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. 2021 Jun 11;187(1):218–235. doi: 10.1093/plphys/kiab268

LATE MERISTEM IDENTITY1 regulates leaf margin development via the auxin transporter gene SMOOTH LEAF MARGIN1

Xiao Wang 1, Juanjuan Zhang 1, Yangyang Xie 1, Xiu Liu 1, Lizhu Wen 1, Hongfeng Wang 1,2, Jing Zhang 1, Jie Li 1, Lu Han 1, Xiaolin Yu 1, Kirankumar S Mysore 3, Jiangqi Wen 3, Chuanen Zhou 1,✉,†
PMCID: PMC8418409  PMID: 34618141

Abstract

Plant leaves have evolved into diverse shapes and LATE MERISTEM IDENTITY1 (LMI1) and its putative paralogous genes encode homeodomain leucine zipper transcription factors that are proposed evolutionary hotspots for the regulation of leaf development in plants. However, the LMI1-mediated regulatory mechanism underlying leaf shape formation is largely unknown. MtLMI1a and MtLMI1b are putative orthologs of LMI1 in the model legume barrelclover (Medicago truncatula). Here, we investigated the role of MtLMI1a and MtLMI1b in leaf margin morphogenesis by characterizing loss-of-function mutants. MtLMI1a and MtLMI1b are expressed along leaf margin in a near-complementary pattern, and they redundantly promote development of leaf margin serrations, as revealed by the relatively smooth leaf margin in their double mutants. Moreover, MtLMI1s directly activate expression of SMOOTH LEAF MARGIN1 (SLM1), which encodes an auxin efflux carrier, thereby regulating auxin distribution along the leaf margin. Further analysis indicates that MtLMI1s genetically interact with NO APICAL MERISTEM (MtNAM) and the ARGONAUTE7 (MtAGO7)-mediated trans-acting short interfering RNA3 (TAS3 ta-siRNA) pathway to develop the final leaf margin shape. The participation of MtLMI1s in auxin-dependent leaf margin formation is interesting in the context of functional conservation. Furthermore, the diverse expression patterns of LMI1s and their putative paralogs within key domains are important drivers for functional specialization, despite their functional equivalency among species.

In barrelclover, the MtLMI1 transcription factors activate transcription of an auxin efflux carrier gene to regulate auxin distribution and control leaf margin development.

Introduction

Leaves play important roles in capturing light energy during photosynthesis, gas exchange, water transport, and environmental adaptation in plants (Tsukaya, 2006). In response to complex environmental conditions such as temperature, hydraulic pressure, light, and herbivory, leaves evolve into different shapes; a leaf blade is simple or compound and leaf margin can be categorized into smooth, serrated, or lobed (Champagne and Sinha, 2004; Nicotra et al., 2011).

Leaves begin as the peg-like primordia from the flanks of the shoot apical meristem (SAM), while leaflets are initiated from the marginal region of the primordia and later form their final shape (Moon and Hake, 2011). The shape of leaves depends on the formation of a correct leaf margin. Auxin transport carrier PIN-FORMED1 (PIN1) has a polar localization and creates auxin activity maxima, which are involved in the development of serrations along the leaf margin, the initiation of leaf and leaflet primordia as well as the midvein and lateral vein formation (Barkoulas et al., 2008; Koenig et al., 2009; Zhou et al., 2011; Kierzkowski et al., 2019). Perturbation of polar auxin transport by N-1-naphthylphthalamic acid (NPA) or mutation in either PIN1 or regulators of auxin response pathway factors results in failure to form leaf serrations and a plant that exhibits toothless leaves (Scanlon, 2003; DeMason and Chawla, 2004; Koenig et al., 2009; Zhou et al., 2011; Ben-Gera et al., 2012; Kierzkowski et al., 2019). On the contrary, ectopic expression of these genes or exogenous auxin application results in more serrations or generates extra leaflets (Barkoulas et al., 2008; Koenig et al., 2009). Much effort has been devoted to the identification of regulators for leaf margin development. Several studies showed that NO APICAL MERISTEM and CUP-SHAPED COTYLEDON genes are involved in the formation of leaf margin (Nikovics et al., 2006; Blein et al., 2008; Hasson et al., 2011; Maugarny-Cales et al., 2019; Serra and Perrot-Rechenmann, 2020). In mouse-ear cress (Arabidopsis thaliana), leaves of cuc2 mutants are smooth. The transcripts of CUC2 are targeted by miR164A; thus, overexpression of miR164 decreases leaf serration number (Nikovics et al., 2006). Moreover, the negative feedback loops involving PIN1 and CUC2 play a key role in the formation of leaf serrations in Arabidopsis. In this model, CUC2 promotes PIN1-dependent auxin activity maxima at the teeth tips; meanwhile, high auxin in the tips confines CUC2 expression to the sinuses, thus repressing the growth of teeth (Nikovics et al., 2006; Bilsborough et al., 2011; Kierzkowski et al., 2019).

Homeodomain leucine zipper (HD-Zip) family members act as transcription factors and are present only in plants. They have been reported to take part in many biological processes, including vascular development, secondary cell wall formation, trichome development, and leaf polarity establishment (Zhao et al., 2015; Merelo et al., 2016; Zhou et al., 2019; Whitewoods et al., 2020). In total, 47 members in the HD-Zip family have been found in Arabidopsis. Based on the sequence characteristics, these HD-Zip genes are grouped into four different classes: HD-Zip I–IV. Among them, 17 genes belong to HD-Zip I subfamily, which function as transcriptional activators and respond to different environmental signals (Schena and Davis, 1992; Henriksson et al., 2005). In Arabidopsis, LATE MERISTEM IDENTITY1 (LMI1) is an HD-Zip I transcription factor and functions as a meristem identity regulator. LMI1 is a target of LEAFY and, together with LEAFY, activates the expression of CAULIFLOWER (CAL). As a growth repressor, LMI1 functions in the formation of bracts as well as serrations, but the mechanism is still unknown (Saddic et al., 2006). In Cardamine hirsuta, ChLMI1 shows a similar expression pattern as LMI1, which is expressed at the leaf margin. ChLMI1 cannot increase the leaf complexity when overexpressed in Arabidopsis, but is able to complement the phenotype of smooth leaf margin in lmi1 mutant (Vlad et al., 2014). The REDUCED COMPLEXITY (RCO) gene, which is the putative paralog of ChLMI1, promotes leaf complexity in C. hirsuta and Capsella species. The combined expression of RCO and SHOOTMERISTEMLESS (STM) reconstructs dissected leaf shape in Arabidopsis (Sicard et al., 2014; Vlad et al., 2014; Kierzkowski et al., 2019). Moreover, the LMI1 putative orthologs in cotton (Gossypium hirsutum L.) and rapeseed (Brassica napus L.) are responsible for the leaf shape and determine the formation of leaf lobes (Andres et al., 2017; Hu et al., 2018).

Independent studies in different species shed light on the roles of LMI1 genes in elaboration of leaf shape. However, thus far, the regulatory mechanism is still unknown. Barrelclover (Medicago truncatula) is a model legume species whose adult leaves are compound and develop serrations along the leaf margin. In M. truncatula, SMOOTH LEAF MARGIN1 (SLM1), the ortholog of PIN1, has been identified and characterized. The loss-of-function slm1 mutant exhibits a smooth leaf margin due to the diffuse auxin distribution, indicating the conserved roles of the auxin/SLM1 module among species (Zhou et al., 2011). Besides, MtNAM acts as a positive factor in promoting the development of leaf margin serrations (Cheng et al., 2012; Zhou et al., 2013). Moreover, we have identified another regulator that plays negative roles in the formation of leaf margin serrations. By analyzing an argonaute7 mutant in M. truncatula, we revealed that the degree of margin indentation is determined by the trans-acting short interfering RNA3 (TAS3 ta-siRNA) pathway, which is partially dependent on the auxin/SLM1 module (Zhou et al., 2013). In this study, we analyzed the developmental roles of LMI1 putative orthologs (MtLMI1s), MtLMI1a and MtLMI1b, in M. truncatula by characterizing the loss-of-function mutants. The genetic evidence in combination with the expression pattern demonstrate that MtLMI1a and MtLMI1b play a redundant role in the formation of leaf margin serration, although their expression domains are near-complementary along the leaf margin. Molecular analyses revealed that MtLMI1s directly activate the expression of SLM1 to regulate the auxin distribution. Furthermore, MtLMI1s genetically interact with MtNAM and the MtAGO7-mediated TAS3 ta-siRNA pathway to elaborate the final shape of the leaf margin, demonstrating that formation of proper leaf margin requires the cooperation of existing conserved regulators.

Results

Identification of MtLMIs and their loss-of-function mutants in M. truncatula

To identify the putative orthologs of LMI1 in M. truncatula, the protein sequence of LMI1 was used as a query in BLAST searches against the protein sequence database of the M. truncatula genome in National Center for Biotechnology Information. Based on the homology analysis, Medtr1g061660 and Medtr7g103340 displayed close relationships with LMI1 and were, therefore, named MtLMI1a and MtLMI1b (collectively called MtLMI1s), respectively (Supplemental Figure S1). Then, the putative LMI1 orthologs from M. truncatula, Pisum sativum, Glycine max, Arachis hypogaea, Solanum lycopersicum, Vitis vinifera, C. hirsuta, as well as A. thaliana, were chosen for further phylogenetic analysis. The results showed that MtLMI1a is close to GmLMI1a/b, while MtLMI1b is clustered with PsLMI1 (TENDRIL-LESS) and GmLMI1c/d, implying different evolutionary relationships among them (Figure 1, A). Previous studies showed that LMI1 belongs to the class I HD-Zip transcription factor subfamily, which is characterized by a DNA-binding homeodomain (HD) motif and a Leu zipper (Zip) motif (Ruberti et al., 1991; Henriksson et al., 2005). According to this, the amino acid sequence alignment of the LMI1 putative orthologs showed two types of conserved domains, including three α-helices of the HD domains and one Leu Zip domain (Supplemental Figure S2). Subsequently, GFP-tagged MtLMI1a and MtLMI1b coding sequences (CDS) driven by the CaMV 35S promoter (35Spro:MtLMI1s-GFP) were generated and transiently expressed in Tobacco (Nicotiana benthamiana) leaves. MtLMI1a-GFP and MtLMI1b-GFP fusion proteins were localized in the nucleus, which supported their roles as transcription factors (Figure 1, B). Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed physical interaction between MtLMI1a and MtLMI1b, implying their potential capability to form heterodimers (Supplemental Figure S3).

Figure 1.

Figure 1

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Molecular characterization and subcellular localization of MtLMI1a and MtLMI1b in M. truncatula. A, Phylogenetic tree analysis of LMI1 putative orthologs in M. truncatula and other species. The scale bar indicates the sequence divergence is 0.1 per unit bar, which represents 10% substitutions per nucleotide position. B, Subcellular localization of MtLMI1s. Free GFP as a control. Scale bars = 20 μm. C and E, Schematic representation of the gene structures of MtLMI1s showing the Tnt1 (the transposable element of tobacco cell type1) insertion sites in mtlmi1a-1, mtlmi1a-2, and mtlmi1b-1. The positions of ATG start and TAA stop codons are shown. Vertical arrows mark the location of Tnt1 retrotransposons in mtlmi1-mutant alleles. Introns are represented by a line and exons are represented by a box. D and F, RT-PCR shows transcript abundance of MtLMI1s in the leaf of wild-type and mtlmi1 mutants. MtACTIN was used as the control.

To explore the functions of MtLMI1s in M. truncatula, a reverse genetic screening was performed on a Tnt1 retrotransposon-tagged mutant collection of M. truncatula (Cheng et al., 2014). Two independent alleles of MtLMI1a (mtlmi1a-1 and mtlmi1a-2) and one allele of MtLMI1b (mtlmi1b-1) were isolated. Sequence analysis showed that a single Tnt1 was inserted in the first exon of MtLMI1a in mtlmi1a-1, and in the 5′-UTR region of MtLMI1a in mtlmi1a-2 (Figure 1, C). Reverse transcription PCR (RT-PCR) data showed that no MtLMI1a transcript was detected in mtlmi1a-1, but a very low transcript level was measured in mtlmi1a-2 (Figure 1, D). As for mtlmi1b-1, a Tnt1 insertion was located at the second exon of MtLMI1b genomic sequence (Figure 1, E). MtLMI1b transcript was completely interrupted by the Tnt1 insertion in mtlmi1b-1 (Figure 1, F). These data indicate that mtlmi1a-1 and mtlmi1b-1 are knockout mutants, and mtlmi1a-2 is a knockdown mutant.

MtLMI1a and MtLMI1b redundantly regulate leaf margin development

Compared with the wild type, loss-of-function mutants of MtLMI1a or MtLMI1b did not exhibit obvious defects in compound leaf pattern and leaf morphology (Figure 2, A–D). To assess functional redundancy between MtLMI1a and MtLMI1b, double mutants were generated. Simultaneous disruption of MtLMI1a and MtLMI1b in both mtlmi1a-1 mtlmi1b-1 and mtlmi1a-2 mtlmi1b-1 resulted in relatively smooth leaf margins compared with that in wild type (Figure 2, A, E, and F). To better characterize the defects in leaf margin of mtlmi1a mtlmi1b double mutants, the numbers of leaf marginal serrations were counted. No differences were evident among wild type, mtlmi1a-1, mtlmi1a-2, and mtlmi1b-1 single mutants, but both mtlmi1a-1 mtlmi1b-1 and mtlmi1a-2 mtlmi1b-1 exhibited fewer serrations than those in both lateral leaflets and the terminal leaflet of wild type (Figure 2, G and H). The ratios of serration numbers versus area of leaves in mtlmi1a-1 mtlmi1b-1 mutant were also decreased compared with those in wild type (Supplemental Figure S4). Furthermore, leaf marginal defects in double mutants were fully rescued by introducing the MtLMI1a or MtLMI1b genomic sequence (Figure 2, I and J). These results indicate that both MtLMI1a and MtLMI1b are required for the development of leaf marginal serrations.

Figure 2.

Figure 2

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Phenotype analysis of leaf margin in mtlmi1a mtlmi1b. A–F, Leaf margin phenotypes of WT (A), mtlmi1a-1 (B), mtlmi1a-2 (C), mtlmi1b-1 (D), mtlmi1a-1 mtlmi1b-1 (E), and mtlmi1a-2 mtlmi1b-1 (F). Scale bars = 1 cm. G and H, Numbers of serrations in lateral leaflets and terminal leaflets in WT, mtlmi1a-1, mtlmi1a-2, mtlmi1b-1, mtlmi1a-1 mtlmi1b-1, and mtlmi1a-2 mtlmi1b-1. Bars represent means ± sd (n = 20). ***P < 0.001. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. I and J, Genetic complementation of the mtlmi1a mtlmi1b mutant. mtlmi1a-1 mtlmi1b-1 line transformed with either MtLMI1 genomic sequence shows normal WT-like leaf margins. Scale bars = 1 cm. K–R, Scanning electron micrographs analysis of the leaf margin in WT (K)–(N) and mtlmi1a-1 mtlmi1b-1 (O)–(R) at the different developmental stages. The formation of leaf marginal serrations was displayed at the early stage (K) and (O), middle stage (L) and (P), later stage (M) and (Q), and became mature (N) and (R). Arrows indicate the deep sinuses in WT (L), while arrowheads point to the less pronounced sinus in mutant (P). Arrow points to the initiating tooth in WT (M), but not in mutant (Q), indicated by arrowhead. Arrows indicate more teeth developed in WT (N), and arrowheads indicate the fewer teeth formed in mutant (R). Green lines indicate the sharp tooth in WT (N) and less pronounced tooth in mtlmi1a-1 mtlmi1b-1 (R). The black and white images in (A)–(F), (I), and (J) were produced by Adobe Photoshop and showed the leaf margin of each lines. The same image of the WT leaf appears in A and Figures 5, A, 8, A. Scale bars = 100 μm in (K)–(M) and (O)–(Q), 1 mm in (N) and (R).

Serrations are initiated as small primordia along the leaf margin during the early stage of leaf development and eventually develop into saw-like structures in mature leaves (Tsukaya, 2006; Zhou et al., 2013). To further characterize the defects in mutants, the formation of leaf marginal serrations in wild-type and mtlmi1a-1 mtlmi1b-1 double mutant was compared at the different developmental stages by scanning electron microscopy (SEM) analysis (Figure 2, K–R). At the early stage, there was no obvious difference between wild type and mutant (Figure 2, K and O). At the middle stage, more pronounced sinuses were formed in wild type, compared with those in mutant (Figure 2, L and P). At the later (Figure 2, M and Q) and mature stages (Figure 2, N and R), more teeth with sharp serration tips were developed in wild type. These observations suggest that MtLMI1s are involved in the regulation of both the number and shape of marginal serrations. Later, the first fully expanded mature leaves of wild type and mtlmi1a-1 mtlmi1b-1 were chosen for further analysis. SEM analysis showed that less pronounced leaf teeth tips were exhibited in the mature leaves of mtlmi1a-1 mtlmi1b-1 compared with those in wild type (Supplemental Figure S5, A, B, D, and E). Moreover, the margin cells at the teeth tips formed the ridge-like structure in wild type (Supplemental Figure S5, A and B). The amount of this kind of margin cells was decreased in mtlmi1a-1 mtlmi1b-1 (Supplemental Figure S5, D and E), implying that the development of teeth tips is abnormal in the double mutant. Similar to wild type, marginal cells in mtlmi1a-1 mtlmi1b-1 were elongated, but less pronounced sinuses in the double mutant were observed (Supplemental Figure S5, C and F), indicating redundant roles of MtLMI1a and MtLMI1b in repression of the degree of leaf margin indentation.

Expression patterns of MtLMI1a and MtLMI1b

To understand the expression patterns of MtLMI1s, a series of assays were performed. First, expression levels of MtLMI1a and MtLMI1b were examined in different organs by RT-quantitative PCR (RT-qPCR) analysis. Both MtLMI1a and MtLMI1b were highly expressed in vegetative buds and flowers, but MtLMI1a showed a high expression level in petioles. The overall transcript level of MtLMI1a was higher than that of MtLMI1b among different organs, except in juvenile leaves (Figure 3, A). Second, MtLMI1a and MtLMI1b promoter-β-glucuronidase (GUS) reporters were separately introduced into wild type. The transgenic plants of MtLMI1apro: GUS and MtLMI1bpro: GUS showed similar expression patterns, which displayed the GUS expression in leaf margin of young leaves. In mature leaves, GUS signals were detected on the whole leaf blades, but were stronger at marginal teeth tips and veins (Figure 3, B). Moreover, GUS signals were also detected in germinating seeds, seed pods, and flowers (Supplemental Figure S6). To gain a better spatial expression pattern, RNA in situ hybridization was performed at different developmental stages. Low MtLMI1a transcripts were detected in the SAM and the tip of leaf primordia at Stage 3, but not at Stage 2 (Figure 3, C). At Stage 4, MtLMI1a mRNA was detected on the developing leaf blade (Figure 3, D). At Stages 5 and 6, MtLMI1a was mainly accumulated in the tips of initiating teeth as well as the leaf vein precursors (Figure 3, E and F). At Stage 8, strong MtLMI1a expression was observed in each leaf marginal tooth in the developed leaves (Figure 3, G). MtLMI1b showed a distinct expression pattern during the development of leaf margin (Figure 3, H–L). MtLMI1b transcript was not detected in SAM and leaf primordia at Stages 2 and 3 (Figure 3, H). Only weak expression of MtLMI1b was detected at the tip of leaf primordia at Stages 4 and 5 (Figure 3, I and J). In contrast with the expression pattern of MtLMI1a, MtLMI1b transcripts accumulated in the leaf sinus between the adjacent serrations and excluded from teeth tips and developing veins at Stages 6 and 8 (Figure 3, K and L). Therefore, MtLMI1a and MtLMI1b showed a near-complementary expression pattern during the initiation and development of leaf marginal serrations (Figure 3, M), implying that MtLMI1a and MtLMI1b may function cooperatively along the leaf margin. The sense probes were hybridized as the controls and did not show any signal (Figure 3, N and O).

Figure 3.

Figure 3

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Expression patterns of MtLMI1a and MtLMI1b in M. truncatula. A, RT-qPCR analysis of MtLMI1s expression in different organs. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates. B, Promoter–GUS fusion studies of MtLMI1s expression in transgenic M. truncatula. The promoters of MtLMI1a- and MtLMI1b-driven GUS are expressed in the unexpanded leaf, fully expanded leaf, and leaf vein. Twenty leaves in each line were observed. Scale bars = 2 mm. (C)–(L) In situ hybridization analysis of MtLMI1s mRNA in WT. Longitudinal sections of SAM and leaf primordia at different developmental stages are shown. Scale bars = 50 μm. Arrows indicate the signals of MtLMI1a. Arrowheads indicate the signals of MtLMI1b. M, A schematic illustration of a near-complementary expression pattern of MtLMI1a and MtLMI1b. N and O, The sense probes were hybridized and used as the controls. Scale bars = 50 μm.

MtLMI1a and MtLMI1b are sufficient for increasing leaf marginal serrations

The aforementioned findings indicate that MtLMI1a and MtLMI1b may play positive roles in regulating the development of leaf marginal serrations. To determine whether increasing activities of MtLMI1a and MtLMI1b are sufficient to produce more serrations along leaf margin, both genes were overexpressed under the control of the CaMV 35S promoter in wild type. Compared with wild type, the expression levels of MtLMI1a were increased by 101- to 451-fold in 35Spro: MtLMI1a-GFP transgenic plants (Figure 4, D). The leaf shape in different 35Spro: MtLMI1a-GFP lines and wild type was compared. 35Spro: MtLMI1a-GFP-1 line, with the highest expression level, displayed severe downward-curled leaves. 35Spro: MtLMI1a-GFP-4 line, with the lowest expression level, showed the slightly curled leaves (Figure 4, A, B, and D). A similar phenotype was also observed in 35Spro: MtLMI1b-GFP transgenic plants (Figure 4, A, C, and G). These observations indicate that ectopic expression of MtLMI1a or MtLMI1b is able to promote the curvature of leaves in a dosage-dependent manner. In addition, the leaf blade area of 35Spro: MtLMI1-GFP transgenic plants was reduced, which is reminiscent of the stf mutants (Tadege et al., 2011). Thus, we checked the expression of STF in wild type, 35Spro: MtLMI1a-GFP-1 and 35Spro: MtLMI1b-GFP-1 transgenic plants. The data showed that expression level of STF was decreased in both transgenic plants, indicating that STF is probably involved in the MtLMI1-mediated leaf blade outgrowth (Supplemental Figure S7). Moreover, the ratios of serration numbers versus leaf area in both terminal leaflets and lateral leaflets were increased in a dosage-dependent manner in plants overexpressing MtLMI1a or MtLMI1b (Figure 4, E, F, H, and I), indicating that ectopic expression of MtLMI1a or MtLMI1b is able to induce serration formation. Taken together, these data suggest that MtLMI1a and MtLMI1b are not only necessary for formation of leaf marginal serrations, but also sufficient for increasing the number of serrations.

Figure 4.

Figure 4

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Phenotype analysis of 35Spro: MtLMI1s-GFP in M. truncatula. A–C, Leaf phenotypes of WT (A), 35Spro: MtLMI1a-GFP-1, 2, 4 (B), and 35Spro: MtLMI1b-GFP-1, 3, 6 (C). Scale bars = 1 cm. D and G, The expression levels of MtLMI1s in the leaves of 28-d-old WT and 35Spro: MtLMI1s-GFP transgenic lines, determined by RT-qPCR. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates. E, F, H, and I, The ratios of leaf serration numbers versus leaf area in lateral leaflets and terminal leaflets in WT and 35Spro: MtLMI1s-GFP transgenic lines. Bars represent means ± sd (n = 20–40). The dot represents the value of one sample. ***P < 0.001. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. ***P < 0.001, *P < 0.05. AD, adaxial side; AB, abaxial side; TL, terminal leaflet; LL, lateral leaflet.

MtLMI1a and MtLMI1b regulate auxin responses in the leaf margin

Our previous study showed that the auxin/SLM1 module, in which SLM1 generates local auxin activity gradients along leaf margin, is tightly associated with leaf serration development in M. truncatula (Zhou et al., 2011). The leaf shape in slm1 mutants was variable. However, slm1-1 mutant always displayed a smooth leaf margin phenotype, compared with wild type (Figure 5, A and B). To investigate the genetic relationship between SLM1 and MtLMI1s, the triple mutant was generated. Simultaneous disruption of SLM1, MtLMI1a, and MtLMI1b led to developmental defects that are similar to the phenotype in the slm1-1 mutant, such as smooth leaf margin and ectopic leaflets (Figure 5, B–D). This finding suggests that slm1 is genetically epistatic to mtlmi1a mtlmi1b in leaf margin development, implying that MtLMI1s control the leaf serration formation in an auxin/SLM1-dependent manner.

Figure 5.

Figure 5

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MtLMI1a and MtLMI1b regulate auxin response in the leaf margin. A–D, Adult leaves of the WT (A), slm1-1 (B), mtlmi1a-1 mtlmi1b-1 (C), and mtlmi1a-1 mtlmi1b-1 slm1-1 (D). The black and white images were produced by Adobe Photoshop and showed the leaf margin of each lines. E–G, DR5: GUS expression in mature leaves of WT (E), mtlmi1a-1 mtlmi1b-1 (F), and slm1-1 (G). Arrows point to the leaf margin tips and arrowheads point to lateral veins, which do not terminate at the margin in (F) and (G). Thirty-six leaves derived from three independent transgenic lines of WT and mutants were analyzed. H, The percent of free-ending veins in WT, mtlmi1a-1 mtlmi1b-1, and slm1-1. I–P, DR5: GFP expression in leaf margin at the different developmental stages. The leaf marginal serrations were visible in the early stage in (I) and (J), middle stage in (K) and (L), and later stage in (M) and (N), and became mature in (O) and (P). Arrows indicate the auxin accumulation in WT and arrowheads indicate the less auxin response in mutant. Scale bars = 50 μm in (I)–(P), 0.5 mm in (E)–(G), and 1 cm in (A)–(D).

To investigate the relationship between MtLMI1s and auxin/SLM1, the auxin response reporter DR5: GUS was introduced into wild-type and mutant plants. In mature leaves, GUS signal was observed in the veins, and no obvious difference in auxin responsiveness was displayed between wild type and mutants. However, the patterning of leaf veins was defective in mutants, based on the distribution of GUS signals. The free-ending veins were observed at the distal end of lateral veins in mtlmi1a-1 mtlmi1b-1 and slm1-1 where the development of serrations was defective (Figure 5, E–G). The percentages of free-ending veins were 4.36% (n = 275) in wild type, 45.5% (n = 246) in mtlmi1a-1 mtlmi1b-1, and 76.2% (n = 181) in slm1-1 (Figure 5, H). This observation suggests that the defects of veins and serrations in mtlmi1a-1 mtlmi1b-1 resemble those of slm1-1. To further elucidate the role of auxin in leaf margin morphogenesis, DR5: GFP was introduced into the mtlmi1a-1 mtlmi1b-1 mutant to show local auxin level in leaves at the early stages. By examining the GFP signals, the auxin responsiveness was observed along the leaf margin, but the relative higher levels of auxin responsiveness were shown in the tips of initiating serrations in wild type (Figure 5, I and K). In the developing serration tips of wild type, much stronger GFP signals were also observed (Figure 5, M and O). These observations indicate that auxin activity gradients are critical for the formation of leaf marginal serrations. Compared with wild type, the GFP signals became weak in the tips of both initiating serrations (Figure 5, J and L) and developing serrations (Figure 5, N and P) in mtlmi1a-1 mtlmi1b-1 mutant, suggesting a diffuse auxin distribution in the leaf marginal tips. These observations indicate that the local auxin responsiveness is partially defective during the leaf margin development in mtlmi1a-1 mtlmi1b-1, leading to the less pronounced leaf tooth tips.

MtLMI1a and MtLMI1b directly activate the transcription of SLM1 in the leaf margin

In M. truncatula, SLM1 plays key roles in auxin distribution. To investigate the regulatory relationship between MtLMI1s and SLM1, a yeast one-hybrid (Y1H) assay was performed (Figure 6, A and B). The pGADT7-Rec-53 plasmid transformed into the YIH (p53-pAbAi) strain functioned as the positive control, while pGADT7-MtLMI1a or pGADT7-MtLMI1b introduced into YIH (p53-pAbAi) strain and YIH (pSLM1-pAbAi) strain transformed with the empty vector functioned as negative controls. The results showed that YIH (pSLM1-pAbAi) strain harboring plasmid pGADT7-MtLMI1a (Figure 6, A) or pGADT7-MtLMI1b (Figure 6, B) grew in the SD selection medium (-Leu, 500 ng/mL AbA), implying that MtLMI1a and MtLMI1b are able to bind to the promoter of SLM1. To further test if MtLMI1a and MtLMI1b can activate the expression of SLM1, transient expression assay was performed. The luciferase reporter construct driven by a 2098-bp SLM1 promoter was co-transformed with MtLMI1a or MtLMI1b effector proteins into Arabidopsis protoplasts. The results showed that luminescence intensity was increased significantly, compared with the GFP control effector protein (Figure 6, C and D). This result implies that MtLMI1a and MtLMI1b are able to recognize the SLM1 promoter in protoplasts and activate its expression. Previous study reported that the binding sites of LMI1 protein are CAATXAT, where X is A or T (Franco-Zorrilla et al., 2014; Vuolo et al., 2018). Thus, the direct associations of MtLMI1a or MtLMI1b with the SLM1 promoter were tested by chromatin immunoprecipitation (ChIP) followed by RT-qPCR. The data showed that three LMI1-binding regions in the promoter of SLM1 were enriched in the chromatin immunoprecipitated with MtLMI1s-GFP, compared with the non-binding site (Figure 6, E). These results suggest that MtLMI1a and MtLMI1b can directly activate the transcription of SLM1 in vivo by binding to the CAATXAT motif.

Figure 6.

Figure 6

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MtLMI1a and MtLMI1b directly activate the expression of SLM1. A and B, Interaction between MtLMI1s and SLM1 promoter tested by Y1H binding assay. C, Schematic representation of reporter and effector constructs used in transient expression assay. D, Transient expression assay in Arabidopsis protoplasts showing activation of SLM1 by the MtLMI1 effectors compared with the GFP control. Bars represent means ± sd of three biological replicates. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. *P < 0.05. E, Quantitative ChIP-PCR analysis of MtLMI1s binding to the promoter of SLM1. The regions tested by ChIP assay are indicated in the schematic representation. P1–P3 represent the LMI1-binding regions and P4 represents the non-binding site of LMI1. MtUBIQUITIN was used for normalization. Bars represent means ± sd of three biological replicates. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. ***P < 0.001, *P < 0.05. F–I, RNA in situ hybridization analysis of SLM1 mRNA in the longitudinal sections of the leaf primordia in WT (F, H) and mtlmi1a-1 mtlmi1b-1 (G, I). Arrows mark the signals of SLM1 at the serrations. Scale bars = 50 μm. J, The expression levels of SLM1 in WT and 35Spro: MtLMI1s-GFP transgenic lines determined by RT-qPCR. Transcript levels were measured by RT-qPCR using vegetative buds of 28-d-old plants. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. ***P < 0.001.

To further confirm the regulatory relationship between MtLMI1s and SLM1, in situ hybridization was performed to monitor the spatial expression change of SLM1 at the leaf margin in wild type and mtlmi1a-1 mtlmi1b-1 mutant. In wild type, SLM1 mRNA was detected in the leaf marginal teeth, which were initiated in the developing leaves at Stage 6 (Figure 6, F) and Stage 8 (Figure 6, H). However, an obvious reduction in the SLM1 expression in leaf margins of mtlmi1a-1 mtlmi1b-1 was observed at the same stages (Figure 6, G and I). Moreover, the expression levels of SLM1 were tested in leaf buds of wild type, 35Spro: MtLMI1s-GFP transgenic plants and mtlmi1a-1 mtlmi1b-1. RT-qPCR data showed that the expression of SLM1 was increased in both MtLMI1a and MtLMI1b overexpressing lines (Figure 6, J), while decreased in mtlmi1a-1 mtlmi1b-1 (Supplemental Figure S8). Taken together, these results demonstrate that MtLMI1a and MtLMI1b directly activate the transcription of SLM1 in leaf margin to regulate the formation of marginal serrations.

MtLMI1s and the TAS3 ta-siRNA pathway antagonistically regulate leaf margin development

MtAGO7 is involved in the development of leaf serrations (Zhou et al., 2013). Loss-of-function mtago7 mutant displays lobed leaves due to the ectopic expression of AUXIN RESPONSE FACTOR3 (MtARF3; Figure 7, A). Moreover, MtAGO7 is involved in the trans-acting siRNA3 (TAS3) pathway, which functions cooperatively with the auxin/SLM1 module to regulate leaf margin formation (Zhou et al., 2013). To investigate the genetic relationship between MtAGO7 and MtLMI1s, the mtago7-1 mtlmi1a-1 mtlmi1b-1 triple mutant was generated and analyzed (Figure 7, B and C). Compared with the lobed leaf margin in mtago7-1, the extent of indentation in leaf margins of triple mutant was less pronounced in triple mutant, indicating that MtLMI1s and MtAGO7 antagonistically regulate the development of leaf marginal serrations (Figure 7, C). Moreover, the expression levels of MtARF3, MtARF4a, and MtARF4b genes were examined in wild type and mutants. The data showed that MtARF3 and MtARF4b were ectopically expressed in both mtago7-1 and mtlmi1a-1 mtlmi1b-1 mtago7-1. However, their expression levels were similar between mtago7-1 and mtlmi1a-1 mtlmi1b-1 mtago7-1 (Figure 7, F). This finding implies that the less pronounced serration in triple mutant is not due to the repression of MtARF3 expression. To further confirm this hypothesis, the MtARF3 cDNA carrying two altered ta-siARF target sites (MtARF3mut) under the regulation of the CaMV 35S promoter (35Spro: MtARF3mut; Zhou et al., 2013) was introduced into mtlmi1a-1 mtlmi1b-1 double mutant. In wild type, overexpression of MtARF3mut can mimic the lobed leaves in mtago7-1 mutant (Figure 7, D and G; Zhou et al., 2013). The leaves of 35Spro: MtARF3mut mtlmi1a-1 mtlmi1b-1 plant showed fewer serrated leaf margins compared with those in 35Spro: MtARF3mut plant (Figure 7, D and E). In addition, MtARF3 was also highly expressed in 35Spro: MtARF3mut mtlmi1a-1 mtlmi1b-1 (Figure 7, G), suggesting that the partial recovery of leaf margin defects in 35Spro: MtARF3mut mtlmi1a-1 mtlmi1b-1 is not due to the down-regulation of MtARF3. Taken together, these observations suggest that MtLMI1s and the MtAGO7-mediated TAS3 ta-siRNA pathway function antagonistically in controlling the formation of leaf marginal serrations.

Figure 7.

Figure 7

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Interaction between the TAS3 ta-siRNA pathway and MtLMI1s in leaf margin development in M. truncatula. A–E, Adult leaves of mtago7-1 (A), mtlmi1a-1 mtlmi1b-1 (B), mtago7-1 mtlmi1a-1 mtlmi1b-1 (C), 35Spro: MtARF3mut (D), and 35Spro: MtARF3mut mtlmi1a-1 mtlmi1b-1 (E). The black and white images in (A)–(E) were produced by Adobe Photoshop and showed the leaf margin of each lines. Scale bars = 1 cm. F, Transcript levels of MtARF3, MtARF4a, and MtARF4b in the WT, mtlmi1a-1 mtlmi1b-1, mtago7-1, and mtago7-1 mtlmi1a-1 mtlmi1b-1. Transcript levels were measured by RT-qPCR using leaves of 28-d-old plants. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates, and the different letters above bars indicate statistically significant differences among samples. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. P < 0.001. G, Transcript levels of MtARF3 in the WT, 35Spro: MtARF3mut, and 35Spro: MtARF3mut mtlmi1a-1 mtlmi1b-1 plants. Transcript levels were measured by RT-qPCR using leaves of 28-d-old plants. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates. ***P < 0.001.

MtLMI1a and MtLMI1b function synergistically with MtNAM in leaf margin formation

NAM/CUC genes play conserved roles in organ boundary development and leaf margin development across different species (Blein et al., 2008). In M. truncatula, the leaflets of mtnam-2 are fused and leaf teeth are relatively smooth, compared with those of wild type (Figure 8, A and B; Cheng et al., 2012; Zhou et al., 2013). To investigate the genetic relationship between MtNAM and MtLMI1s, the mtnam-2 mtlmi1a-1 mtlmi1b-1 triple mutant was generated. The leaf margin in the triple mutant was smoother than those in either mtnam-2 or mtlmi1a-1 mtlmi1b-1 (Figure 8, A–D). Additionally, the ratios of serration numbers versus area of leaves in the triple mutant were decreased (Figure 8, E) compared with those in parents. These findings suggest that MtLMI1s and MtNAM play redundant roles in regulating the formation of leaf margin in M. truncatula. Furthermore, the expression of MtNAM was measured in leaf buds of wild type and mtlmi1a-1 mtlmi1b-1 by RT-qPCR. The data showed that the transcript of MtNAM was slightly reduced in the mutant (Figure 8, F). RNA in situ hybridization was performed to monitor the spatial expression change of MtNAM at the leaf margin. MtNAM mRNA was detected in the boundary of leaflet primordia, but not in leaf marginal teeth (Supplemental Figure S9), indicating the very low expression level of MtNAM in leaf margin. However, the expression levels of MtNAM were increased in 35Spro: MtLMI1a-GFP-1 and 35Spro: MtLMI1b-GFP-1 plants (Figure 8, G), supporting the idea that ectopic expression of MtLMIs can activate MtNAM. Therefore, based on the genetic evidence, MtLMI1s act synergistically with MtNAM in leaf margin development through at least partially parallel pathways.

Figure 8.

Figure 8

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Interaction between MtNAM and MtLMI1s in leaf margin development in M. truncatula. A and B, Leaf margin phenotypes of WT (A) and mtnam-2 (B). Closeup views of leaf margin (boxed region) were shown in right panel of each figure. Arrows point to the serrations in WT. Arrowheads point to the less pronounced serration tips in mtnam-2. C and D, Leaf margin phenotypes of mtlmi1a-1 mtlmi1b-1 (C) and mtnam-2 mtlmi1a-1 mtlmi1b-1 (D). The black and white images in (A)–(D) were produced by Adobe Photoshop and showed the leaf margin of each lines. Scale bars = 1 cm. E, The ratios of leaf serration numbers versus leaf area in WT, mtnam-2, mtlmi1a-1 mtlmi1b-1, and mtnam-2 mtlmi1a-1 mtlmi1b-1 (n = 15). The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. ***P < 0.001, **P < 0.01. F, Expression levels of MtNAM in leaf buds of WT and mtlmi1a-1 mtlmi1b-1 plants. Bars represent means ± sd of three biological replicates. G, Expression levels of MtNAM in overexpression of MtLMI1a or MtLMI1b transgenic plants. Transcript levels were measured by RT-qPCR using leaves of 28-d-old plants. MtUBIQUITIN was used as the internal control. Bars represent means ± sd of three biological replicates. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant. **P < 0.01. Scale bars = 1 cm in (A)–(D) and 150 μm in closeups (A) and (B).

Discussion

The expression pattern diversity of LMI1s and their putative paralogs in key domains is an important driver of functional specialization across species

Leaf shape varies dramatically across plant species and this diversity reflects natural selection operating on leaf function. The outline of the leaf margin is an important trait of leaf shape. In this study, we characterized two regulators, MtLMI1a and MtLMI1b, in controlling the formation of leaf margin in M. truncatula. LMI1 and its putative paralogous genes have been proposed as evolutionary hotspots for leaf development, which is caused by their different expression levels and locations. In Arabidopsis, LMI1 is strongly expressed in leaves, where LMI1 promotes the leaf marginal serrations and represses the blade outgrowth from the petiole (Saddic et al., 2006). Meanwhile, the ancestral paralog of LMI1 in the crucifer family, RCO, acquired a novel expression domain at the base of leaf, which divides the leaf into distinct leaflets (Sicard et al., 2014; Vlad et al., 2014). Although LMI1 and RCO play a conserved function in growth repression, the loss of RCO in Arabidopsis during evolution results in leaf simplification, while the lmi1 mutant displays smooth leaf margin. Such species-specific activity of RCO requires the evolution of an enhancer element, indicating the important roles of cis-regulatory divergence of genes (Vuolo et al., 2016). Furthermore, modification of LMI1 expression levels is critical for the determination of leaf shape in other species (Chang et al., 2016; Andres et al., 2017). In cotton, a tandem duplication in the promoter of GhLMI1-D1b leads to elevated expression, resulting in deeply lobed leaf margin (Andres et al., 2017). In rapeseed, the promoter variations in BnA10.LMI1 determine the depth of leaf lobes (Hu et al., 2018). Among these species, increased activity of LMI1 putative orthologs results in lobed leaves, indicating their positive roles in leaf serration formation. In M. truncatula, neither the mutant mtlmi1a, nor mtlmi1b, shows a strong phenotype. However, mtlmi1a mtlmi1b double mutant exhibits a relatively smooth leaf margin, indicating their redundant roles in marginal serration formation. Moreover, MtLMI1a and MtLMI1b act as growth repressors, which are evidenced by small and curled leaves in their overexpression lines. Therefore, consistent with their putative orthologs in other species, both MtLMI1a and MtLMI1b are required for the proper development of leaf marginal serrations.

The elaboration of compound leaves and leaf marginal serrations is context dependent (Efroni et al., 2010). In both simple-leafed and compound-leafed plants, formation of leaflets or serrations relies on their initiation time. If the initiation of primordia occurs after the leaflets have become flattened and determined, serrations, instead of leaflets, will form (Floyd and Bowman, 2010). In C. hirsuta, expression of ChLMI1 is confined to the leaf margin. By comparison, RCO is expressed in the proximal leaf blade, which is a highly proliferative region, and thus induces the formation of compound leaves. The distinct expression domains of ChLMI1 and RCO lead to their roles in different developmental processes (Sicard et al., 2014; Vlad et al., 2014; Vuolo et al., 2016). In our study, in situ hybridization analysis showed that expression regions of MtLMI1a and MtLMI1b exhibit a near-complementary pattern along the leaf margin. MtLMI1a and MtLMI1b expression is detected in the leaflet margin, which is differentiated from a common leaf primordium (Zhou et al., 2011). Thus, the restriction of MtLMI1a and MtLMI1b expression domains determines that they are involved in leaf margin formation instead of compound leaf patterning. Additionally, LMI1 and its putative orthologs are involved in the development of other plant organs. As a growth repressor, LMI1 not only elaborates leaf serration, but also suppresses the transformation of stipule into leaflet in Arabidopsis (Vuolo et al., 2018). Moreover, the closely related gene of LMI1 in pea, Tl, is expressed in tendrils, where it causes growth arrest of leaf blades. Loss-of-function tl mutant transforms tendrils into leaflets, suggesting that Tl affects organ identity in specific contexts (Gourlay et al., 2000; Hofer et al., 2009). In our study, mtlmi1a mtlmi1b double mutants display normal compound leaf patterning and stipules, and defects only in leaf margin. This phenotype also supported the idea that the leaf margin activity is independent from leaflet formation (Du et al., 2020). Thus, LMI1 genes play roles in a context-dependent manner. Overall, our data suggest that diversity of expression patterns of LMI1s and of their putative paralogs in key domains within the leaf is an important driver of functional specialization, even if the proteins themselves are functionally equivalent among species.

MtLMI1s regulate leaf serration formation by directly activating the expression of SLM1

The auxin efflux carrier PIN1/SLM1 controls polar auxin transport, which plays a key role in the elaboration of leaf margin (Barkoulas et al., 2008; Koenig et al., 2009; Bilsborough et al., 2011; Zhou et al., 2011). In our previous study, the auxin responsiveness was observed in developing serrations of the mature leaf at the very late developmental stage. The results suggest that formation of serrations on the leaf margin correspond with auxin activity maxima, which is mediated by SLM1 in M. truncatula, at the tip of serrations (Zhou et al., 2011). In addition, TAS3 pathway functions as a repressor in the formation of marginal indentation. The genetic evidence showed that TAS3 pathway and MtNAM antagonistically regulate the formation of sinuses (Zhou et al., 2013). In this study, MtLMI1a and MtLMI1b are involved in this developmental process by directly activating the expression of SLM1, leading to proper auxin distribution along leaf margin to promote the initiation of leaf marginal teeth (Figure 9). The further observations of DR5:GFP indicate that auxin responsiveness is partially defective in leaf margin development in mtlmi1a-1 mtlmi1b-1, which is consistent with the role of auxin/SLM1 module in the formation of serrations. The expression domains of MtLMI1a and MtLMI1b display a near-complementary pattern along the leaf margin, implying that they may function cooperatively to generate the proper auxin distribution at the different domains of the leaf margin. However, it is notable that only mtlmi1a mtlmi1b double mutant shows the defects in leaf margin formation. The possible reason is that other regulators are also involved and play redundant roles in the regulation of auxin distribution in this developmental process. A previous study proposed a fine model for leaf margin development in Arabidopsis. In this model, both CUC2 and PIN1 are required for the establishment of proper auxin activity gradients in the formation of marginal serrations (Bilsborough et al., 2011). In M. truncatula, leaf teeth of mtnam-2 are relatively smooth, indicating that MtNAM is involved in the leaf margin formation (Figure 8, B). However, MtNAM mRNA was not detected in the leaf marginal teeth by RNA in situ hybridization, probably due to its limited sensitivity in detecting low copy numbers of nucleic acids. RT-qPCR data showed that the expression of MtNAM was slightly down-regulated in mtlmi1a-1 mtlmi1b-1. Additionally, ectopic expression of MtLMI1s could increase the transcript level of MtNAM, and MtNAM was involved in promoting the sinus. Thus, MtNAM is possibly under the regulation of MtLMIs, although such regulation is relatively weak. Moreover, we found that MtNAM is redundant with MtLMI1a and MtLMI1b based on the genetic evidence, namely that the mtnam mtlmi1a mtlmi1b triple mutant displays fewer serrations than either mtnam or mtlmi1a mtlmi1b (Figure 8, B–E). Based on this notion, neither mtlmi1a nor mtlmi1b shows the obvious defects in leaf morphology. Taken together, MtLMI1s play roles not only in initiation of teeth, but also in formation of marginal indentation (Figure 9).

Figure 9.

Figure 9

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A proposed model illustrating the functional roles of MtLMI1s and other regulators in leaf margin development. MtLMI1s directly activate the expression of SLM1 to regulate the auxin distribution along leaf margin, which is critical for the initiation of marginal teeth. MtLMI1s act synergistically with MtNAM in leaf margin development through at least partially parallel pathways and the expression level of MtNAM can be induced in MtLMI1s-overexpressing plants. Moreover, MtLMI1s and the MtAGO7-mediated TAS3 ta-siRNA pathway antagonistically regulate the leaf margin development. Solid arrows represent the direct regulation and dashed arrow represents the indirect regulation between genes.

In M. truncatula, elaboration of leaf margin formation also requires the determination of degree of marginal indentation, which is regulated by the MtAGO7-mediated TAS3 ta-siRNA pathway through the suppression of MtARF3 expression (Zhou et al., 2013). In this study, the leaf margin of mtago7 mtlmi1a mtlmi1b and 35Spro:MtARF3mut mtlmi1a mtlmi1b plants exhibited the intermediate phenotype between parents, suggesting the antagonistic effect of MtLMI1s and the TAS3 ta-siRNA pathway on leaf margin notch formation (Figure 9). A previous study reported that members of HD-Zip I protein family play roles in plant developmental pathways (Aoyama et al., 1995). In Arabidopsis, ARABIDOPSIS HOMEOBOX PROTEIN 5 (AtHB5) and AtHB6 function redundantly and negatively in regulating the expression of INDOLE-3-ACETIC ACID 12 (IAA12) during embryogenesis, and then, IAAs negatively regulate the auxin response through interacting with ARFs (De Smet et al., 2013; Leyser, 2018). Furthermore, IAA9 and IAA8 were reported to redundantly influence the formation of leaf serrations (Koenig et al., 2009), and specific combinations of IAAs and ARFs were thought to be involved in auxin responses (Weijers et al., 2005; Mutte et al., 2018). These studies imply possible interactions between LMI1 putative orthologs and the auxin-related pathway in plant developmental regulation. Therefore, identification of LMI1 partners will be essential for elaboration of leaf margin development and will help to provide insight into the roles of LMI1 putative orthologs among species.

Materials and methods

Plant materials and growth conditions

Barrelclover (M. truncatula) ecotype R108 was used in this study. Plants were grown at 24°C ± 2°C under long-day conditions (16-h light and 8-h dark), with a relative humidity of 70%–80%. mtlmi1a-1, mtlmi1a-2, and mtlmi1b-1 were identified from a tobacco (Nicotiana tabacum) Tnt1 retrotransposon-tagged mutant collection of M. truncatula (Supplemental Table S1; Tadege et al., 2008). Nicotiana benthamiana was grown in soil at 22°C ± 2°C under long-day conditions, and mouse-ear cress (A. thaliana) was grown in a growth chamber at 20°C under short-day conditions (8-h light and 16-h dark).

Microscopy observations and histochemical GUS assay

For subcellular localization analysis, the 35Spro: MtLMI1s-GFP and the 35Spro: GFP were used in N. benthamiana leaves through Agrobacterium-mediated infiltration. The infiltrated leaves were incubated for 48 h before observation, and the GFP signals were observed under confocal microscopy (Zeiss). For GUS staining, samples were collected and the GUS activity was histochemically detected as described previously (Wang et al., 2019). For SEM, 56-d-old leaves were collected and fixed in 3% glutaraldehyde fixation solution (w/v) and incubated in 4°C overnight. Then, the leaves were washed, dehydrated, and critical point-dried. Finally, the samples were coated with gold for 240 s and examined under a Quanta 250 FEG scanning electron microscope (FEI).

In situ hybridization analysis

For RNA in situ hybridization, the 260-bp CDS of MtLMI1a and 446-bp CDS of MtLMI1b were amplified as probes. The PCR products were cloned into pGEM-T vector (Promega) in correct direction to generate the probes. The probe of SLM1 was generated in previous study (Zhou et al., 2011). The probe of MtNAM was generated according to the previous study (Cheng et al., 2012). RNA in situ hybridization was performed on vegetative buds of 30-d-old wild-type or mtlmi1a-1 mtlmi1b-1 plants as previously described (Zhou et al., 2011).

RT-PCR and RT-qPCR analysis and statistical analysis

Total RNA was extracted from leaves and vegetative buds of 28-d-old plants using Trizol-RT Reagent (Molecular Research Center, Inc.). The first-strand cDNA was synthesized using Synthesis Kit (Roche). Quantitative PCR analyses were performed using SYBR green reagent (Roche) as the reporter dye with a CFX connect real-time PCR detection system (Bio-Rad). The primers used for PCR are listed in Supplemental Table S2. The one-way ANOVA followed by a post hoc Tukey’s test was used to estimate if the difference is significant in analysis of gene expression level and plant phenotype.

Plasmids and plant transformation

To generate the transgenic plants and observe the subcellular localization, 35Spro: MtLMI1s-GFP vectors were constructed and the full length CDS without stop codon of MtLMI1s was obtained by PCR amplification and inserted into pENTR/D-TOPO cloning vector (Invitrogen), and then recombined with destination vector pEarleyGate 103, using the Gateway LR recombination reactions (Invitrogen). As the control, pEarleyGate 103 was also introduced to generate the transgenic plants expressing 35Spro: GFP. To generate the MtLMI1spro: GUS constructs, 2236-bp promoter region of MtLMI1a and 1531-bp promoter region of MtLMI1b were amplified and transferred into the Gateway vector pBGWFS7. To obtain the constructs used for functional complementation of the mtlmi1a mtlmi1b mutant, a 5154-bp fragment was amplified from M. truncatula R108 genomic DNA and ligated to the pCAMBIA3301 vector (after digestion with SacI and PstI). The construct contained a 2191-bp promoter sequence, the entire gene coding sequence, and an 831-bp terminator sequence of MtLMI1a. Similarly, a 4473-bp fragment was amplified from M. truncatula R108 genomic DNA using the primers gMtLMI1b-F and gMtLMI1b-R, and was ligated to the pCAMBIA3301 vector (after digestion with SacI and PstI). The construct contained a 2146-bp promoter sequence, the entire gene coding sequence, and a 342-bp terminator sequence of MtLMI1b. DR5: GUS and DR5: GFP were generated using the DR5 in the pUC19 construct (Ulmasov et al., 1997). The DR5 reporter transgenic lines in M. truncatula were derived from our previous study (Zhou et al., 2011). The primers used in this study are listed in Supplemental Table S2. All the final binary vectors were introduced into the Agrobacterium tumefaciens EHA105 strain. Leaves of M. truncatula R108 were used for transformation (Cosson et al., 2006).

Microscopy methodology

DR5-GFP images were captured by a laser scanning confocal microscope (LSM900, Zeiss, German). GFP signals were exited at 488 nm and collected at 512–548 nm. Approximately 70 leaves at the early developmental stages derived from three independent transgenic lines of wild type and mutants were examined.

Luciferase assay in A. thaliana

A 2098-bp SLM1 promoter was cloned into the KpnI–SalI cloning site of pGreenII 0800-Luc vector to generate the reporter plasmid. The CDS of MtLMI1s was cloned separately into the Gateway vector pBI221 to generate effector plasmid. Isolation of Arabidopsis protoplasts and transformation were performed according to the protocol described previously (Yoo et al., 2007). For each transformation, 2 μg of reporter plasmid and 8 μg of effector plasmid were co-transformed. LUC and REN activities were measured using a Dual-Luciferase reporter kit (Promega).

ChIP-qPCR analysis

The leaves of 45-d-old transgenic plants expressing 35Spro: MtLMI1s-GFP and 35Spro: GFP were used for the ChIP assay with some modifications (Kaufmann et al., 2010). The chromatin was sheared to an average length of 500-bp by ultrasonic treatment. GFP-Trap_A agarose beads (ChromoTek) were used for the immunoprecipitation. The precipitated DNA was detected by qPCR. The related primers are listed in Supplemental Table S2.

Y1H assay

The CDS of MtLM1a/b was cloned separately into the pGADT7 vector, while the promoter sequence of SLM1 was inserted into the SmaI–XhoI cloning site of pAbAi vector. The pSLM1-pAbAi plasmid was integrated into the YIHGold yeast genome. The yeast cells were grown in SD/-Ura medium to ensure that the yeast cells were successfully transformed and then generated the YIH (pSLM1-pAbAi) strain. The pGADT7-MtLMI1a or pGADT7-MtLMI1b plasmids were introduced into the positive YIH (pSLM1-pAbAi) strain separately, and grown in the SD/-Leu selection medium supplemented with 500 ng/mL AbA (Clontech) to identify the interaction. The procedure was carried out following the manufacturer’s protocol as described in the Yeast Protocols Handbook (Clontech).

Y2H and BiFC assays

The Y2H assay was performed using the Matchmaker Gold System (Clontech). The CDS of MtLMI1a was cloned into the pGADT7 vector and the CDS of MtLMI1b was cloned into the pGBKT7 vector using the Gateway system (Invitrogen). The bait and prey plasmids were cotransformed into the yeast strain AH109 (Clontech). Protein–protein interactions were tested by stringent (SD/-Leu/-Trp/-His) selection supplied with 3-amino-1,2,4-triazole and X-α-Gal according to the manufacturer’s protocol (Clontech). Plates were incubated in the dark at 30°C for 3 d. For the BiFC assays, MtLMI1a was cloned to pEarleyGate202-YC, while MtLMI1b was cloned into pEarleyGate201-YN using the Gateway system (Invitrogen). MtLMI1a-cYFP and MtLMI1b-nYFP were introduced into the A. tumefaciens EHA105 strain. Various combinations of transformed cells were simultaneously infiltrated into 21-d-old N. benthamiana leaves. The fluorescent signals were observed 48–60 h after infiltration under confocal microscopy (Zeiss). All primers used are listed in Supplemental Table S2.

Phylogenetic analysis

The phylogenetic trees were constructed with the maximum-likelihood method using the MEGA software suite (http://www.megasoftware.net/) as described previously (Tamura et al., 2011).

Accession numbers

Sequence data from this article can be found in the GenBank and M. truncatula Genome Project v4.0 under the following accession numbers: MtLMI1a, Medtr1g061660; MtLMI1b, Medtr7g103340; SLM1, AAT48630; MtAGO7, XM_003613868; MtNAM, JF929904; MtARF3, Medtr2g014770; MtARF4a, Medtr4g060470; MtARF4b, Medtr2g093740; GmLMI1a, XP_003535265; GmLMI1b, XP_003542577; GmLMI1c, XP_003553578; GmLMI1d, XP_003521430; TENDRIL-LESS, ACI42911; AhLMI1, XP_025635003; SlLMI1, XP_004246902; VvLMI1, XP_002283931; ChLMI1, AHW98968; RCO, AHW98969.1.

Supplemental data

The following supplemental materials are available.

Supplemental Figure S1. Phylogenetic analysis of MtLMI1a, MtLMI1b, LMI1, and the blasted proteins in M. truncatula.

Supplemental Figure S2. Multiple alignment of LMI1 and its homologs.

Supplemental Figure S3. Physical interaction between MtLMI1a and MtLMI1b.

Supplemental Figure S4. The ratios of leaflet serration numbers versus leaflet area in WT and mtlmi1a-1 mtlmi1b-1 double mutant.

Supplemental Figure S5. Scanning electron micrograph analysis of leaf margin in mature leaves of WT and mtlmi1a-1 mtlmi1b-1.

Supplemental Figure S6. Promoter–GUS fusion studies of MtLM1a/b expression in transgenic M. truncatula.

Supplemental Figure S7. The expression levels of STF in WT and 35Spro: MtLMI1a/b-GFP-1 determined by RT-qPCR.

Supplemental Figure S8. The expression levels of SLM1 in WT and mtlmi1a-1 mtlmi1b-1 determined by RT-qPCR.

Supplemental Figure S9. RNA in situ hybridization analysis of MtNAM mRNA in WT and mtlmi1a-1 mtlmi1b-1.

Supplemental Table S1. List of mutant alleles.

Supplemental Table S2. Primers used in this study.

Supplementary Material

kiab268_Supplementary_Data
Click here for additional data file. (1.8MB, pdf)

Acknowledgments

The authors would like to thank Haiyan Yu from the State Key Laboratory of Microbial Technology of Shandong University for help and guidance in microscopy.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31671507 and 31871459) and Shandong Province (ZR2019MC013 and ZR2020KC018), project for Scientific Research Innovation Team of Young Scholar in Colleges and Universities of Shandong Province (2019KJE008), project for Innovation and Entrepreneurship Leader of Qingdao (19-3-2-3-zhc). Development of M. truncatula Tnt1 mutant population was, in part, funded by the National Science Foundation, USA (DBI-0703285 and IOS-1127155).

Conflict of interest statement. The authors have no conflicts of interest to declare.

 

X.W. and C.Z. conceived the study and designed the experiments. X.W., J.J.Z., Y.X., X.L., L.W., J.Z., and J.L. performed the research. K.S.M. and J.W. contributed to the generation of Tnt1-tagged mutants. X.W., H.W., L.H., X.Y., K.S.M., J.W., and C.Z. analyzed the data, provided the critical discussion on the work, and edited the manuscript. X.W. and C.Z. wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Chuanen Zhou (czhou@sdu.edu.cn).

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