Dual regulations of cell cycle regulator DPa by auxin in Arabidopsis root distal stem cell maintenance.

The plant hormone auxin plays a key role to maintain root stem cell identity which is essential for root development. However, the molecular mechanism by which auxin regulates root distal stem cell (DSC) identity is not well understood. In this study, we revealed that the cell cycle factor DPa is a vital regulator in the maintenance of root DSC identity through multiple auxin signaling cascades. On the one hand, auxin positively regulates the transcription of DPa via AUXIN RESPONSE FACTOR 7 and ARF19. On the other hand, auxin enhances the protein stability of DPa through MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3)/MPK6-mediated phosphorylation. Consistently, mutation of the identified three threonine residues (Thr10, Thr25, and Thr227) of DPa to nonphosphorylated form alanine (DPa3A) highly decreased the phosphorylation level of DPa, which decreased its protein stability and affected the maintenance of root DSC identity. Taken together, this study provides insight into the molecular mechanism of how auxin regulates root distal stem cell identity through the dual regulations of DPa at both transcriptional and posttranslational levels.

PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity.

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development.

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4 (MKK4) and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA (YDA), also known as MAPK KINASE KINASE4 (MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade (YDA-MKK4/MKK5-MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5-MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern (PAMP) treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9) approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5-MPK3/MPK6 module in both plant immunity and growth/development.

MPK3/MPK6-mediated phosphorylation of ERF72 positively regulates resistance to Botrytis cinerea through directly and indirectly activating the transcription of camalexin biosynthesis enzymes.

Ethylene response factor (ERF) Group VII members generally function in regulating plant growth and development, abiotic stress responses, and plant immunity in Arabidopsis; however, the details of the regulatory mechanism by which Group VII ERFs mediate plant immune responses remain elusive. Here, we characterized one such member, ERF72, as a positive regulator that mediates resistance to the necrotrophic pathogen Botrytis cinerea. Compared with the wild-type (WT), the erf72 mutant showed lower camalexin concentration and was more susceptible to B. cinerea, while complementation of ERF72 in erf72 rescued the susceptibility phenotype. Moreover, overexpression of ERF72 in the WT promoted camalexin biosynthesis and increased resistance to B. cinerea. We identified the camalexin-biosynthesis genes PAD3 and CYP71A13 and the transcription factor WRKY33 as target genes of ERF72. We also determined that MPK3 and MPK6 phosphorylated ERF72 at Ser151 and improved its transactivation activity, resulting in increased camalexin concentration and increased resistance to B. cinerea. Thus, ERF72 acts in plant immunity to coordinate camalexin biosynthesis both directly by regulating the expression of biosynthetic genes and indirectly by targeting WRKK33.

MKK4/5-MPK3/6 Cascade Regulates Agrobacterium-Mediated Transformation by Modulating Plant Immunity in Arabidopsis.

Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity.

The YDA-MKK4/MKK5-MPK3/MPK6 Cascade Functions Downstream of the RGF1-RGI Ligand-Receptor Pair in Regulating Mitotic Activity in Root Apical Meristem.

The mitotic activity of root apical meristem (RAM) is critical to primary root growth and development. Previous studies have identified the roles of ROOT GROWTH FACTOR 1 (RGF1), a peptide ligand, and its receptors, RGF1 INSENSITIVEs (RGIs), a clade of five leucine-rich-repeat receptor-like kinases, in promoting cell division in the RAM, which determines the primary root length. However, the downstream signaling components remain elusive. In this study, we identify a complete mitogen-activated protein kinase (MAPK or MPK) cascade, composed of YDA, MKK4/MKK5, and MPK3/MPK6, that functions downstream of the RGF1-RGI ligand-receptor pair. Similar to the rgi1/2/3/4/5 quintuple mutant, loss-of-function mutants of MPK3 and MPK6, MKK4 and MKK5, or YDA show a short-root phenotype, which is associated with reduced mitotic activity and lower expression of PLETHORA 1 (PLT1)/PLT2 in the RAM. Furthermore, MPK3/MPK6 activation in response to exogenous RGF1 treatment is impaired in the rgi1/2/3/4/5 quintuple, yda single, and mkk4 mkk5 double mutants. Epistatic analyses demonstrated that the expression of constitutively active MKK4, MKK5, or YDA driven by the RGI2 promoter can rescue the short-root phenotype of the rgi1/2/3/4/5 mutant. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the RGF1-RGI ligand-receptor pair and upstream of PLT1/PLT2 to modulate the stem cell population and primary root growth in Arabidopsis.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions.

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding. The root primordia form in the source tissue, and during emergence the adventitious roots penetrate the inner cell layers and the epidermis; however, the mechanisms underlying this emergence remain largely unexplored. Here, we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), the MAP kinases MPK3 and MPK6, and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots (J-ARs) from the root-hypocotyl junctions in Arabidopsis thaliana. ABI4 negatively regulates J-AR emergence, preventing the accumulation of reactive oxygen species and death of epidermal cells, which would otherwise facilitate J-AR emergence. Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4. MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence. We propose that this "double-check" mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.

Co-regulation of indole glucosinolates and camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6 signaling pathways.

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)-induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium-dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss-of-function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5-VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid-inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5-VK, leading to high-level accumulation of camalexin and 4-methoxyindole-3-yl-methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.

MPK3- and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis.

Low temperatures affect plant growth, development, productivity, and ecological distribution. Expression of the C-repeat-binding factor (CBF) transcription factors is induced by cold stress, which in turn activates downstream cold-responsive (COR) genes that are required for the acquisition of freezing tolerance. Inducer of CBF expression 1 (ICE1) is a master regulator of CBFs, and ICE1 stability is crucial for its function. However, the regulation of ICE1 is not well understood. Here, we report that mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ICE1, which reduces its stability and transcriptional activity. Consistently, the mpk3 and mpk6 single mutants and the mpk3 mpk6 double mutants show enhanced freezing tolerance, whereas MPK3/MPK6 activation attenuates freezing tolerance. Phosphor-inactive mutations of ICE1 complement freezing sensitivity in the ice1-2 mutant. These combined results indicate that MPK3/MPK6 phosphorylate and destabilize ICE1, which negatively regulates CBF expression and freezing tolerance in plants.

Mild osmotic stress promotes 4-methoxy indolyl-3-methyl glucosinolate biosynthesis mediated by the MKK9-MPK3/MPK6 cascade in Arabidopsis.

KEY MESSAGE: MKK9-MPK3/MPK6 cascade positively regulates IGSs' biosynthetic genes. Glucosinolates (GSs), secondary metabolites well known for their roles in plant defense, have been implicated to play an important role in plant abiotic stress response; however, the exact role in these processes and the underlying regulatory mechanisms remain elusive. Mitogen-activated protein kinase (MAPK) cascades are extensively involved in plant abiotic stress response. In this study, we examined the levels of four indolic glucosinolates (IGSs) in the shoots of Arabidopsis seedlings under mild osmotic stress conditions and found that 4-methoxy indolyl-3-methyl glucosinolate (4MI3G) accumulated and that MPK3 and MPK6 were activated. Loss of MPK3 or MPK6 function led to a reduction in mild osmotic stress-induced 4MI3G. Further analyses revealed that MKK9 acts upstream of MPK3 and MPK6 to promote 4MI3G accumulation. The level of 4MI3G induced by mild osmotic stress was reduced in the mkk9 mutant. Conversely, 4MI3G increased in MKK9 DD , a constitutively activate mutant of MKK9. Gene expression analyses indicated that the activated MKK9-MPK3/MPK6 cascade upregulates the IGS biosynthetic genes. Moreover, the lack of MYB51, the transcription factor controlling biosynthetic genes responsible for synthesizing the IGS core structure, or CYP81F2, the enzyme catalyzing core structure modification to 4MI3G, significantly reduced mild osmotic stress- and MKK9 DD -induced 4MI3G. Thus, our study demonstrates that mild osmotic stress promotes 4MI3G biosynthesis and the accumulation in Arabidopsis through activation of the MKK9-MPK3/MPK6 cascade and provides an MAPK-mediated signaling pathway for the IGS response to abiotic stress in plants.

Putrescine regulating by stress-responsive MAPK cascade contributes to bacterial pathogen defense in Arabidopsis.

Polyamines in plants are involved in various physiological and developmental processes including abiotic and biotic stress responses. We investigated the expression of ADCs, which are key enzymes in putrescine (Put) biosynthesis, and roles of Put involving defense response in Arabidopsis. The increased expression of ADC1 and ADC2, and the induction of Put were detected in GVG-NtMEK2(DD) transgenic Arabidopsis, whereas, their performance was partially compromised in GVG-NtMEK2(DD)/mpk3 and GVG-NtMEK2(DD)/mpk6 mutant following DEX treatment. The expression of ADC2 was highly induced by Pst DC3000 inoculation, while the transcript levels of ADC1 were slightly up-regulated. Compared to the WT plant, Put content in the adc2 knock-out mutant was reduced after Pst DC3000 inoculation, and showed enhanced susceptibility to pathogen infection. The adc2 mutant exhibited reduced expression of PR-1 after bacterial infection and the growth of the pathogen was about 4-fold more than that in the WT plant. Furthermore, the disease susceptibility of the adc2 mutant was recovered by the addition of exogenous Put. Taken together, these results suggest that Arabidopsis MPK3 and MPK6 play a positive role in the regulation of Put biosynthesis, and that Put contributes to bacterial pathogen defense in Arabidopsis.