Potential but limited redundant roles of MtPIN4, MtPIN5 and MtPIN10/SLM1 in the development of Medicago truncatula

Auxin polar transport is crucial in regulating plant growth and patterning. As auxin efflux carriers, the PIN FORMED (PIN) proteins are responsible for transportation of auxin out of the cell. There are eight and ten PIN members in Arabidopsis (AtPIN) and Medicago truncatula (MtPIN), respectively. Compared with MtPIN10/SMOOTH LEAF MARGIN1 (SLM1), MtPIN4 exhibits a closer relationship with AtPIN1 based phylogenetic analysis. In addition, the gene structure and distribution of transmembrane segments of MtPIN4, MtPIN5 and MtPIN10/SLM1 are similar, implying possible redundant roles among them. However, analysis using Gene Expression Atlas revealed different expression patterns among MtPIN4, MtPIN5 and MtPIN10/SLM1. Loss of function of MtPIN10/SLM1 in M. truncatula resulted in pleiotropic phenotypes in different organs, which are similar with the defects in the pin1 mutant of Arabidopsis, suggesting that the MtPIN10/SLM1 is a putative ortholog of AtPIN1. MtPIN4, MtPIN5 and MtPIN10/SLM1 may have limited redundant functions in the development of M. truncatula. The creation of double and triple mutants will help to elucidate their potential roles in auxin transport and plant development.     

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.     

Weeds of change: Cardamine hirsuta as a new model system for studying dissected leaf development

Cardamine hirsuta, a small crucifer closely related to the model organism Arabidopsis thaliana, offers high genetic tractability and has emerged as a powerful system for studying the genetic basis for diversification of plant form. Contrary to A. thaliana, which has simple leaves, C. hirsuta produces dissected leaves divided into individual units called leaflets. Leaflet formation requires activity of Class I KNOTTED1-like homeodomain (KNOX) proteins, which also promote function of the shoot apical meristem (SAM). In C. hirsuta, KNOX genes are expressed in the leaves whereas in A. thaliana their expression is confined to the SAM, and differences in expression arise through cis-regulatory divergence of KNOX regulation. KNOX activity in C. hirsuta leaves delays the transition from proliferative growth to differentiation thus facilitating the generation of lateral growth axes that give rise to leaflets. These axes reflect the sequential generation of cell division foci across the leaf proximodistal axis in response to auxin activity maxima, which are generated by the PINFORMED1 (PIN1) auxin efflux carriers in a process that resembles organogenesis at the SAM. Delimitation of C. hirsuta leaflets also requires the activity of CUP SHAPED COTYLEDON (CUC) genes, which direct formation of organ boundaries at the SAM. These observations show how species-specific deployment of fundamental shoot development networks may have sculpted simple versus dissected leaf forms. These studies also illustrate how extending developmental genetic studies to morphologically divergent relatives of model organisms can greatly help elucidate the mechanisms underlying the evolution of form.     

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.     

Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato

The enormous variation in architecture of flowering plants is based to a large extent on their ability to form new axes of growth throughout their life span. Secondary growth is initiated from groups of pluripotent cells, called meristems, which are established in the axils of leaves. Such meristems form lateral organs and develop into a side shoot or a flower, depending on the developmental status of the plant and environmental conditions. The phytohormone auxin is well known to play an important role in inhibiting the outgrowth of axillary buds, a phenomenon known as apical dominance. However, the role of auxin in the process of axillary meristem formation is largely unknown. In this study, we show in the model species Arabidopsis thaliana and tomato (Solanum lycopersicum) that auxin is depleted from leaf axils during vegetative development. Disruption of polar auxin transport compromises auxin depletion from the leaf axil and axillary meristem initiation. Ectopic auxin biosynthesis in leaf axils interferes with axillary meristem formation, whereas repression of auxin signaling in polar auxin transport mutants can largely rescue their branching defects. These results strongly suggest that depletion of auxin from leaf axils is a prerequisite for axillary meristem formation during vegetative development.     

Studies of aberrant phyllotaxy1 Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.     

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.     

Multiple MONOPTEROS-dependent pathways are involved in leaf initiation

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.     

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.     

STM/BP-Like KNOXI Is Uncoupled from ARP in the Regulation of Compound Leaf Development in Medicago truncatula

Class I KNOTTED-like homeobox (KNOXI) genes are critical for the maintenance of the shoot apical meristem. The expression domain of KNOXI is regulated by ASYMMETRIC LEAVES1/ROUGHSHEATH2/PHANTASTICA (ARP) genes, which are associated with leaf morphology. In the inverted repeat-lacking clade (IRLC) of Fabaceae, the orthologs of LEAFY (LFY) function in place of KNOXI to regulate compound leaf development. Here, we characterized loss-of-function mutants of ARP (PHAN) and SHOOTMERISTEMLESS (STM)- and BREVIPEDICELLUS (BP)-like KNOXI in the model IRLC legume species Medicago truncatula. The function of ARP genes is species specific. The repression of STM/BP-like KNOXI genes in leaves is not mediated by PHAN, and no suppression of PHAN by STM/BP-like KNOXI genes was observed either, indicating that STM/BP-like KNOXI genes are uncoupled from PHAN in M. truncatula. Furthermore, comparative analyses of phenotypic output in response to ectopic expression of KNOXI and the M. truncatula LFY ortholog, SINGLE LEAFLET1 (SGL1), reveal that KNOXI and SGL1 regulate parallel pathways in leaf development. We propose that SGL1 probably functions in a stage-specific manner in the regulation of the indeterminate state of developing leaves in M. truncatula.     

Control of compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medicago truncatula

Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

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. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

RNAi Phenotypes and the Localization of a Protein::GUS Fusion Imply a Role for Medicago truncatula PIN Genes in Nodulation

The symbiosis between legumes and rhizobia results in the development of a new plant organ, the nodule. A role for polar auxin transport in nodule development in Medicago truncatula has been demonstrated using molecular genetic tools. The expression of a DR5::GUS auxin-responsive promoter in uninoculated M. truncatula roots mirrored that reported in Arabidopsis, and expression of the construct in nodulating roots confirmed results reported in white clover. The localization of a root-specific PIN protein (MtPIN2) in normal roots, developing lateral roots and nodules provided the first evidence that a PIN protein is expressed in nodules. Reduced levels of MtPIN2, MtPIN3, and MtPIN4 mRNAs via RNA interference demonstrated that plants with reduced expression of various MtPINs display a reduced number of nodules. The reported results show that in M. truncatula, PIN proteins play an important role in nodule development, and that nodules and lateral roots share some early auxin responses in common, but they rapidly differentiate with respect to auxin and MtPIN2 protein distribution.     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.     

The Stem Cell Niche in Leaf Axils Is Established by Auxin and Cytokinin in Arabidopsis

Plants differ from most animals in their ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems harboring new stem cell niches. In seed plants, this is achieved by axillary meristems, which are established in the axil of each leaf base and develop into lateral branches. Here, we describe the initial processes of Arabidopsis thaliana axillary meristem initiation. Using reporter gene expression analysis, we find that axillary meristems initiate from leaf axil cells with low auxin through stereotypical stages. Consistent with this, ectopic overproduction of auxin in the leaf axil efficiently inhibits axillary meristem initiation. Furthermore, our results demonstrate that auxin efflux is required for the leaf axil auxin minimum and axillary meristem initiation. After lowering of auxin levels, a subsequent cytokinin signaling pulse is observed prior to axillary meristem initiation. Genetic analysis suggests that cytokinin perception and signaling are both required for axillary meristem initiation. Finally, we show that cytokinin overproduction in the leaf axil partially rescue axillary meristem initiation-deficient mutants. These results define a mechanistic framework for understanding axillary meristem initiation.