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. 2019 Dec 20;182(3):1481–1493. doi: 10.1104/pp.19.01434

AtMOB1 Genes Regulate Jasmonate Accumulation and Plant Development1,[OPEN]

Zhiai Guo a, Xiaozhen Yue a,b, Xiaona Cui a,b, Lizhen Song a, Youfa Cheng a,b,c,2,3
PMCID: PMC7054864  PMID: 31862839

A core component of the Hippo pathway plays important roles in regulating jasmonate accumulation and plant development in Arabidopsis.

Abstract

The MOB1 proteins are highly conserved in yeasts, animals, and plants. Previously, we showed that the Arabidopsis (Arabidopsis thaliana) MOB1A gene (AtMOB1A/NCP1) plays critical roles in auxin-mediated plant development. Here, we report that AtMOB1A and AtMOB1B redundantly and negatively regulate jasmonate (JA) accumulation and function in Arabidopsis development. The two MOB1 genes exhibited similar expression patterns, and the MOB1 proteins displayed similar subcellular localizations and physically interacted in vivo. Furthermore, the atmob1a atmob1b (mob1a/1b) double mutant displayed severe developmental defects, which were much stronger than those of either single mutant. Interestingly, many JA-related genes were up-regulated in mob1a/1b, suggesting that AtMOB1A and AtMOB1B negatively regulate the JA pathways. mob1a/1b plants accumulated more JA and were hypersensitive to exogenous JA treatments. Disruption of MYC2, a key gene in JA signaling, in the mob1a/1b background partially alleviated the root defects and JA hypersensitivity observed in mob1a/1b. Moreover, the expression levels of the MYC2-repressed genes PLT1 and PLT2 were significantly decreased in the mob1a/1b double mutant. Our results showed that MOB1A/1B genetically interact with SIK1 and antagonistically modulate JA-related gene expression. Taken together, our findings indicate that AtMOB1A and AtMOB1B play important roles in regulating JA accumulation and Arabidopsis development.

The Hippo signaling pathway was first described in animals. It plays pivotal roles in controlling cell proliferation, apoptosis, organ growth, and tissue homeostasis (Pan, 2010). Dysregulation of the pathway causes various cancers (Harvey et al., 2013). The core components of the pathway, including the Ste20-like kinases MST1/2, the AGC kinase NDR/LATS, and the kinase regulators MOB1 and Sav, form a kinase cascade. Sav interacts with MST1/2 and activates its kinase activity. MST1/2 phosphorylates NDR/LATS kinase and MOB1. Subsequently, MOB1 interacts with NDR/LATS and regulates kinase activity of the latter. The activated NDR/LATS in turn phosphorylates and inactivates the transcriptional coactivator YAP/TAZ (Hansen et al., 2015).

The MOB1 proteins are highly conserved from yeast (Saccharomyces cerevisiae) to plants and animals (Lai et al., 2005; Cui et al., 2016). The yeast MOB1 is an essential gene required for completion of mitosis and maintenance of ploidy (Luca and Winey, 1998). In Drosophila (Drosophila melanogaster), disruption of MOB1/Mats results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth (Lai et al., 2005). In humans (Homo sapiens), among the 7 homologs of yeast MOB (hMOB1A, hMOB1B, hMOB2A, hMOB2B, hMOB2C, hMOB3, and hMOB4), hMOB1A and hMOB1B share more than 95% sequence identity/similarity. A biochemical characterization of hMOBs showed that only hMOB1A and hMOB1B interact with both LATS1 and LATS2 to regulate cell proliferation and apoptosis (Chow et al., 2010). In mouse (Mus musculus), the mob1a/1b double mutant showed cancer susceptibility and embryonic lethality (Nishio et al., 2012). Mob1a/1b double mutation in mouse liver results in the death of more than half of mutant mice within 3 weeks of birth. All survivors eventually develop liver cancers and die by age 60 weeks (Nishio et al., 2016). In addition, tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficient mice had chondrodysplasia (Goto et al., 2018).

The Arabidopsis (Arabidopsis thaliana) genome contains four MOB1 genes: AtMOB1A, AtMOB1B, AtMOB1C, and AtMOB1D (Citterio et al., 2006; Vitulo et al., 2007; Cui et al., 2016). AtMOB1A is required for tissue patterning of the root tip, and sporophyte and gametophyte development (Galla et al., 2011; Pinosa et al., 2013). Recently, we reported that AtMOB1A plays critical roles in auxin-mediated plant development (Cui et al., 2016). The atmob1a mutant genetically interacts with mutants in auxin biosynthesis, signaling, and transport in many developmental processes. Interestingly, the defects of atmob1a can be fully rescued by the Drosophila MOB1 gene Mats, suggesting conserved gene functions among different species (Cui et al., 2016). It was shown that AtMOB1A and AtMOB1B interact with SIK1, a Hippo/STE20 homolog, and regulate cell proliferation and expansion in Arabidopsis (Xiong et al., 2016). These findings demonstrate that AtMOB1A plays important roles in plant development. However, the functions of other Arabidopsis MOB1 genes remain to be elucidated.

Jasmonates (JA) are a group of phytohormones including jasmonic acid and its derivatives. They are important in regulating plant growth and development, and plant responses to biotic and abiotic stresses. Jasmonic acid is synthesized from α-linolenic acid via the octadecanoid pathway in plastids and peroxisomes. Following synthesis, jasmonic acid is exported from the peroxisomes into the cytoplasm, where it is conjugated with Ile to produce bioactive JA-Ile. In the JA signaling pathway, JA-Ile promotes the interaction between the JA receptor COI1 and JAZ proteins. JAZ can be ubiquitinated and degraded by the 26S proteasome, leading to the release of MYC2, the major transcription factor of JA-mediated gene expression. Consequently, the JA responsive gene expression and JA responses are activated (Huang et al., 2017).

Here we show that the mob1a/1b double mutant displays severe developmental defects at the seedling stage. AtMOB1B expression was similar to that of AtMOB1A, and the two MOB1 proteins interacted with each other in vivo. Transcriptomic analysis indicated that the expression levels of many genes in the JA pathways were increased in the mob1a/1b double mutant. JA contents were also significantly increased in mob1a/1b. Consistently, mob1a/1b displayed hypersensitivity to exogenous JA treatments and the expression of MYC2, which encodes a key transcription factor in JA signaling, was increased. Moreover, myc2 partially suppressed the developmental defects of mob1a/1b. We found that the expression levels of MYC2-repressed PLT1 and PLT2 in the mob1a/1b double mutant were significantly decreased. We conclude that AtMOB1A and AtMOB1B regulate JA accumulation, and plant growth and development.

RESULTS

AtMOB1A and AtMOB1B Redundantly Control Plant Development

There are four MOB1 genes in Arabidopsis, namely AtMOB1A, AtMOB1B, AtMOB1C, and AtMOB1D (Citterio et al., 2006; Vitulo et al., 2007; Cui et al., 2016). AtMOB1A and AtMOB1B form a subgroup, whereas AtMOB1C and AtMOB1D form another clade (Fig. 1A). Previously, we reported that the loss-of-function MOB1A mutants, mob1a-1 and mob1a-2, developed short roots and displayed small floral organs and reduced fertility compared with wild type (Cui et al., 2016).

Figure 1.

Figure 1.

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Analysis of loss-of-function atmob1mutants. A, A phylogenetic tree of AtMOB1 family proteins. B, Schematic representation of AtMOB1 gene structures and positions of the T-DNA insertion. The 5-bp deletion in mob1c-2 mutant was designated as “Del-agGAG” in AtMOB1C. C and D, 6-d-old seedlings of Col-0, mob1a-2, mob1b-1, mob1a-2/b-1+/−, and mob1a-2/b-1 mutants. E, Electron micrograph of a mob1a-2/b-1 double-mutant seedling. F and G, Adult plants (F) and siliques (G) of Col-0, mob1a-2, mob1b-1, and mob1a-2/b-1+/−· H to K, 6-d-old seedlings of mob1a-2/b-1 (H), mob1a-2/b-1/c-1 (I), mob1a-2/b-1/d-1 (J), and mob1a-2/b-1/c-2/d-1 (K). L, 6-d-old seedlings of Col-0, mob1c-1, mob1d-1, and mob1c-2/d-1. Scale bars = 5 mm (C and D) and 100 µm (E and H–K).

To define functions of the other AtMOB1 genes, we obtained transfer DNA (T-DNA) insertion mutants, denoted mob1b-1, mob1c-1, and mob1d-1 (Fig. 1B), from the Nottingham Arabidopsis Stock Center. Reverse transcription-PCR (RT-PCR) analysis indicated that all of the mutations appeared null (Supplemental Fig. S1A). However, no obvious developmental defects were observed in the single mutants, suggesting that the MOB1 genes may have overlapping functions. We then generated the double and triple mutant combinations (Fig. 1, C–J). Because MOB1C (At5g20430) and MOB1D (At5g20440) are located in tandem on Chromosome V, it is impossible to generate the double mutant or the quadruple mutant by genetic crossing. We thus constructed the mob1a-2 mob1b-1 mob1c-2 mob1d-1 quadruple mutant (mob1a-2/b-1/c-2/d-1) using CRISPR/Cas9 gene editing technology (Gao et al., 2016; Zeng et al., 2018; Fig. 1K; Supplemental Fig. S1B) in the mob1a-2+/−/mob1b-1/mob1d-1 (mob1a-2+/−/b-1/d-1) background.

The seedlings of mob1a-2/b-1+/− displayed smaller cotyledons, shorter roots, and a longer hypocotyl than single mutants and wild type (Fig. 1, D and E). The siliques of mob1a-2/b-1+/− adult plants were very short and completely sterile (Fig. 1, F and G). On the other hand, the mob1a-2+/−/b-1 mutant resembled mob1b-1. These results indicated that AtMOB1B became haploid insufficient in the mob1a mutant background. The mob1a-2/b-1 seedlings were very tiny, and their development was significantly retarded. The 21-d-old double-mutant plants had much smaller leaves and shorter roots compared with wild type and the single mutants (Supplemental Fig. S2). To confirm that the phenotypes were caused by the mutations in AtMOB1A and AtMOB1B, we carried out genetic complementation experiments. Transformation of mob1a-2/b-1 with the AtMOB1A genomic fragment resulted in plants that were similar to mob1b-1. We also found that mob1a-2/b-1 plants harboring the AtMOB1B transgene behaved similarly to mob1a. Our results demonstrate that the strong developmental defects observed in the mob1a-2/b-1 mutant are caused by the disruption of both AtMOB1A and AtMOB1B (Supplemental Fig. S3).

The mob1a-1/b-1/c-1 and mob1a-2/b-1/d-1 triple mutants and the mob1a-2/b-1/c-2/d-1 quadruple mutant displayed phenotypes similar to that of mob1a-2/b-1 (Fig. 1, H–J). Moreover, the mob1c-2/d-1 double mutant segregated from the mob1/a-2/b-1/c-2/d-1 quadruple mutant similar to the mob1c-1 and mob1d-1 single mutants and wild-type plants (Fig. 1L). These results suggest that AtMOB1A plays a more predominant role than AtMOB1B. AtMOB1A and AtMOB1B appear to be more important in regulating plant development than AtMOB1C and AtMOB1D under our growth conditions. We therefore focused on AtMOB1A and AtMOB1B in this work.

AtMOB1A and AtMOB1B Show Similar Expression Patterns

To investigate the expression patterns of AtMOB1A and AtMOB1B, we made constructs containing AtMOB1A or AtMOB1B genomic DNA sequences fused with the GUS gene, which were then expressed in wild-type plants. AtMOB1A-GUS and AtMOB1B-GUS were expressed with very similar patterns in cotyledons, true leaves, trichomes, root hairs, primary and lateral roots of seedlings, and floral organs (Supplemental Fig. S4, A–F and H–M).

We also generated transgenic plants containing AtMOB1B genomic DNA sequence fused with the GFP gene. AtMOB1B-GFP was expressed in the roots, and the fusion protein was localized to the nucleus, cytoplasm, and plasma membrane, similar to AtMOB1A-GFP (Supplemental Fig. S4, G and N). The subcellular localization of AtMOB1B-GFP was similar to that of AtMOB1A-GFP (Cui et al., 2016). These results are consistent with our hypothesis that AtMOB1A and AtMOB1B redundantly regulate plant development.

AtMOB1A and AtMOB1B Are Necessary for Cell Proliferation

The seedlings of the mob1a-2/b-1 mutant had very short roots. The lengths of the root elongation zone and the meristem zone were dramatically decreased in the mob1a-2/b-1 mutant (Fig. 2, A–C). The cell numbers of the elongation zone and the meristem zone were also significantly reduced in the double mutant (Fig. 2, D and E). Moreover, the root cap columella of mob1a-2/b-1 was much smaller (Fig. 2, F and G). These results suggest that cell division activities are abnormal in mob1a-2/b-1. To test this hypothesis, we used CYCB1;1:GUS, which is a widely used marker for the G2/M phase of the cell cycle (Colón-Carmona et al., 1999). We introduced this marker into the mob1a-2/b-1 double and single mutant backgrounds by genetic crossing. The expression of CYCB1;1:GUS was decreased significantly in mob1a-2 and slightly in mob1b-1 single mutants, and was barely detectable in the mob1a-2/b-1 double mutant (Fig. 2, H and I). These results suggest that both MOB1A and MOB1B are important for cell proliferation in Arabidopsis roots.

Figure 2.

Figure 2.

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The mob1a-2 b-1 mutant displays strong defects in root tips. A, The root tips of 6-d-old seedlings of Col-0, mob1a-2, mob1b-1, and mob1a-2/b-1 mutants are shown. The meristem zone is marked with two arrowheads. B to E, Measurements of the lengths of root elongation zone (B) and meristem (C), and cell numbers in elongation zone (D) and meristem (E). F, Root tips of 6-d-old seedlings stained with FM4-64. G, The columella root cap cell of 6-d-old seedlings revealed by Lugol staining. H, CYCB1;1:GUS expression in the root of 6-d-old seedlings. I, Quantification of cells with CYCB1;1:GUS signal in (H). Data represent means ± sd. Different letters represent the significance at the P < 0.001 level (one-way ANOVA, lsd test); n > 20. Scale bars = 200 µm (A) and 50 µm (F–H).

AtMOB1A Physically Interacts with AtMOB1B

We tested whether AtMOB1A and AtMOB1B physically interact with each other. We carried out an coimmunoprecipitation (Co-IP) assay in Nicotiana benthamiana leaves, which were cotransformed with yellow fluorescent protein (YFP)-tagged AtMOB1B and FLAG-tagged AtMOB1A. The proteins were immuno-precipitated using anti-GFP beads and detected with FLAG antibody. Our results clearly indicated that AtMOB1A and AtMOB1B physically interact in N. benthamiana leaves (Fig. 3A).

Figure 3.

Figure 3.

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Physical interaction between AtMOB1A and AtMOB1B proteins in vivo. A, Co-IP assay of AtMOB1A and AtMOB1B. AtMOB1B-GFP was immunoprecipitated by using anti-GFP agarose beads, followed by immunoblot with anti-Flag antibody, and AtMOB1A-Flag was detected. B, LCI assays showing the interaction between MOB1A and MOB1B in N. benthamiana leaf cells. Left shows the combinations of agrobacteria containing the indicated plasmids used to coinfiltrate into different leaf regions shown at right. Empty cLUC and nLUC vectors were used as negative controls. The experiments were carried out with three independent biological repeats.

We also performed immunoprecipitation mass spectrometry (IP-MS) experiments using Arabidopsis seedlings transformed with 35S::AtMOB1A-FLAG. Total protein was extracted from seedlings and immunoprecipitated with anti-FLAG antibody beads. The candidate interacting proteins were then identified by mass spectrometry. A total of 11 AtMOB1A/B peptide sequences were detected as AtMOB1A-interacting proteins. Although nine of these were shared by AtMOB1A/B, the other two were AtMOB1B specific (Supplemental Table S1), indicating that AtMOB1A and AtMOB1B interacted in the IP-MS assay in Arabidopsis.

To further confirm the interaction, we carried out the firefly luciferase complementation imaging (LCI) assay (Chen et al., 2008). The results indicated that AtMOB1A directly interacts with AtMOB1B in N. benthamiana leaves (Fig. 3B). Combined, these experiments show that AtMOB1A interacts with AtMOB1B in vivo.

AtMOB1A and AtMOB1B Negatively Regulate the JA Pathways

To identify differentially expressed genes (DEGs) between the mob1a-2/b-1 mutant and wild type, we performed RNA-sequencing (RNA-seq) analysis using 10-d-old seedlings of mob1a-2/b-1 and Col-0, with three biological repeats included of each genotype. A total of 1202 DEGs were identified, including 725 up-regulated and 477 down-regulated genes (Supplemental Table S2). Gene Ontology (GO) analysis revealed that many genes in the JA biosynthetic, metabolic, and signaling pathways were significantly enriched (Fig. 4A). In addition, genes involved in several JA-related biological processes were also enriched, including glucosinolate biosynthetic and metabolic processes, and responses to insects and wounding (Huang et al., 2017; Wasternack and Song, 2017; Fig. 4A; Supplemental Table S3).

Figure 4.

Figure 4.

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Expression of genes involved in JA biosynthesis, metabolism, and signaling are altered in the mob1a-2/b-1 double mutant. A, Functional assignment of the DEGs by GO enrichment analysis. Bars = –lg (P value), and the P values were adjusted by the Bonferroni approach. B to D, DEGs in jasmonic acid biosynthesis (B), metabolic processes of JA and JA-Ile (C), and JA signaling pathway and response to JA (D). The heatmap indicates the ratio being up-regulated. Scale colors represent log2(ratio). α-LeA, α-linolenic acid; 13-HPOT, 13-hydroperoxylinoleic acid; 12,13-EOT, 12,13-epoxyoctadecatrienoic acid; OPDA, 12-oxophytodienoic acid; OPC-8, 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid; PLA1, phospholipase A1; 13-LOX, 13-lipoxygenase; AOS, allene oxide synthase; AOC, allene oxide cyclase; OPR, OPDA reductase; OPCL, OPC-8-CoA ligase; ST2A, 12-OH-JA sulfotransferase; JOX, jasmonic acid oxidase; JAR1, jasmonoyl Ile syntase; ILL6 and IAR3, IAA-amino acid hydrolase; CYP94C1; 12-OH-JA-Ile carboxylase; JAZ, jasmonate-zim-domain protein; MYC2, bHLH zip transcription factor; VSP2, vegetative storage protein 2; TAT3, Tyr aminotransferase 3; JR2, jasmonic acid responsive 2; RAP2.6L, ERF/AP2 transcription factor; ANAC081, Arabidopsis NAC domain containing protein 81. E to G, Relative expression of the DEGs in JA biosynthesis (E), metabolism (F), and signaling (G) in Col-0, mob1a-2, mob1b-1, and mob1a-2/b-1. Ten-day-old seedlings were collected for RNA extraction and RT-qPCR analysis. ACTIN2 was used as an internal control. The expression levels of the indicated genes in Col-0 were set to 1. Error bars = SD of three biological repeats. Different letters indicate significant difference at P < 0.001 (one-way ANOVA, Tukey post-test).

We identified 89 JA-related DEGs in mob1a-2/b-1, which include 83 up-regulated and 6 down-regulated genes (Supplemental Table S4). First, the expression of many pivotal genes in JA biosynthesis, including the four 13-LOXs, AOS, AOC, OPR3, and OPCL were dramatically up-regulated (Fig. 4B). Second, JAR1, ILL6, IAR3, ST2A, JOX, CYP94B3, and CYP94C1 were also up-regulated, which encode enzymes that catalyze a number of key metabolic processes of JA and JA-Ile, including hydrolysis, sulfation, hydroxylation, and carboxylation (Fig. 4C). Third, five JAZs and MYC2 in the JA signaling pathway were markedly up-regulated (Fig. 4D). Moreover, some genes involved in response to JA, such as VSP2, TAT3, and JR2, were also up-regulated (Fig. 4D; Supplemental Table S4).

To validate the RNA-seq results, we carried out RT-quantitative PCR (qPCR) analysis. The results confirmed the increased expression of the up-regulated DEGs in mob1a-2/b-1 compared with that in the single mutants and wild type (Fig. 4, E–G). Therefore, AtMOB1A and AtMOB1B likely negatively regulate JA biosynthetic, metabolic, and signaling pathways.

JA Content Is Significantly Increased in the mob1a-2/b-1 Double Mutant

The up-regulation of many genes involved in JA biosynthesis, metabolism, and signaling in the mob1a-2/b-1 double mutant may lead to an alteration of JA concentrations. To test this hypothesis, we measured endogenous JA contents using the gas chromatography-mass spectrometry (GC-MS) method. Indeed, JA content in the mob1a-2/b-1 mutant was dramatically increased compared with that in the single mutants and wild-type plants. JA content was similar in the single mutants and wild type (Fig. 5A). These results suggest that AtMOB1A and AtMOB1B negatively regulate JA accumulation.

Figure 5.

Figure 5.

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AtMOB1A and AtMOB1B negatively regulate JA accumulation. A, Measurement of the JA contents in seedlings of Col-0, mob1a-2, mob1b-1, and mob1-2/b-1 mutants by using GC-MS. Seedlings were grown on half-strength Murashige and Skoog medium for 14 d after germination. Data represent means ± sd of three independent biological repeats. Different letters represent statistical significance at the P < 0.001 level (one-way ANOVA, lsd test). B and C, 6-d-old seedlings of Col-0, myc2-2, mob1a-2/b-1, and myc2-2 mob1a-2/b-1 mutants. Seedlings were grown on half-strength Murashige and Skoog medium for 6 d after germination. Scale bars = 2 mm (B) and 1 mm (C). D, Measurement of root length of Col-0, myc2-2, mob1a-2/b-1, and myc2-2 mob1a-2/b-1 mutants. Data represent means ± sd (n ≥ 20) with significant differences determined by Student’s t test. ***P < 0.001 compared with mob1-2 b-1. E, mob1a-2/b-1 mutants were hypersensitive to exogenous JA treatment. Five-day-old seedlings grown on half-strength Murashige and Skoog plates were transferred to the half-strength Murashige and Skoog plates without (Control, top) or with (JA, bottom) 100 µM Me-JA and grown for 2 weeks. Scale bars = 2 mm. F, Measurement of chlorophyll contents. The chlorophyll contents of indicated mutants are relative to that in Col-0, which was set to 1.0. Data represent means ± sd (n = 15). Student’s t test. ***P < 0.001 compared with control.

The mob1a-2/b-1 Double Mutant Is Hypersensitive to Exogenous Me-JA Treatment

It is known that JA can promote leaf senescence (He et al., 2002; Xiao et al., 2004; Shan et al., 2011). We tested whether the mob1 single and double mutants responded to exogenous JA treatments differently than wild type. The 5-d-old seedlings of wild type, mob1a-2, and mob1b-1 single mutants, and the mob1a-2/b-1 double mutant were transferred onto half-strength Murashige and Skoog plates containing different concentration (0, 10, 25, 50, 100, and 200 µm) of Me-JA and grown for a further 14 d. The leaves of the double mutant became senescent and yellow when treated with Me-JA at 100 µm and 200 µm, whereas the wild type and the single mutants remained green (Supplemental Fig. S5). In addition, RNA-seq results indicated that several senescence-associated genes displayed altered expression in the double mutant (Supplemental Table S5), including up-regulated Senescence-associated gene 21 (SAG21/AT4G02380), Senescence/dehydration-associated protein-related A/(AT4G15450), and ERD7/Senescence/dehydration-associated protein-related (AT2G17840), and down-regulated Rubisco Activase (RCA), and Senescence-associated family protein (AT1G66330). These results are consistent with the observed increase in JA content in the double mutant.

Disruption of MYC2 Partially Rescues the Developmental Defects and JA Hypersensitivity of mob1a-2/b-1

The increased expression of JA-related genes and JA content in the mob1a-2/b-1 double mutant suggested that AtMOB1A and AtMOB1B negatively regulate JA accumulation. To test the biological relevance of these observations, we analyzed the genetic interaction between myc2-2 and the mob1a-2/b-1 double mutant. MYC2 encodes a key downstream transcription factor that regulates diverse aspects of JA responses, and its expression was also found to be up-regulated in mob1a-2/b-1 (Supplemental Table S4; Fig. 4G). We introduced the myc2-2 mutation into the mob1a-2/b-1 background by genetic crossing. The myc2-2 mob1a-2/b-1 triple mutant developed longer roots than the mob1a-2/b-1 double mutant (Fig. 5, B–D), indicating that the myc2-2 mutation can partially suppress the root defects of mob1a-2/b-1.

Because MYC2 plays critical roles in activating JA-induced leaf senescence (Qi et al., 2015), we examined JA-induced senescence in the myc2-2 mob1a-2/b-1 triple mutant. It was clear that the myc2-2 mutation repressed the JA hypersensitivity of the mob1a-2/b-1 double mutant. Both the morphological defects and decreased chlorophyll content of mob1a-2/b-1 were suppressed by myc2-2. (Fig. 5, E and F). These results suggest that the root defects and JA hypersensitivity of the mob1a-2/b-1 double mutant are partially caused by overaccumulation of JA, and are dependent on MYC2-mediated JA signaling.

Expression Levels of PLT1 and PLT2 Are Decreased in the mob1a-2/b-1 Double Mutant

PLETHORA (PLT) 1 and PLT2 encode proteins belonging to the AP2 class of transcription factors, and are essential for root stem cell niche patterning (Aida et al., 2004; Galinha et al., 2007). It is known that MYC2 represses the expression of PLT1 and PLT2, which restricts root meristem activity and inhibits primary root growth (Chen et al., 2011). To investigate the expression of PLT1 and PLT2 in mob1a-2/b-1, we introduced markers pPLT1:cyan fluorescent protein (pPLT1:CFP) and pPLT2:CFP (Galinha et al., 2007) into the mutant backgrounds by genetic crossing. The expression levels of pPLT1:CFP and pPLT2:CFP were significantly decreased in the mob1a-2/b-1 double mutant compared with that in the single mutants and wild type (Fig. 6, A–D). We further introduced the pPLT1:PLT-YFP and pPLT2:PLT-YFP fusions (Galinha et al., 2007) into the mutant backgrounds by genetic crossing, and found that, in agreement, the resulting protein levels were also significantly decreased in mob1a-2/b-1 compared with that in the single mutants and wild type (Fig. 6, E–H). To examine the root stem cell identity, we introduced the quiescent center marker pWOX5::GFP into the mutant backgrounds by genetic crossing. The GFP signals were detected in the quiescent center cells (Supplemental Fig. S6), indicating that the root stem cell identity was still maintained. These results suggest that up-regulated MYC2 expression represses PLT1/2 expression in the mob1a-2/b-1 double mutant, which at least partially accounts for the root developmental defects.

Figure 6.

Figure 6.

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Expression levels of PLT1 and PLT2 are reduced in mob1a-2/b-1. A, C, E, and G, Representative expression of PLT1:CFP (A), PLT2:CFP (C), PLT1:PLT1-YFP (E), and PLT2:PLT2-YFP (G) in the root tips of 6-d-old Col-0, mob1a -2, mob1b-1, and mob1a-2/b-1 mutant seedlings. Scale bars = 50 µm. B, D, F, and H, Quantification of CFP (A, C) and YFP (E, G) fluorescence, respectively. The fluorescence strength of Col-0 was set to 1. Data represent means ± sd (n = 15). Different letters represent statistical significance at the P < 0.001 level (one-way ANOVA, lsd test).

Genetic Interaction Between mob1a-2/b-1 and sik1 Mutants

It was previously reported that the Hippo/STE20 homolog SIK1 interacts with MOB1 to regulate cell proliferation and cell expansion in Arabidopsis (Xiong et al., 2016). sik1 mutant plants are smaller than wild type (Xiong et al., 2016), and the JA levels in the sik1 mutant are decreased compared with wild type (Zhang et al., 2018). We generated a sik1 mob1a-2/b-1 triple mutant by genetic crossing, and found that seedlings of the resulting triple mutant were smaller than mob1a-2/b-1 double-mutant seedlings (Fig. 7, A–C). Because JA levels are increased in mob1a-2/b-1 but decreased in sik1 mutant, we examined the expression of several JA-responsive genes in these mutant backgrounds. The expression levels of JAZ1, JAZ2, JAZ5, JAZ9, JAZ10, and MYC2 were increased in mob1a-2/b-1 but significantly decreased in the sik1 mutant. The expression levels of these genes were increased in the sik1 mob1a-2/b-1 triple mutant compared with in the sik1 mutant (Fig. 7D). These results suggest that changes to JA-responsive gene expression caused by the mob1a-2/b-1 mutations were partially alleviated by the sik1 mutation in the sik1 mob1a-2/b-1 triple mutant, consistent with the observed differences in JA levels in mob1a-2/b-1 and sik1.

Figure 7.

Figure 7.

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Genetic interaction between MOB1A/B and SIK1. A and B, 12-d-old seedlings of Col-0, sik1-1, mob1a-2/b-1, and mob1a-2/b-1 sik1-1 mutants. C, Close-up of seedlings in (B). Note the triple mutant was smaller than the double mutant. Scale bars = 2 mm. D, Relative expression of JA signaling-related genes in 12-d-old seedlings of Col-0, mob1a-2/b-1, sik1-1, and mob1a-2/b-1 sik1-1. ACTIN2 was used as an internal control. The expression levels of the indicated genes in Col-0 were set to 1. Different letters indicate significant difference at P < 0.001 (n = 3, one-way ANOVA, Tukey post-test).

DISCUSSION

In this paper, we report that AtMOB1A and AtMOB1B genetically and physically interact to control Arabidopsis development. The mob1a-2/b-1 double mutant was reduced in size with severe developmental defects, and the expression levels of many genes in the JA biosynthetic, metabolic, signaling, and responses pathways were up-regulated. Consistent with these observations, mob1a-2/b-1 accumulated much more JA than the single mutants and wild type, and the double mutant was hypersensitive to JA treatment. Disruption of the key JA signaling gene MYC2 partially alleviated mob1a-2/b-1 root defects and JA hypersensitivity. mob1a-2/b-1 was associated with decreased expression of PLT1/2, suggesting that altered expression of these critical root development genes partially accounts for the observed root defects.

AtMOB1A and AtMOB1B Interact to Regulate Growth and Development

Previously, it has been shown that AtMOB1A is important for many plant development processes (Citterio et al., 2006; Pinosa et al., 2013; Cui et al., 2016) and that AtMOB1A plays critical roles in auxin-mediated development (Cui et al., 2016). Although the mob1a single mutant plants displayed strong developmental defects and the mob1b phenotype was similar to wild type, mob1a-2/b-1 showed an enhanced mutant phenotype compared with mob1a-2 and mob1b-1, indicating that AtMOB1A and AtMOB1B have unique and overlapping functions. Interestingly, such genetic interaction and redundant or overlapping function of MOB1A/B were also observed in mouse. The mouse mob1a/b double mutant exhibited embryonic lethality or severe cancer susceptibility (Nishio et al., 2012). Mob1a/1b double mutation in mouse liver resulted in death within 3 weeks of birth or liver cancers and death by age 60 weeks (Nishio et al., 2016).

On the other hand, AtMOB1A and AtMOB1B proteins physically interact in vivo. The interaction between hMOB1A and hMOB1B was also reported in humans (Wang et al., 2014), suggesting that the interaction between these two proteins is also conserved. Previously, it was reported that Arabidopsis Ser/Thr kinase 1 (SIK1) is a Hippo homolog and that AtMOB1A and AtMOB1B interact with SIK1 (Xiong et al., 2016). These genetic and physical interactions suggest that SIK1, AtMOB1A, and AtMOB1B form a large protein complex. Interestingly, the sik1 mob1a-2/b-1 triple mutant displayed stronger developmental defects compared with the mob1a-2/b-1 double mutant, suggesting that there might be additional components involved in regulating the affected developmental processes. There are 10 SIK1-like genes in the MAP4 Kinase family (Zhang et al., 2018). It is possible that other members of the MAP4 family play redundant roles with SIK1. Recently, it was shown that SIK1 associates with, phosphorylates, and stabilizes the central immune regulator BIK1 (Zhang et al., 2018). On the other hand, MOB1s are adaptor/scaffolding proteins. It is not clear whether SIK1 phosphorylates MOB1 and/or MOB1 activates SIK1 kinase activity.

Because the phenotypes of the mob1a-2/b-1 double mutant are stronger than those of the mob1a-2 and mob1b-1 single mutants, it is likely that AtMOB1B and AtMOB1A proteins also form homodimer/oligomers. Also, because the mob1a-2 mutant displayed strong developmental defects, whereas mob1b-1 was largely normal, it is likely that AtMOB1A plays the dominant role. Indeed, a total of 11 AtMOB1A/B peptide sequences were detected as AtMOB1A-interacting proteins. Nine of these were shared by AtMOB1A/B, and the remaining two were AtMOB1B specific (Supplemental Table S1). These results indicate AtMOB1A and AtMOB1B interacted in the IP-MS assay in Arabidopsis, and further suggest that AtMOB1A and AtMOB1B form homodimer/oligomers.

AtMOB1A and AtMOB1B Modulate JA Accumulation

JAs are important in regulating plant growth and development as well as plant responses to biotic and abiotic stresses. JA inhibits primary root growth and promotes leaf senescence (Huang et al., 2017). We found that mob1a-2/b-1 mutant plants had a very small growth habit, with small cotyledons and short roots. It was reported that JA treatment markedly reduces the expression of CYCB1;1:GUS and represses cell division activity in Arabidopsis root meristems (Chen et al., 2011). We showed that the expression of CYCB1;1:GUS was dramatically decreased in mob1a-2/b-1 mutant plants (Fig. 2), which is consistent with their higher JA level (Fig. 5). These results suggest that the short root phenotype of the mob1a-2/b-1 double mutant could be partially caused by JA-induced repression of root cell division.

The expression levels of many genes involved in JA biosynthetic, metabolic, and signaling pathways were increased, and JA content was elevated in mob1a-2/b-1. Moreover, the expression of MYC2 was increased and the expression of PLT1/2 was decreased in the double-mutant plants. These observations are consistent with the findings that JA reduces the expression levels of PLT1 and PLT2, which is mediated by the direct binding of MYC2 to the promoters of PLT1 and PLT2 to repress their expression (Chen et al., 2011). Moreover, disruption of MYC2 in the mob1a-2/b-1 background partially alleviated the short-root phenotype, suggesting that the root defects were at least partially caused by increased endogenous JA. The mob1a-2/b-1 double mutant was hypersensitive to exogenous Me-JA treatment in terms of leaf senescence. JA-repressed RCA, which plays an important role in JA-induced leaf senescence (Shan et al., 2011), was among the few down-regulated DEGs in mob1a-2/b-1 plants. Our RNA-seq analysis also revealed that expression of some JA responsive genes was altered in the mob1a-2/b-1 double mutant, such as the up-regulation of VPS2, TAT, and LOX3, the most prominent marker genes responding to wounding activated by MYC2 in the JA signaling pathway (Titarenko et al., 1997; Lorenzo et al., 2004). More importantly, the JA hypersensitivity of the mob1a-2/b-1 double mutant was markedly alleviated by the myc2 mutation in myc2-2 mob1a-2/b-1 plants. These results suggest that the elevated JA content in the mob1a-2/b-1 double mutant caused the JA-related phenotypes. Intriguingly, disruption of MYC2 only mildly suppressed the extreme short-root phenotype observed in mob1a-2/b-1, suggesting that other factors besides JA pathways, such as auxin signaling, are also involved in regulating root growth in the mob1a-2/b-1 double mutant. This hypothesis is consistent with our previous report that AtMOB1A plays a role in auxin-mediated development (Cui et al., 2016). It is likely that AtMOB1B plays similar roles in modulating auxin signaling.

Interestingly, it was reported that the expression of JAZ genes (JAZ1, JAZ2, JAZ5, JAZ6, JAZ9, and JAZ12) and MYC genes (MYC2, MYC3, and MYC4) was repressed in the sik1 mutant (Xiong et al., 2016), suggesting that SIK1 also plays roles in JA-related development. However, because the expression of some of the JAZs and MYCs in the mob1a-2/b-1 double mutant was up-regulated, it seems that AtMOB1A/B and SIK1 play different roles in modulating JA pathways. Indeed, JA levels were decreased in the sik1 mutant compared with wild type (Zhang et al., 2018), whereas they were increased in mob1a-2/b-1 (Fig. 5A). These findings suggest that SIK1 promotes and MOB1A/B represses JA levels. Consistently, the expression levels of JAZs and MYC2 were increased in the sik1 mob1a-2/b-1 triple mutant compared with in the sik1mutant (Fig. 7). It is intriguing how components in the same protein complex would antagonistically modulate JA levels. The sik1 mob1a-2/b-1 triple-mutant plants showed compromised regulation of JA-related genes, but displayed more severe developmental defects than sik1 or the mob1a-2/b-1 double mutant. These results suggest that MOB1 and SIK1 may function similarly in controlling cell proliferation, but may have opposite roles in regulating JA levels and JA-related gene expression. The exact molecular mechanisms for such complex regulation are not currently understood. It was reported that the sik1 mutant exhibited significantly higher levels of basal salicylic acid and decreased levels of JA (Zhang et al., 2018). Since SIK1 plays a role in antibacterial immunity response (Zhang et al., 2018) and MOB1 and SIK1 physically interact, it is possible that MOB1 plays a role in biotic stress responses, perhaps through JA signaling.

AtMOB1A and AtMOB1B Act as Key Regulators in Auxin- and JA-Mediated Plant Growth

Previously, we reported that AtMOB1A plays critical roles in auxin-mediated plant development. We isolated the mob1a/ncp1 mutant as an enhancer of pid, and showed that it had strong genetic interactions with mutants in auxin biosynthesis, polar transport, and signaling (Cui et al., 2016). Disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our previous results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling (Cui et al., 2016). Our results presented in this paper clearly indicate that AtMOB1A and AtMOB1B also act as key regulators of JA-mediated plant growth. It is likely that AtMOB1A and AtMOB1B physically interact with each other and are components of a large protein complex, which includes SIK1 (Xiong et al., 2016). AtMOB1A and AtMOB1B promote auxin signaling and cell proliferation; however, the two proteins repress JA accumulation. Thus, the AtMOB1-containing protein complex likely regulates the cross talk between auxin- and JA-mediated plant growth and development. A molecular framework for JA-induced inhibition of root growth through interaction with auxin pathways is well established, in which MYC2-mediated repression of PLT expression integrates JA action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance (Chen et al., 2011). Auxin upregulates PLT1 and PLT2 transcripts and positively regulates stem cell niche maintenance and meristem activity (Aida et al., 2004), and JA downregulates PLT1 and PLT2 expression and negatively regulates stem cell niche maintenance and meristem activity (Chen et al., 2011). We showed that PLT1/2 expression is repressed in the mob1a-2/b-1 double mutant, which could be an integrative outcome of elevated JA content and reduced auxin signaling. It would be interesting to further explore the mechanisms by which AtMOB1A/B control JA accumulation and the cross talk with auxin signaling.

MATERIALS AND METHODS

Plant Materials and Growth Condition

All Arabidopsis (Arabidopsis thaliana) materials used in this work were in the Col-0 ecotype background. Seeds were surface sterilized for 15 min in 70% (v/v) and then 100% (v/v) ethyl alcohol. The seeds were then sowed on half-strength Murashige and Skoog medium containing 0.8% (w/v) agar. The plates were transferred to 4°C for 3 d in darkness for vernalization. Plants were grown at 22°C under a 16-h light/8-h dark cycle. The T-DNA insertion lines, including mob1 a-2 (GK_719G04), mob1 b-1 (SALK_062070), and mob1 d-1 (SALK_053800), were purchased from the NASC. The mob1 a-2 (GK_719G04) mutant was genotyped as described previously (Cui et al., 2016). For genotyping mob1 b-1 (SALK_062070), the primers 5′-GGA​TGA​AGT​GTT​TGA​AGC-3′ and 5′-GCTGAGTAATGG TTGTGA-3′ combined with JMLB1 were used. For genotyping mob1 d-1 (SALK_053800), the primers 5′-GGG​CAA​AGT​CCA​AAT​CCT-3′ and 5′-CCG​CTT​CAC​GAA​ATC​CTC-3′ combined with JMLB1 were used.

DNA Constructs and Plant Transformation

For the expression patterns of ATMOB1B, the plasmid was constructed with its genomic DNA fragment containing the coding region alongside up- and down-stream regulatory sequences with the GFP or GUS gene inserted before the stop code. The AtMOB1B gene was divided into parts A and B. The part A was amplified using primers 5′-CGG​GGT​ACC​GCA​GGA​ATA​CTA​TTG​GGC​CTG-3′ and 5′-ACT​GGG​CCC​GTA​AGG​TGC​AAT​GAT​AGA​TTC-3′. The part B was amplified using primers 5′-ACT​GGG​CCC​ACC​AAA​CAA​AAC​CCA​AAT​CCT​C-3′ and 5′-ACT​GTC​GAC​GGC​TCC​AAC​ACA​AAA​TAC​CTC-3′. The two PCR products were double digested with KpnI and Apa I, or ApaI and SalI, respectively, and cloned into the KpnI-SalI sites of vector pPZP211 to generate pPZP211-gAtMOB1B construct. The GUS or GFP gene was inserted immediately before the stop code using restriction site of ApaI. The pPZP211-gAtMOB1B-GUS or pPZP211-gAtMOB1B-GFP construct was transformed into Agrobacterium tumefaciens strain GV3101, and then Arabidopsis plants were transformed by floral dipping. The transgenic seedlings were selected on half-strength Murashige and Skoog plates with 50 μg/mL kanamycin. The pPZP211-gAtMOB1A (Cui et al., 2016) and pPZP211-gAtMOB1B-GFP constructs were used in the complementation experiments of the mob1a-2/b-1 mutants (Supplemental Fig. S3).

Phenotypic Analysis, Statistical Analysis, and Microscopy

Seedlings and roots were photographed, and their lengths were measured using National Institutes of Health ImageJ software (https://imagej.nih.gov/ij/). Seedlings were mounted in HCG (water-Chloral hydrate-Glycerine) solution (Chloral hydrate:water:glycerol = 8:3:1) and the root meristem cells analyzed on a Leica microsystems DM4500B microscope. Statistical significance was evaluated by Student’s t test analysis or one-way ANOVA analysis followed by lsd test (SPSS). Histochemical staining for GUS activity in plants was performed as described previously (Cui et al., 2016). For the Lugol staining, roots were incubated in the Lugol solution for 3–5 min, and then washed in water once, and mounted in HCG solution for microscopy analysis. GFP, YFP, CFP, and FM4-64 fluorescence was imaged under a confocal laser scanning microscope Olympus FV1000MPE following the manufacturer’s instructions. The fluorescence intensities were measured using ImageJ for quantification analysis.

Co-IP Assays and Mass Spectrometry

For the Co-IP assay of AtMOB1A and AtMOB1B, pSuper1300:AtMOB1A-Flag and pEarleyGate 104-35S:YFP-AtMOB1B plasmids were constructed and introduced into Agrobacterium tumefaciens strain GV3101; then Nicotiana benthamiana leaves were transformed by injection. Leaves were ground in liquid nitrogen, and proteins were extracted with same volume of extraction buffer (100 mm HEPES [pH 7.5], 5 mm EDTA, 5 mm EGTA, 10 mm NaF, 5% [v/v] Glycerol, 10 mm Na3VO4, 10 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.1% [v/v] Triton X-100, protease inhibitor cocktail [Sigma]). Samples were mixed twice quickly following incubating on ice for 30 min and then centrifuged at 14,000 g at 4°C for 30 min. The supernatant was incubated with anti-Flag agarose (Sigma) or Anti-GFP-mAb agarose (MBL) in IP buffer (20 mm Tris-HCl [pH 7.5], 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 mm NaF, 10 mm β-glycerophosphate, 0.1% [v/v] Triton X-100, protease inhibitor cocktail [Sigma]) at 4°C for 2 h with gentle shaking. The agarose was collected and washed three times with PBS, boiled in 2×SDS loading buffer for 5 min, and examined by immunoblot analysis with anti-GFP (CWBIO) or anti-Flag (Abmart) antibodies.

For Mass Spectrometry, pSuper1300:AtMOB1A-Flag vector was introduced into Agrobacterium. Arabidopsis plants were transformed by the floral dipping method. T4 generation transgenic plants were used for extracting total protein and IP using the method described above. The AtMOB1A interacting proteins were examined by Mass Spectrometry.

LCI Assay

The LCI assay was performed as previously described (Chen et al., 2008). A. tumefaciens bacteria strain GV3101 containing pCAMBIA1300-AtMOB1A-nLUC, pCAMBIA1300-cLUC-AtMOB1B, pCAMBIA1300-nLUC, and pCAMBIA1300-cLUC were injected into N. benthamiana leaves. The empty cLUC and nLUC vectors were used as negative controls. The plants were incubated in darkness overnight, and the leaves were harvested after 2 to 3 d. Leaves were incubated in d-Luciferin, Potassium Salt (Goldbio) solution in darkness for 3 min, and the luciferase signals were analyzed with a Tanon 5200 Chemiluminescent Imaging System (Tanon). The imaging exposure time was 3 min. Eight leaves were analyzed for each experiment, with three biological replicates in total.

RNA Extraction, RT-qPCR, and RNA-Seq

The samples were collected from 10-d-old whole seedlings of Col-0, mob1a-2, mob1b-1, and mob1a-2 b-1. Total RNA was extracted using a Biozol kit (Biomiga) according to the manufacturer’s instructions. The first-strand complementary DNA synthesis was performed using M-MLV Reverse Transcriptase (Promega). The qPCR analysis was performed using a light cycle 96 (Roche) and SYBR Green I (Takara). ACTIN2 (AT3G18780) was used as an internal control. All experiments were performed with three independent biological replicates. Statistical significance was evaluated by one-way ANOVA analysis (multiple comparison by Tukey post-test). The primers used for RT-qPCR analysis are listed in Supplemental Table S6.

For RNA-seq, mRNA enrichment, complementary DNA library construction, and single-end sequencing were performed by BGI (www.genomics.org.cn). Filtered Clean-reads were mapped to the reference genome (TAIR10) available at The Arabidopsis Information Resource (http://www.arabidopsis.org). The gene expression quantitation was calculated using the RSEM tool (Li and Dewey, 2011). The DEGs were selected according the threshold value fold change ≥2 (log2 ratio ≥1.0) and deviation probability ≥ 0.7 through comparing the gene expression quantitation of mutants and wild-type. GO enrichment analysis was carried out using AmiGO 2 program (http://amigo.geneontology.org/amigo).

Measurement of Endogenous JA by GC-MS

Fourteen-day-old whole plants of every genotype were collected in triplicate replicates. The samples were homogenized quickly in liquid N2; then powdered samples were weighed dry-frozen. Samples were extracted with 80% (v/v) methanol containing 0.2 ng [2H6]JA as an internal standard and incubated overnight at 4°C. After centrifugation at 5,976 g for 5 min, the supernatant layer was collected in new glass tubes and dried with nitrogen gas. The samples were dissolved in 2.5% (v/v) ethyl acetate, and the supernatant were dried with nitrogen gas. Following the addition of 23% (v/v) methanol and incubation for 2 h at −20°C, samples were centrifuged at 10,625 g for 7 min and the supernatant was dried with nitrogen gas. Samples were dissolved in 30 µL bis(trimethylsilyl)trifluoroacetamide with 3 µL pyridine and incubated for 30 min at 80°C, then analyzed using GC-MS (7890A-7000B, Agilent). The amount of JA present in plant samples was calculated based on the internal standards and weight of the tissues and retention time.

Measurement of Chlorophyll Content

Measurement of chlorophyll content was performed as previously described (Qi et al., 2015). The leaves were detached and weighted. For chlorophyll extraction, the leaves were incubated in 80% (v/v) acetone in the dark. Absorbances were measured at 645 and 663 nm using a spectrophotometer (Beckman Coulter DU-800). Chlorophyll contents were calculated and expressed as a ratio of the chlorophyll content of Col-0 treated with mock.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers JAZ1 (AT1G19180), JAZ2 (AT1G74950), JAZ3 (AT3G17860), JAZ4 (AT1G48500), JAZ5 (AT1G17380), JAZ6 (AT1G72450), JAZ7 (AT2G34600), JAZ8 (AT1G30135), JAZ9 (AT1G70700), JAZ10 (AT5G13220), JAZ11 (AT3G43440), and JAZ12 (AT5G20900).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Yunde Zhao for critical reading of the manuscript, Dr. Daoxin Xie and Dr. Susheng Song for providing the myc2 mutant seeds, Dr. Qingqiu Gong for sik1 mutant seeds, Dr. Yuxin Hu for the Chemiluminescent Imaging System, Dr. Jianmin Zhou for the LCI system vectors, and Dr. Li-jia Qu for insightful discussion. We thank Lu Wang and Jingquan Li for their excellent technical assistance, and the Plant Science Facility of the Institute of Botany, Chinese Academy of Sciences.

Footnotes

1

This work was supported by the projects from the National Natural Science Foundation of China (NSFC) (31970309; 31171389; 91217310; 91017008; 31270330), National Key Research and Development Program of China (2016YFD0100400), National Basic Research Program of China (2014CB943400), and One-hundred Talent Project of the Chinese Academy of Sciences (STS Program KFJ-STS-ZDTP-076 to Y.C.).

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References

  1. Aida M, Beis D, Heidstra R, Willemsen V, Blilou I, Galinha C, Nussaume L, Noh YS, Amasino R, Scheres B (2004) The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche. Cell 119: 109–120 [DOI] [PubMed] [Google Scholar]
  2. Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, Tang X, Zhou JM (2008) Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol 146: 368–376 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen Q, Sun J, Zhai Q, Zhou W, Qi L, Xu L, Wang B, Chen R, Jiang H, Qi J, et al. (2011) The basic helix-loop-helix transcription factor MYC2 directly represses PLETHORA expression during jasmonate-mediated modulation of the root stem cell niche in Arabidopsis. Plant Cell 23: 3335–3352 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chow A, Hao Y, Yang X (2010) Molecular characterization of human homologs of yeast MOB1. Int J Cancer 126: 2079–2089 [DOI] [PubMed] [Google Scholar]
  5. Citterio S, Piatti S, Albertini E, Aina R, Varotto S, Barcaccia G (2006) Alfalfa Mob1-like proteins are involved in cell proliferation and are localized in the cell division plane during cytokinesis. Exp Cell Res 312: 1050–1064 [DOI] [PubMed] [Google Scholar]
  6. Colón-Carmona A, You R, Haimovitch-Gal T, Doerner P (1999) Technical advance: Spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J 20: 503–508 [DOI] [PubMed] [Google Scholar]
  7. Cui X, Guo Z, Song L, Wang Y, Cheng Y (2016) NCP1/AtMOB1A plays key roles in auxin-mediated Arabidopsis development. PLoS Genet 12: e1005923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galinha C, Hofhuis H, Luijten M, Willemsen V, Blilou I, Heidstra R, Scheres B (2007) PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature 449: 1053–1057 [DOI] [PubMed] [Google Scholar]
  9. Galla G, Zenoni S, Marconi G, Marino G, Botton A, Pinosa F, Citterio S, Ruperti B, Palme K, Albertini E, et al. (2011) Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L. Gene 484: 1–12 [DOI] [PubMed] [Google Scholar]
  10. Gao X, Chen J, Dai X, Zhang D, Zhao Y (2016) An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol 171: 1794–1800 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goto H, Nishio M, To Y, Oishi T, Miyachi Y, Maehama T, Nishina H, Akiyama H, Mak TW, Makii Y, et al. (2018) Loss of Mob1a/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD-dependent repression of SOX9. Development 145: dev159244. [DOI] [PubMed] [Google Scholar]
  12. Hansen CG, Moroishi T, Guan KL (2015) YAP and TAZ: A nexus for Hippo signaling and beyond. Trends Cell Biol 25: 499–513 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. Nat Rev Cancer 13: 246–257 [DOI] [PubMed] [Google Scholar]
  14. He Y, Fukushige H, Hildebrand DF, Gan S (2002) Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence. Plant Physiol 128: 876–884 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang H, Liu B, Liu L, Song S (2017) Jasmonate action in plant growth and development. J Exp Bot 68: 1349–1359 [DOI] [PubMed] [Google Scholar]
  16. Lai ZC, Wei X, Shimizu T, Ramos E, Rohrbaugh M, Nikolaidis N, Ho LL, Li Y (2005) Control of cell proliferation and apoptosis by mob as tumor suppressor, mats. Cell 120: 675–685 [DOI] [PubMed] [Google Scholar]
  17. Li B, Dewey CN (2011) RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12: 323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lorenzo O, Chico JM, Sánchez-Serrano JJ, Solano R (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16: 1938–1950 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luca FC, Winey M (1998) MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy. Mol Biol Cell 9: 29–46 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nishio M, Hamada K, Kawahara K, Sasaki M, Noguchi F, Chiba S, Mizuno K, Suzuki SO, Dong Y, Tokuda M, et al. (2012) Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice. J Clin Invest 122: 4505–4518 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nishio M, Sugimachi K, Goto H, Wang J, Morikawa T, Miyachi Y, Takano Y, Hikasa H, Itoh T, Suzuki SO, et al. (2016) Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice. Proc Natl Acad Sci USA 113: E71–E80 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pan D. (2010) The hippo signaling pathway in development and cancer. Dev Cell 19: 491–505 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pinosa F, Begheldo M, Pasternak T, Zermiani M, Paponov IA, Dovzhenko A, Barcaccia G, Ruperti B, Palme K (2013) The Arabidopsis thaliana Mob1A gene is required for organ growth and correct tissue patterning of the root tip. Ann Bot 112: 1803–1814 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Qi T, Wang J, Huang H, Liu B, Gao H, Liu Y, Song S, Xie D (2015) Regulation of jasmonate-induced leaf senescence by antagonism between bHLH subgroup IIIe and IIId factors in Arabidopsis. Plant Cell 27: 1634–1649 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shan X, Wang J, Chua L, Jiang D, Peng W, Xie D (2011) The role of Arabidopsis Rubisco activase in jasmonate-induced leaf senescence. Plant Physiol 155: 751–764 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Titarenko E, Rojo E, León J, Sánchez-Serrano JJ (1997) Jasmonic acid-dependent and -independent signaling pathways control wound-induced gene activation in Arabidopsis thaliana. Plant Physiol 115: 817–826 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vitulo N, Vezzi A, Galla G, Citterio S, Marino G, Ruperti B, Zermiani M, Albertini E, Valle G, Barcaccia G (2007) Characterization and evolution of the cell cycle-associated mob domain-containing proteins in eukaryotes. Evol Bioinform Online 3: 121–158 [PMC free article] [PubMed] [Google Scholar]
  28. Wang W, Li X, Huang J, Feng L, Dolinta KG, Chen J (2014) Defining the protein-protein interaction network of the human hippo pathway. Mol Cell Proteomics 13: 119–131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wasternack C, Song S (2017) Jasmonates: Biosynthesis, metabolism, and signaling by proteins activating and repressing transcription. J Exp Bot 68: 1303–1321 [DOI] [PubMed] [Google Scholar]
  30. Xiao S, Dai L, Liu F, Wang Z, Peng W, Xie D (2004) COS1: An Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence. Plant Cell 16: 1132–1142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xiong J, Cui X, Yuan X, Yu X, Sun J, Gong Q (2016) The Hippo/STE20 homolog SIK1 interacts with MOB1 to regulate cell proliferation and cell expansion in Arabidopsis. J Exp Bot 67: 1461–1475 [DOI] [PubMed] [Google Scholar]
  32. Zeng W, Dai X, Sun J, Hou Y, Ma X, Cao X, Zhao Y, Cheng Y (2018) Modulation of auxin signaling and development by polyadenylation machinery. Plant Physiol 179: 686–699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhang M, Chiang YH, Toruno TY, Lee D, Ma M, Liang X, Lal NK, Lemos M, Lu YJ, Ma S, et al. (2018) The MAP4 kinase SIK1 ensures robust extracellular ROS burst and antibacterial immunity in plants. Cell Host Microbe 24: 379–391 [DOI] [PMC free article] [PubMed] [Google Scholar]

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