Arabidopsis MORE AXILLARY GROWTH2 (MAX2) is a key component in the strigolactone (SL) and karrikin (KAR) signaling pathways and regulates the degradation of SUPPRESSOR OF MAX2 1/ SMAX1-like (SMAX1/SMXL) proteins, which are transcriptional co-repressors that regulate plant architecture, as well as abiotic and biotic stress responses. The max2 mutation reduces resistance against Pseudomonas syringae pv. tomato (Pst). To uncover the mechanism of MAX2-mediated resistance, we evaluated the resistance of various SL and KAR signaling pathway mutants. The resistance of SL-deficient mutants and of dwarf 14 (d14) was similar to that of the wild-type, whereas the resistance of the karrikin insensitive 2 (kai2) mutant was compromised, demonstrating that the KAR signaling pathway, not the SL signaling pathway, positively regulates the immune response. We measured the resistance of smax1 and smxl mutants, as well as the double, triple, and quadruple mutants with max2, which revealed that both the smax1 mutant and smxl6/7/8 triple mutant rescue the low resistance phenotype of max2 and that SMAX1 accumulation diminishes resistance. The susceptibility of smax1D, containing a degradation-insensitive form of SMAX1, further confirmed the SMAX1 function in the resistance. The relationship between the accumulation of SMAX1/SMXLs and disease resistance suggested that the inhibitory activity of SMAX1 to resistance requires SMXL6/7/8. Moreover, the exogenous application of KAR2 enhanced resistance against Pst, but KAR-induced resistance depended on salicylic acid (SA) signaling. Inhibition of karrikin signaling delayed SA-mediated defense responses and inhibited pathogen-induced protein biosynthesis. Together, we propose that the MAX2-KAI2-SMAX1 complex regulates resistance with the assistance of SMXL6/7/8 and SA signaling and that SMAX1/SMXLs possibly form a multimeric complex with their target transcription factors to fine tune immune responses.

Both herbivory and jasmonic acid (JA) activate the biosynthesis of defensive metabolites in maize, but the mechanism underlying this remains unclear. We generated maize mutants in which ZmMYC2a and ZmMYC2b, two transcription factor genes important in JA signaling, were individually or both knocked out. Genetic and biochemical analyses were used to elucidate the functions of ZmMYC2 proteins in the maize response to simulated herbivory and JA. Compared with the wild‐type (WT) maize, the double mutant myc2ab was highly susceptible to insects, and the levels of benzoxazinoids and volatile terpenes, and the levels of their biosynthesis gene transcripts, were much lower in the mutants than in the WT maize after simulated insect feeding or JA treatment. Moreover, ZmMYC2a and ZmMYC2b played a redundant role in maize resistance to insects and JA signaling. Transcriptome and Cleavage Under Targets and Tagmentation‐ Sequencing (CUT&Tag‐Seq) analysis indicated that ZmMYC2s physically targeted 60% of the JA‐ responsive genes, even though only 33% of these genes were transcriptionally ZmMYC2‐dependent. Importantly, CUT&Tag‐Seq and dual luciferase assays revealed that ZmMYC2s transactivate the benzoxazinoid and volatile terpene biosynthesis genes IGPS1/3, BX10/11/12/14, and TPS10/2/3/4/ 5/8 by directly binding to their promoters. Furthermore, several transcription factors physically targeted by ZmMYC2s were identified, and these are likely to function in the regulation of benzoxazinoid biosynthesis. This work reveals the transcriptional regulatory landscapes of both JA signaling and ZmMYC2s in maize and provides comprehensive mechanistic insight into how JA signaling modulates defenses in maize responses to herbivory through ZmMYC2s.

Inorganic phosphate (Pi) is often limited in soils due to precipitation with iron (Fe) and aluminum (Al). To scavenge heterogeneously distributed phosphorus (P) resources, plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe‐P or Al‐P oxides. In this study, we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE‐RESISTANT 1 (BZR1) expression in Arabidopsis thaliana. Exogenous 2,4‐epibrassinolide treatment or constitutive activation of BZR1 (in the bzr1‐D mutant) significantly reduced primary root growth inhibition under Pi‐starvation conditions by downregulating ALUMINUM‐ACTIVATED MALATE TRANSPORTER 1 (ALMT1) expression and malate secretion. Furthermore, AtBZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1 (STOP1) on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis. The ratio of nuclear‐localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis. In addition, BZR1‐inhibited malate secretion is conserved in rice (Oryza sativa). Our findings provide insight into plant mechanisms for optimizing the secretion of malate, an important carbon resource, to adapt to Pi‐deficiency stress.

Abscisic acid (ABA) is a key regulator of plant responses to abiotic stresses, such as drought. Abscisic acid receptors and coreceptors perceive ABA to activate Snf1-related protein kinase2s (SnRK2s) that phosphorylate downstream effectors, thereby activating ABA signaling and the stress response. As stress responses come with fitness penalties for plants, it is crucial to tightly control SnRK2 kinase activity to restrict ABA signaling. However, how SnRK2 kinases are inactivated remains elusive. Here, we show that NUCLEAR PORE ANCHOR (NUA), a nuclear pore complex (NPC) component, negatively regulates ABA-mediated inhibition of seed germination and post-germination growth, and drought tolerance in Arabidopsis thaliana. The role of NUA in response to ABA depends on SnRK2.2 and SnRK2.3 for seed germination and on SnRK2.6 for drought. NUA does not directly inhibit the phosphorylation of these SnRK2s or affects their abundance. However, the NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, negatively regulates ABA signaling by directly interacting with and inhibiting SnRK2 phosphorylation and protein levels. More importantly, we demonstrated that SnRK2.6 can be SUMOylated in vitro, and ESD4 inhibits its SUMOylation. Taken together, we identified NUA and ESD4 as SnRK2 kinase inhibitors that block SnRK2 activity, and reveal a mechanism whereby NUA and ESD4 negatively regulate plant responses to ABA and drought stress possibly through SUMOylation-dependent regulation of SnRK2s.

The oomycete pathogen Phytophthora sojae is a causal agent of soybean root rot. Upon colonization of soybeans, P. sojae secretes various RXLR effectors to suppress host immune responses, supporting successful infection. Previous research has demonstrated that the RXLR effector Avh94 functions as a virulence effector, but the molecular mechanism underlying its role in virulence remains unknown. Here, we demonstrate that Avh94 overexpression in plants and pathogens promotes Phytophthora infection. Avh94 interacts with soybean JAZ1/2, which is a repressor of jasmonic acid (JA) signaling. Avh94 stabilizes JAZ1/2 to inhibit JA signaling and silencing of JAZ1/2 enhances soybean resistance against P. sojae. Moreover, P. sojae lines overexpressing Avh94 inhibit JA signaling. Furthermore, exogenous application of methyl jasmonate improves plant resistance to Phytophthora. Taken together, these findings suggest that P. sojae employs an RXLR effector to hijack JA signaling and thereby promote infection.

The phytohormones ethylene (ET) and jasmonate (JA) regulate plant development, growth, and defense responses; however, the molecular basis for their signaling crosstalk is unclear. Here, we show that JA-ZIM-domain (JAZ) proteins, which repress JA signaling, repress trichome initiation/branching and anthocyanin accumulation, and inhibit the transcriptional activity of the basic helix-loop-helix (bHLH)-MYB members (GLABRA3 (GL3)-GL1 and TRANSPARENT TESTA 8 (TT8)-MYB75) of WD-repeat/bHLH/MYB (WBM) complexes. The ET-stabilized transcription factors ETHYLENE-INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1) were found to bind to several members of WBM complexes, including GL3, ENHANCER OF GLABRA3 (EGL3), TT8, GL1, MYB75, and TRANSPARENT TESTA GLABRA1 (TTG1). This binding repressed the transcriptional activity of the bHLH-MYB proteins and inhibited anthocyanin accumulation, trichome formation, and defenses against insect herbivores while promoting root hair formation. Conversely, the JA-activated bHLH members GL3, EGL3, and TT8 of WBM complexes were able to interact with and attenuate the transcriptional activity of EIN3/EIL1 at the HOOKLESS1 promoter, and their overexpression inhibited apical hook formation. Thus, this study demonstrates a molecular framework for signaling crosstalk between JA and ET in plant development, secondary metabolism, and defense responses.

Glycogen synthase kinase 3 (GSK3) proteins play key roles in brassinosteroid (BR) signaling during plant growth and development by phosphorylating various substrates. However, how GSK3 protein stability and activity are themselves modulated is not well understood. Here, we demonstrate in vitro and in vivo that C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (OsCPL3), a member of the RNA Pol II CTD phosphatase-like family, physically interacts with OsGSK2 in rice (Oryza sativa). OsCPL3 expression was widely detected in various tissues and organs including roots, leaves and lamina joints, and was induced by exogenous BR treatment. OsCPL3 localized to the nucleus, where it dephosphorylated OsGSK2 at the Ser-222 and Thr-284 residues to modulate its protein turnover and kinase activity, in turn affecting the degradation of BRASSINAZOLE-RESISTANT 1 (BZR1) and BR signaling. Loss of OsCPL3 function resulted in higher OsGSK2 abundance and lower OsBZR1 levels, leading to decreased BR responsiveness and alterations in plant morphology including semi-dwarfism, leaf erectness and grain size, which are of fundamental importance to crop productivity. These results reveal a previously unrecognized role for OsCPL3 and add another layer of complexity to the tightly controlled BR signaling pathway in plants.

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.

Jasmonic acid (JA) is a key regulator of plant defense responses. Although the transcription factor MYC2, the master regulator of the JA signaling pathway, orchestrates a hierarchical transcriptional cascade that regulates the JA responses, only a few transcriptional regulators involved in this cascade have been described. Here, we identified the basic helix-loop-helix (bHLH) transcription factor gene in tomato (Solanum lycopersicum), METHYL JASMONATE (MeJA)-INDUCED GENE (SlJIG), the expression of which was strongly induced by MeJA treatment. Genetic and molecular biology experiments revealed that SlJIG is a direct target of MYC2. SlJIG knockout plants generated by gene editing had lower terpene contents than the wild type from the lower expression of TERPENE SYNTHASE (TPS) genes, rendering them more appealing to cotton bollworm (Helicoverpa armigera). Moreover, SlJIG knockouts exhibited weaker JA-mediated induction of TPSs, suggesting that SlJIG may participate in JA-induced terpene biosynthesis. Knocking out SlJIG also resulted in attenuated expression of JA-responsive defense genes, which may contribute to the observed lower resistance to cotton bollworm and to the fungus Botrytis cinerea. We conclude that SlJIG is a direct target of MYC2, forms a MYC2-SlJIG module, and functions in terpene biosynthesis and resistance against cotton bollworm and B. cinerea.

Drought is a critical environmental factor which constrains plant survival and growth. Genetic engineering provides a credible strategy to improve drought tolerance of plants. Here, we generated transgenic poplar lines expressing the isopentenyl transferase gene (IPT) under the driver of PtRD26 promoter (PtRD26_pro-IPT). PtRD26 is a senescence and drought-inducible NAC transcription factor. PtRD26_pro-IPT plants displayed multiple phenotypes, including improved growth and drought tolerance. Transcriptome analysis revealed that auxin biosynthesis pathway was activated in the PtRD26_pro-IPT plants, leading to an increase in auxin contents. Biochemical analysis revealed that ARABIDOPSIS RESPONSE REGULATOR10 (PtARR10), one of the type-B ARR transcription factors in the cytokinin pathway, was induced in PtRD26_pro-IPT plants and directly regulated the transcripts of YUCCA4 (PtYUC4) and YUCCA5 (PtYUC5), two enzymes in the auxin biosynthesis pathway. Overexpression of PtYUC4 enhanced drought tolerance, while simultaneous silencing of PtYUC4/5 evidently attenuated the drought tolerance of PtRD26_pro-IPT plants. Intriguingly, PtYUC4/5 displayed a conserved thioredoxin reductase activity that is required for drought tolerance by deterring reactive oxygen species accumulation. Our work reveals the molecular basis of cytokinin and auxin interactions in response to environmental stresses, and shed light on the improvement of drought tolerance without a growth penalty in trees by molecular breeding.

ETHYLENE RESPONSE FACTOR1 (ERF1) is a key component in ethylene signaling, playing crucial roles in both biotic and abiotic stress responses. Here, we demonstrate that ERF1 also has an important role during floral initiation in Arabidopsis thaliana. Knockdown or knockout of ERF1 accelerated floral initiation, whereas overexpression of ERF1 dramatically delayed floral transition. These contrasting phenotypes were correlated with opposite transcript levels of FLOWERING LOCUS T (FT). Chromatin immunoprecipitation (ChIP) assays revealed that ERF1 associates with genomic regions of the FT gene to repress its transcription. ft-10/ERF1RNAi plants showed a similar flowering phenotype to the ft-10 mutant, whereas the flowering of FTox/ERF1ox mimicked that of FTox plants, suggesting that ERF1 acts upstream of FT during floral initiation. Similarly, altered floral transition in ethylene-related mutants was also correlated with FT expression. Further analysis suggested that ERF1 also participates in delay in flowering-time control mediated by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Thus, ERF1 may act as a negative modulator of flowering-time control by repressing FT transcription in Arabidopsis.

Trichomes function in plant defenses against biotic and abiotic stresses; examination of glabrous lines, which lack trichomes, has revealed key aspects of trichome development and function. Tests of allelism in 51 glabrous rice (Oryza sativa) accessions collected worldwide identified OsSPL10 and OsWOX3B as regulators of trichome development in rice. Here, we report that OsSPL10 acts as a transcriptional regulator controlling trichome development. Haplotype and transient expression analyses revealed that variation in the approximately 700-bp OsSPL10 promoter region is the primary cause of the glabrous phenotype in the indica cultivar WD-17993. Disruption of OsSPL10 by genome editing decreased leaf trichome density and length in the NIL-HL6 background. Plants with genotype OsSPL10^WD-17993/HL6 generated by crossing WD-17993 with NIL-HL6 also had fewer trichomes in the glumes. HAIRY LEAF6 (HL6) encodes another transcription factor that regulates trichome initiation and elongation, and OsSPL10 directly binds to the HL6 promoter to regulate its expression. Moreover, the transcript levels of auxin-related genes, such as OsYUCCA5 and OsPIN-FORMED1b, were altered in OsSPL10 overexpression and RNAi transgenic lines. Feeding tests using locusts (Locusta migratoria) demonstrated that non-glandular trichomes affect feeding by this herbivore. Our findings provide a molecular framework for trichome development and an ecological perspective on trichome functions.

Among the five members of AUX1/LAX genes coding for auxin carriers in rice, only OsAUX1 and OsAUX3 have been reported. To understand the function of the other AUX1/LAX genes, two independent alleles of osaux4 mutants, osaux4-1 and osaux4-2, were constructed using the CRISPR/Cas9 editing system. Homozygous osaux4-1 or osaux4-2 exhibited shorter primary root (PR) and longer root hair (RH) compared to the wild-type Dongjin (WT/DJ), and lost response to indoleacetic acid (IAA) treatment. OsAUX4 is intensively expressed in roots and localized on the plasma membrane, suggesting that OsAUX4 might function in the regulation of root development. The decreased meristem cell division activity and the downregulated expression of cell cycle genes in root apices of osaux4 mutants supported the hypothesis that OsAUX4 positively regulates PR elongation. OsAUX4 is expressed in RH, and osaux4 mutants showing longer RH compared to WT/DJ implies that OsAUX4 negatively regulates RH development. Furthermore, osaux4 mutants are insensitive to Pi starvation (-Pi) and OsAUX4 effects on the -Pi response is associated with altered expression levels of Pi starvation-regulated genes, and auxin distribution/contents. This study revealed that OsAUX4 not only regulates PR and RH development but also plays a regulatory role in crosstalk between auxin and -Pi signaling.

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H₂O₂), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H₂O₂ generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3–MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H₂O₂ to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.

In plants, recognition of small secreted peptides, such as damage/danger‐associated molecular patterns (DAMPs), regulates diverse processes, including stress and immune responses. Here, we identified an SGPS (Ser‐Gly‐Pro‐Ser) motif‐containing peptide, Nicotiana tabacum NtPROPPI, and its two homologs in Nicotiana benthamiana, NbPROPPI1 and NbPROPPI2. Phytophthora parasitica infection and salicylic acid (SA) treatment induced NbPROPPI1/2 expression. Moreover, SignalP predicted that the 89‐amino acid NtPROPPI includes a 24‐amino acid N‐terminal signal peptide and NbPROPPI1/2‐GFP fusion proteins were mainly localized to the periplasm. Transient expression of NbPROPPI1/2 inhibited P. parasitica colonization, and NbPROPPI1/2 knockdown rendered plants more susceptible to P. parasitica. An eight‐amino‐acid segment in the NbPROPPI1 C‐terminus was essential for its immune function and a synthetic 20‐residue peptide, NbPPI1, derived from the C‐terminus of NbPROPPI1 provoked significant immune responses in N. benthamiana. These responses led to enhanced accumulation of reactive oxygen species, activation of mitogen‐activated protein kinases, and up‐regulation of the defense genes Flg22‐induced receptor‐like kinase (FRK) and WRKY DNA‐binding protein 33 (WRKY33). The NbPPI1‐induced defense responses require Brassinosteroid insensitive 1‐associated receptor kinase 1 (BAK1). These results suggest that NbPPI1 functions as a DAMP in N. benthamiana; this novel DAMP provides a potentially useful target for improving plant resistance to Pytophthora pathogens.

Protein–protein interaction (PPI) network analysis is an effective method to identify key proteins during plant development, especially in species for which basic molecular research is lacking, such as apple (Malus domestica). Here, an MdPPI network containing 30 806 PPIs was inferred in apple and its quality and reliability were rigorously verified. Subsequently, a root‐growth subnetwork was extracted to screen for critical proteins in root growth. Because hormone‐related proteins occupied the largest proportion of critical proteins, a hormone‐related sub‐subnetwork was further extracted from the root‐growth subnetwork. Among these proteins, auxin‐related M. domestica TRANSPORT INHIBITOR RESISTANT 1 (MdTIR1) served as the central, high‐degree node, implying that this protein exerts essential roles in root growth. Furthermore, transgenic apple roots overexpressing an MdTIR1 transgene displayed increased primary root elongation. Expression analysis showed that MdTIR1 significantly upregulated auxin‐responsive genes in apple roots, indicating that it mediates root growth in an auxin‐dependent manner. Further experimental validation revealed that MdTIR1 interacted with and accelerated the degradation of MdIAA28, MdIAA43, and MdIAA46. Thus, MdTIR1‐mediated degradation of MdIAAs is critical in auxin signal transduction and root growth regulation in apple. Moreover, our network analysis and high‐degree node screening provide a novel research technique for more generally characterizing molecular mechanisms.

Plant interphase cortical microtubules (cMTs) mediate anisotropic cell expansion in response to environmental and developmental cues. In Arabidopsis thaliana, KATANIN 1 (KTN1), the p60 catalytic subunit of the conserved MT‐severing enzyme katanin, is essential for cMT ordering and anisotropic cell expansion. However, the regulation of KTN1‐mediated cMT severing and ordering remains unclear. In this work, we report that the Arabidopsis IQ67 DOMAIN (IQD) family gene ABNORMAL SHOOT 6 (ABS6) encodes a MT‐associated protein. Overexpression of ABS6 leads to elongated cotyledons, directional pavement cell expansion, and highly ordered transverse cMT arrays. Genetic suppressor analysis revealed that ABS6‐mediated cMT ordering is dependent on KTN1 and SHADE AVOIDANCE 4 (SAV4). Live imaging of cMT dynamics showed that both ABS6 and SAV4 function as positive regulators of cMT severing. Furthermore, ABS6 directly interacts with KTN1 and SAV4 and promotes their recruitment to the cMTs. Finally, analysis of loss‐of‐function mutant combinations showed that ABS6, SAV4, and KTN1 work together to ensure the robust ethylene response in the apical hook of dark‐grown seedlings. Together, our findings establish ABS6 and SAV4 as positive regulators of cMT severing and ordering, and highlight the role of cMT dynamics in fine‐tuning differential growth in plants.

Pre‐mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre‐mRNA splicing (rdm16‐4). The rdm16‐4 mutant displays a short‐root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16‐4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16‐4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full‐length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short‐root phenotype of rdm16‐4. This reveals that the RDM16‐mediated alternative splicing of cytokinin signaling components contributes to root growth.

In eukaryotes, N‐ethylmaleimide‐sensitive factor (NSF) is a conserved AAA+ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes. Here, we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF, AtNSF, which plays an essential role in the regulation of leaf serration. The AtNSF knock‐down mutant, atnsf‐1, exhibited more serrations in the leaf margin. Moreover, polar localization of the PIN‐FORMED1 (PIN1) auxin efflux transporter was diffuse around the margins of atnsf‐1 leaves and root growth was inhibited in the atnsf‐1 mutant. More PIN1‐GFP accumulated in the intracellular compartments of atnsf‐1 plants, suggesting that AtNSF is required for intracellular trafficking of PIN between the endosome and plasma membrane. Furthermore, the serration phenotype was suppressed in the atnsf‐1 pin1‐8 double mutant, suggesting that AtNSF is required for PIN1‐mediated polar auxin transport to regulate leaf serration. The CUP‐SHAPED COTYLEDON2 (CUC2) transcription factor gene is up‐regulated in atnsf‐1 plants and the cuc2‐3 single mutant exhibits smooth leaf margins, demonstrating that AtNSF also functions in the CUC2 pathway. Our results reveal that AtNSF regulates the PIN1‐generated auxin maxima with a CUC2‐mediated feedback loop to control leaf serration.

MED25 has been implicated as a negative regulator of the abscisic acid (ABA) signaling pathway. However, it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response. Here, we used affinity purification followed by mass spectrometry to uncover Mediator subunits that associate with MED25 in transgenic plants. We found that at least 26 Mediator subunits, belonging to the head, middle, tail, and CDK8 kinase modules, were co‐purified with MED25 in vivo. Interestingly, the tail module subunit MED16 was identified to associate with MED25 under both mock and ABA treatments. We further showed that the disruption of MED16 led to reduced ABA sensitivity compared to the wild type. Transcriptomic analysis revealed that the expression of several ABA‐responsive genes was significantly lower in med16 than those in wild type. Furthermore, we discovered that MED16 may possibly compete with MED25 to interact with the key transcription factor ABA INSENSITIVE 5 (ABI5) to positively regulate ABA signaling. Consistently, med16 and med25 mutants displayed opposite phenotypes in ABA response, cuticle permeability, and differential ABI5‐mediated EM1 and EM6 expression. Together, our data indicate that MED16 and MED25 differentially regulate ABA signaling by antagonistically affecting ABI5‐mediated transcription in Arabidopsis.