Compound Leaf Development Research Articles

Genomic analysis of a spontaneous unifoliate mutant reveals gene candidates associated with compound leaf development in Vigna unguiculata [L] Walp

Molecular mechanisms which underpin compound leaf development in some legumes have been reported, but there is no previous study on the molecular genetic control of compound leaf formation in Vigna unguiculata (cowpea), an important dryland legume of African origin. In most studied species with compound leaves, class 1 KNOTTED-LIKE HOMEOBOX genes expressed in developing leaf primordia sustain morphogenetic activity, allowing leaf dissection and the development of leaflets. Other genes, such as, SINGLE LEAFLET1 in Medicago truncatula and Trifoliate in Solanum lycopersicum, are also implicated in regulating compound leaf patterning. To set the pace for an in-depth understanding of the genetics of compound leaf development in cowpea, we applied RNA-seq and whole genome shotgun sequence datasets of a spontaneous cowpea unifoliate mutant and its trifoliate wild-type cultivar to conduct comparative reference-based gene expression, de novo genome-wide isoform switch, and genome variant analyses between the two genotypes. Our results suggest that genomic variants upstream of LATE ELONGATED HYPOCOTYL and down-stream of REVEILLE4, BRASSINOSTERIOD INSENSITIVE1 and LATERAL ORGAN BOUNDARIES result in down-regulation of key components of cowpea circadian rhythm central oscillator and brassinosteroid signaling, resulting in unifoliate leaves and brassinosteroid-deficient-like phenotypes. We have stated hypotheses that will guide follow-up studies expected to provide more insights.

Rewiring of a KNOXI regulatory network mediated by UFO underlies the compound leaf development in Medicago truncatula

Class I KNOTTED-like homeobox (KNOXI) genes are parts of the regulatory network that control the evolutionary diversification of leaf morphology. Their specific spatiotemporal expression patterns in developing leaves correlate with the degrees of leaf complexity between simple-leafed and compound-leafed species. However, KNOXI genes are not involved in compound leaf formation in several legume species. Here, we identify a pathway for dual repression of MtKNOXI function in Medicago truncatula. PINNATE-LIKE PENTAFOLIATA1 (PINNA1) represses the expression of MtKNOXI, while PINNA1 interacts with MtKNOXI and sequesters it to the cytoplasm. Further investigations reveal that UNUSUAL FLORAL ORGANS (MtUFO) is the direct target of MtKNOXI, and mediates the transition from trifoliate to pinnate-like pentafoliate leaves. These data suggest a new layer of regulation for morphological diversity in compound-leafed species, in which the conserved regulators of floral development, MtUFO, and leaf development, MtKNOXI, are involved in variation of pinnate-like compound leaves in M. truncatula.

GRAS transcription factor PINNATE-LIKE PENTAFOLIATA2 controls compound leaf morphogenesis in Medicago truncatula.

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation.

The role of Class II KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXⅡ) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress the leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations show that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXI-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (ortholog of Arabidopsis class M KNOX) and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for the MtKNOX4-mediated compound leaf formation. These findings suggest that among the members of MtKNOXII, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula. This article is protected by copyright. All rights reserved.

SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

Transcriptome Analyses Reveal the Key Regulators of Tomato Compound Leaf Development

Leaves are one of the organs involved in plant assimilation and transpiration. Different leaf development processes can result in different leaf shapes. Tomato plants have typical compound leaves. It is helpful to explore the regulatory factors affecting the leaf development and morphogenesis of tomatoes to cultivate varieties with high photosynthetic efficiency. We used the typical tomato leaf shape mutants Petroselinum (Pts), Trifoliate (tf2), and Entire (e), which showed a gradual decrease in leaflet number and compound leaf complexity. Transcriptome sequencing was performed to analyze the key differentially expressed genes (DEGs) among the 3 groups, which revealed 2393, 1366, and 1147 DEGs in Pts/VF36, tf2/CR, and e/AC, respectively. We found 86 overlapping DEGs among the 3 groups. In addition, we found that the mutation of Pts, tf2, and e affected not only leaf morphology but also the wax, fatty acid, and abscisic acid pathways during growth and development. An RT-qPCR analysis during leaf primordium development revealed three transcription factors (bHLH079, WRKY44, and WRKY76) and three hormone-regulated genes (IAA-amino acid hydrolase, Gibberellin2ox7, and Gibberellin20ox) that were differentially expressed in the transcriptome. Using virus-induced gene silencing (VIGS), we observed the leaf shape of VIGS plants and found that bHLH079, IAA-amino acid hydrolase, Gibberellin2ox7, Gibberellin20ox, WRKY44, and WRKY76 were the endogenous regulators influencing tomato compound leaf development. This study provides a promising direction for revealing the molecular regulation mechanism underlying compound leaf development in tomatoes.

A GT-1 and PKc domain-containing transcription regulator SIMPLE LEAF1 controls compound leaf development in woodland strawberry.

Leaves are strikingly diverse in terms of shapes and complexity. The wild and cultivated strawberry plants mostly develop trifoliate compound leaves, yet the underlying genetic basis remains unclear in this important fruit crop in Rosaceae. Here, we identified two EMS mutants designated simple leaf1 (sl1-1 and sl1-2) and one natural simple-leafed mutant monophylla in Fragaria vesca. Their causative mutations all reside in SL1 (FvH4_7g28640) causing premature stop codon at different positions in sl1-1 and sl1-2 and an eight-nucleotide insertion (GTTCATCA) in monophylla. SL1 encodes a transcription regulator with the conserved DNA-binding domain GT-1 and the catalytic domain of protein kinases PKc. Expression of SL1pro::SL1 in sl1-1 completely restored compound leaf formation. The 35S::SL1 lines developed palmate-like leaves with four or five leaflets at a low penetrance. However, overexpressing the truncated SL1ΔPK caused no phenotypes, probably due to the disruption of homodimerization. SL1 is preferentially expressed at the tips of leaflets and serrations. Moreover, SL1 is closely associated with the auxin pathway and works synergistically with FveLFYa in leaf morphogenesis. Overall, our work uncovered a new type of transcription regulator that promotes compound leaf formation in the woodland strawberry and shed new lights on the diversity of leaf complexity control.

Effects of gibberellins on important agronomic traits of horticultural plants.

Horticultural plants such as vegetables, fruits, and ornamental plants are crucial to human life and socioeconomic development. Gibberellins (GAs), a class of diterpenoid compounds, control numerous developmental processes of plants. The roles of GAs in regulating growth and development of horticultural plants, and in regulating significant progress have been clarified. These findings have significant implications for promoting the quality and quantity of the products of horticultural plants. Here we review recent progress in determining the roles of GAs (including biosynthesis and signaling) in regulating plant stature, axillary meristem outgrowth, compound leaf development, flowering time, and parthenocarpy. These findings will provide a solid foundation for further improving the quality and quantity of horticultural plants products.

The VIL gene CRAWLING ELEPHANT controls maturation and differentiation in tomato via polycomb silencing.

VERNALIZATION INSENSITIVE 3-LIKE (VIL) proteins are PHD-finger proteins that recruit the repressor complex Polycomb Repressive Complex 2 (PRC2) to the promoters of target genes. Most known VIL targets are flowering repressor genes. Here, we show that the tomato VIL gene CRAWLING ELEPHANT (CREL) promotes differentiation throughout plant development by facilitating the trimethylation of Histone H3 on lysine 27 (H3K27me3). We identified the crel mutant in a screen for suppressors of the simple-leaf phenotype of entire (e), a mutant in the AUX/IAA gene ENTIRE/SlIAA9, involved in compound-leaf development in tomato. crel mutants have increased leaf complexity, and suppress the ectopic blade growth of e mutants. In addition, crel mutants are late flowering, and have delayed and aberrant stem, root and flower development. Consistent with a role for CREL in recruiting PRC2, crel mutants show drastically reduced H3K27me3 enrichment at approximately half of the 14,789 sites enriched in wild-type plants, along with upregulation of many underlying genes. Interestingly, this reduction in H3K27me3 across the genome in crel is also associated with gains in H3K27me3 at a smaller number of sites that normally have modest levels of the mark in wild-type plants, suggesting that PRC2 activity is no longer limiting in the absence of CREL. Our results uncover a wide role for CREL in plant and organ differentiation in tomato and suggest that CREL is required for targeting PRC2 activity to, and thus silencing, a specific subset of polycomb targets.

Molecular Characterization of the miR156/MsSPL Model in Regulating the Compound Leaf Development and Abiotic Stress Response in Alfalfa.

Plant leaf patterns and shapes are spectacularly diverse. Changing the complexity of leaflet numbers is a valuable approach to increase its nutrition and photosynthesis. Alfalfa (Medicago sativa) is the most important forage legume species and has diversified compound leaf patterns, which makes it a model species for studying compound leaf development. However, transcriptomic information from alfalfa remains limited. In this study, RNA-Seq technology was used to identify 3746 differentially expressed genes (DEGs) between multifoliate and trifoliate alfalfa. Through an analysis of annotation information and expression data, SPL, one of the key regulators in modifiable plant development and abiotic stress response, was further analyzed. Here, thirty MsSPL genes were obtained from the alfalfa genome, of which 16 had the putative miR156 binding site. A tissue expression pattern analysis showed that the miR156-targeted MsSPLs were divided into two classes, namely, either tissue-specific or widely expressed in all tissues. All miR156-targeted SPLs strongly showed diversification and positive roles under drought and salt conditions. Importantly, miR156/MsSPL08 was significantly suppressed in multifoliate alfalfa. Furthermore, in the paralogous mutant of MsSPL08 isolated from Medicago truncatula, the phenotypes of mutant plants reveal that miR156/MsSPL08 is involved not only involved the branches but also especially regulates the number of leaflets. The legume is a typical compound leaf plant; the ratio of the leaflet often affects the quality of the forage. This study sheds light on new functions of SPL genes that regulate leaflet number development.

Developmental Analysis of Compound Leaf Development in Arachis hypogaea.

Leaves are the primary photosynthetic structures, while photosynthesis is the direct motivation of crop yield formation. As a legume plant, peanut (Arachis hypogaea) is one of the most economically essential crops as well as an important source of edible oil and protein. The leaves of A. hypogaea are in the tetrafoliate form, which is different from the trifoliate leaf pattern of Medicago truncatula, a model legume species. In A. hypogaea, an even-pinnate leaf with a pair of proximal and distal leaflets was developed; however, only a single terminal leaflet and a pair of lateral leaflets were formed in the odd-pinnate leaf in M. truncatula. In this study, the development of compound leaf in A. hypogaea was investigated. Transcriptomic profiles revealed that the common and unique differentially expressed genes were identified in a proximal leaflet and a distal leaflet, which provided a research route to understand the leaf development in A. hypogaea. Then, a naturally occurring mutant line with leaf developmental defects in A. hypogaea was obtained, which displayed a pentafoliate form with an extra terminal leaflet. The characterization of the mutant indicated that cytokinin and class I KNOTTED-LIKE HOMEOBOX were involved in the control of compound leaf pattern in A. hypogaea. These results expand our knowledge and provide insights into the molecular mechanism underlying the formation of different compound leaf patterns among species.

Super determinant1A, a RAWULdomain-containing protein, modulates axillary meristem formation and compound leaf development in tomato.

Shoot branching and complex leaf development relies on the establishment of boundaries that precedes the formation of axillary meristems (AMs) and leaflets. The tomato (Solanum lycopersicum) super determinant mutant is compromised in both processes, due to a mutation in Sde1A. Sde1A encodes a protein with a RAWUL domain, which is also present in Polycomb Group Repressive Complex 1 (PRC1) RING finger proteins and WD Repeat Domain 48 proteins. Genetic analysis revealed that Sde1A and Bmi1A cooperate, whereas Bmi1C antagonizes both activities, indicating the existence of functionally opposing PRC1 complexes that interact with Sde1A. Sde1A is expressed at early stages of boundary development in a small group of cells in the center of the leaf-axil boundary, but its activity is required for meristem formation at later stages. This suggests that Sde1A and Bmi1A promote AM formation and complex leaf development by safeguarding a pool of cells in the developing boundary zones. Genetic and protein interaction analyses showed that Sde1A and Lateral suppressor (Ls) are components of the same genetic pathway. In contrast to ls, sde1a mutants are not compromised in inflorescence branching, suggesting that Sde1A is a potential target for breeding tomato cultivars with reduced side-shoot formation during vegetative development.

Coordinating the morphogenesis-differentiation balance by tweaking the cytokinin-gibberellin equilibrium.

Morphogenesis and differentiation are important stages in organ development and shape determination. However, how they are balanced and tuned during development is not fully understood. In the compound leaved tomato, an extended morphogenesis phase allows for the initiation of leaflets, resulting in the compound form. Maintaining a prolonged morphogenetic phase in early stages of compound-leaf development in tomato is dependent on delayed activity of several factors that promote differentiation, including the CIN-TCP transcription factor (TF) LA, the MYB TF CLAU and the plant hormone Gibberellin (GA), as well as on the morphogenesis-promoting activity of the plant hormone cytokinin (CK). Here, we investigated the genetic regulation of the morphogenesis-differentiation balance by studying the relationship between LA, CLAU, TKN2, CK and GA. Our genetic and molecular examination suggest that LA is expressed earlier and more broadly than CLAU and determines the developmental context of CLAU activity. Genetic interaction analysis indicates that LA and CLAU likely promote differentiation in parallel genetic pathways. These pathways converge downstream on tuning the balance between CK and GA. Comprehensive transcriptomic analyses support the genetic data and provide insights into the broader molecular basis of differentiation and morphogenesis processes in plants.

The role of WOX1 genes in blade development and beyond.

This article comments on:Wang C, Zhao B, He L, Zhou S, Liu Ye, Zhao W, Guo S, Wang R, Bai Q, Li Y, Wang D, Wu Q, Yang Y, Yan J, Liu Yu, Tadege M, Chen J. 2021. The WOX family transcriptional regulator SlLAM1 controls compound leaf and floral organ development in Solanum lycopersicum. Journal of Experimental Botany 72, 1822–1835.

Coordination of differentiation rate and local patterning in compound-leaf development.

The variability in leaf form in nature is immense. Leaf patterning occurs by differential growth, taking place during a limited window of morphogenetic activity at the leaf marginal meristem. While many regulators have been implicated in the designation of the morphogenetic window and in leaf patterning, how these effectors interact to generate a particular form is still not well understood. We investigated the interaction among different effectors of tomato (Solanum lycopersicum) compound-leaf development, using genetic and molecular analyses. Mutations in the tomato auxin response factor SlARF5/SlMP, which normally promotes leaflet formation, suppressed the increased leaf complexity of mutants with extended morphogenetic window. Impaired activity of the NAC/CUC transcription factor GOBLET (GOB), which specifies leaflet boundaries, also reduced leaf complexity in these backgrounds. Analysis of genetic interactions showed that the patterning factors SlMP, GOB and the MYB transcription factor LYRATE (LYR) coordinately regulate leaf patterning by modulating in parallel different aspects of leaflet formation and shaping. This work places an array of developmental regulators in a morphogenetic context. It reveals how organ-level differentiation rate and local growth are coordinated to sculpture an organ. These concepts are applicable to the coordination of pattering and differentiation in other species and developmental processes.

The WOX family transcriptional regulator SlLAM1 controls compound leaf and floral organ development in Solanum lycopersicum.

Plant-specific WOX family transcription factors play important roles ranging from embryogenesis to lateral organ development. The WOX1 transcription factors, which belong to the modern clade of the WOX family, are known to regulate outgrowth of the leaf blade specifically in the mediolateral axis; however, the role of WOX1 in compound leaf development remains unknown. Phylogenetic analysis of the whole WOX family in tomato (Solanum lycopersicum) indicates that there are 10 members that represent the modern, intermediate, and ancient clades. Using phylogenetic analysis and a reverse genetic approach, in this study we identified SlLAM1 in the modern clade and examined its function and tissue-specific expression pattern. We found that knocking out SlLAM1 via CRISPR/Cas9-mediated genome editing led to narrow leaves and a reduced number of secondary leaflets. Overexpression of tomato SlLAM1 could rescue the defects of the tobacco lam1 mutant. Anatomical and transcriptomic analyses demonstrated that floral organ development, fruit size, secondary leaflet initiation, and leaf complexity were altered due to loss-of-function of SlLAM1. These findings demonstrate that tomato SlLAM1 plays an important role in the regulation of secondary leaflet initiation, in addition to its conserved function in blade expansion.

The 3-ketoacyl-CoA synthase WFL is involved in lateral organ development and cuticular wax synthesis in Medicago truncatula.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

Leaflet initiation and blade expansion are separable in compound leaf development

Compound leaves are composed of multiple separate blade units termed leaflets. In tomato (Solanum lycopersicum) compound leaves, auxin promotes both leaflet initiation and blade expansion. However, it is unclear how these two developmental processes interact. With highly variable complexity, tomato compound leaves provide an ideal system to address this question. In this study, we obtained and analyzed mutants of the WUSCHEL-RELATED HOMEOBOX (WOX) family gene SlLAM1 from tomato, whose orthologs in tobacco (Nicotiana sylvestris) and other species are indispensable for blade expansion. We show that SlLAM1 is expressed in the middle and marginal domains of leaves, and is required for blade expansion in leaflets. We demonstrate that sllam1 mutants cause a delay of leaflet initiation and slightly alter the arrangement of first-order leaflets, whereas the overall leaflet number is comparable to that of wild-type leaves. Analysis of the genetic interactions between SlLAM1 and key auxin signaling components revealed an epistatic effect of SlLAM1 in determining the final leaf form. Finally, we show that SlLAM1 is also required for floral organ growth and affects the fertility of gametophytes. Our data suggest that SlLAM1 promotes blade expansion in multiple leaf types, and leaflet initiation can be largely uncoupled from blade expansion during compound leaf morphogenesis.

Lateral Leaflet Suppression 1 (LLS1), encoding the MtYUCCA1 protein, regulates lateral leaflet development in Medicago truncatula.

In species with compound leaves, the positions of leaflet primordiuminitiation are associated with local peaks of auxin accumulation. However, the role of auxin during the late developmental stages and outgrowth of compound leaves remains largely unknown. Using genome resequencing approaches, we identified insertion sites at four alleles of the LATERAL LEAFLET SUPPRESSION1 (LLS1) gene, encoding the auxin biosynthetic enzyme YUCCA1 in Medicago truncatula. Linkage analysis and complementation tests showed that the lls1 mutant phenotypes were caused by the Tnt1 insertions that disrupted the LLS1 gene. The transcripts of LLS1 can be detected in primordia at early stages of leaf initiation and later in the basal regions of leaflets, and finally in vein tissues at late leaf developmental stages. Vein numbers and auxin content are reduced in the lls1-1 mutant. Analysis of the lls1 sgl1 and lls1 palm1 double mutants revealed that SGL1 is epistatic to LLS1, and LLS1 works with PALM1 in an independent pathway to regulate the growth of lateral leaflets. Our work demonstrates that the YUCCA1/YUCCA4 subgroup plays very important roles in the outgrowth of lateral leaflets during compound leaf development of M.truncatula, in addition to leaf venation.

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.