and Vein Development by Auxin

Leaves are the main photosynthetic organs of vascular plants and show considerable diversity in their geometries, ranging from simple spoonlike forms to complex shapes with individual leaflets, as in compound leaves. Leaf vascular tissues, which act as conduits of both nutrients and signaling information, are organized in networks of different architectures that usually mirror the surrounding leaf shape. Understanding the processes that endow leaves and vein networks with ordered and closely aligned shapes has captured the attention of biologists and mathematicians since antiquity. Recent work has suggested that the growth regulator auxin has a key role in both initiation and elaboration of final morphology of both leaves and vascular networks. A key feature of auxin action is the existence of feedback loops through which auxin regulates its own transport. These feedbacks may facilitate the iterative generation of basic modules that underlies morphogenesis of both leaves and vasculature.Leaf form and vascular patterns provide some of the most impressive examples of the complexity of biological shapes generated in nature. A common feature of the development of the leaf lamina and vein networks is the repeated use of basic modules. For example, the iterative emergence of marginal leaf-shape elements, such as serrations, lobes, and leaflets (Fig. 1A–D), and the arrangement of successive orders of branched veins result in different types of leaf geometries and vascular patterns, respectively. Intriguingly, there is also congruence of leaf shape and vein layouts, such that, at least superficially, the pattern of vasculature formation is well aligned with the final geometry of the leaf lamina. These observations raise the questions of (1) what are the specific signaling pathways that sculpt leaf shape and vascular patterns, (2) to what degree lamina growth and vascular development share common genetic control, and finally (3) how coordination between leaf and vascular development is achieved and impacts on generation of final leaf shape and vein arrangement. Over the past 15 years, genetic approaches have led to substantial increase in our understanding of leaf and vascular development, and have provided good evidence that regulated activity of the small indolic growth regulator auxin provides important spatial cues for both processes. Such roles of auxin in different facets of leaf and vascular development is the focus of our article.Open in a separate windowFigure 1.Axes of leaf asymmetry and diversity of leaf shape. (A) A simple, serrated leaf of the Columbia ecotype of Arabidopsis thaliana. The proximo–distal (P–D) and medio–lateral (M–L) axes are indicated in the image. The asterisk marks one marginal serration. (B) The lobed leaf of the Arabidopsis thaliana relative Arabidopsis lyrata. The asterisk depicts the position of one lobe. Lobes are deep serrations, so the definition of an outgrowth as a serration or lobe is somewhat arbitrary. (C) The dissected leaf of Cardamine hirsuta. The asterisk marks a lateral leaflet. Leaflets are clearly defined as distinct units of the same leaf, which connect with the rachis (R) via a structure called a petiolule (Pu). (D) The dissected leaf of the cultivated tomato. Tomato demonstrates additional orders of dissection with respect to Cardamine hirsuta leaf and produces both primary leaflets (black asterisk) and secondary leaflets (red asterisk). (E) Scanning electron micrograph of the shoot apex of tomato. The white asterisk marks a leaf primordium (1) initiating from the meristem. The adaxial (yellow) and abaxial (orange) domains are marked on the subsequent developing leaf (2). Tomato is a compound leaf plant where leaflets are formed from the leaf blade soon after leaf initiation (a developing leaflet is marked by an arrow in leaf 3). Images in panels A–D are leaf silhouettes. Scale bars: (A–D) 1 cm, (E) 100 µm.     

Responses of plant vascular systems to auxin transport inhibition.

To assess the role of auxin flows in plant vascular patterning, the development of vascular systems under conditions of inhibited auxin transport was analyzed. In Arabidopsis, nearly identical responses evoked by three auxin transport inhibitor substances revealed an enormous plasticity of the vascular pattern and suggest an involvement of auxin flows in determining the sites of vascular differentiation and in promoting vascular tissue continuity. Organs formed under conditions of reduced auxin transport contained increased numbers of vascular strands and cells within those strands were improperly aligned. In leaves, vascular tissues became progressively confined towards the leaf margin as the concentration of auxin transport inhibitor was increased, suggesting that the leaf vascular system depends on inductive signals from the margin of the leaf. Staged application of auxin transport inhibitor demonstrated that primary, secondary and tertiary veins became unresponsive to further modulations of auxin transport at successive stages of early leaf development. Correlation of these stages to anatomical features in early leaf primordia indicated that the pattern of primary and secondary strands becomes fixed at the onset of lamina expansion. Similar alterations in the leaf vascular responses of alyssum, snapdragon and tobacco plants suggest common functions of auxin flows in vascular patterning in dicots, while two types of vascular pattern alterations in Arabidopsis auxin transport mutants suggest that at least two distinct primary defects can result in impaired auxin flow. We discuss these observations with regard to the relative contributions of auxin transport, auxin sensitivity and the cellular organisation of the developing organ on the vascular pattern.     

MORPHOLOGY AND VASCULATURE OF THE LEAF OF POTATO (SOLANUM TUBEROSUM)

The leaf and stem of the potato plant (Solanum tuberosum L. cv. Russet Burbank) were studied by light microscopy to determine their morphology and vasculature; scanning electron microscopy provided supplemental information on the leaf's morphology. The morphology of the basal leaves of the potato shoot is quite variable, ranging from simple to pinnately compound. The upper leaves of the shoot are more uniform, being odd pinnate with three major pairs of lateral leaflets and a number of folioles. The primary vascular system of the stem is comprised of six bundles, three large and three small ones. The three large bundles form a highly interconnected system through a repeated series of branchings and arch-producing mergers. Two of the three large bundles give rise to short, lateral leaf traces at each node. Each of the small bundles in the stem is actually a median leaf trace which extends three internodes before diverging into a leaf. The three leaf traces enter the petiole through a single gap; thus the nodel anatomy is three-trace unilacunar. Upon entering the petiole, each of the laterals splits into an upper and a lower lateral. Whereas the upper laterals diverge entirely into the first pair of leaflets, the lower laterals feed all of the lateral leaflets through a series of bifurcations. Prior to their entering the terminal leaflet, the lower laterals converge on the median bundle to form a single vascular crescent which progresses acropetally into the terminal leaflet as the midvein, or primary vein. In the midrib, portions of the midvein diverge outward and continue as secondaries to the margin on either side of the lamina. Near the tip of the terminal leaflet, the midvein consists of a single vascular bundle which is a continuation of the median bundle. Six to seven orders of veins occur in the terminal leaflet.