The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

Phase separation-based visualization of protein-protein interactions and kinase activities in plants.

Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity.

A Nitrogen-specific Interactome Analysis Sheds Light on the Role of the SnRK1 and TOR Kinases in Plant Nitrogen Signaling.

Nitrogen (N) is of utmost importance for plant growth and development. Multiple studies have shown that N signaling is tightly coupled with carbon (C) levels, but the interplay between C/N metabolism and growth remains largely an enigma. Nonetheless, the protein kinases Sucrose Non-fermenting 1 (SNF1)-Related Kinase 1 (SnRK1) and Target Of Rapamycin (TOR), two ancient central metabolic regulators, are emerging as key integrators that link C/N status with growth. Despite their pivotal importance, the exact mechanisms behind the sensing of N status and its integration with C availability to drive metabolic decisions are largely unknown. Especially for SnRK1, it is not clear how this kinase responds to altered N levels. Therefore, we first monitored N-dependent SnRK1 kinase activity with an in vivo Separation of Phase-based Activity Reporter of Kinase (SPARK) sensor, revealing a contrasting N-dependency in Arabidopsis thaliana (Arabidopsis) shoot and root tissues. Next, using affinity purification (AP) and proximity labeling (PL) coupled to mass spectrometry (MS) experiments, we constructed a comprehensive SnRK1 and TOR interactome in Arabidopsis cell cultures during N-starved and N-repleted growth conditions. To broaden our understanding of the N-specificity of the TOR/SnRK1 signaling events, the resulting network was compared to corresponding C-related networks, identifying a large number of novel, N-specific interactors. Moreover, through integration of N-dependent transcriptome and phosphoproteome data, we were able to pinpoint additional N-dependent network components, highlighting for instance SnRK1 regulatory proteins that might function at the crosstalk of C/N signaling. Finally, confirmation of known and identification of novel SnRK1 interactors, such as Inositol-Requiring 1 (IRE1A) and the RAB GTPase RAB18, indicate that SnRK1, present at the ER, is involved in N signaling and autophagy induction.

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination.

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCC­E­F subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.

SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB/bHLH/TTG1 MBW complex.

Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal, but also protect different tissues against high light, UV and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB- and bHLH-type transcription factors and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is not only useful, but also carbon- and energy-intensive and non-vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon- and energy-depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post-translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.

Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

The Mitochondrial DNA-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination.

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

The role of HEXOKINASE1 in Arabidopsis leaf growth.

KEY MESSAGE: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine ß-synthase domains, as an interacting protein of HXK1.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GSyellow, which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GSyellow tag in the dicot Arabidopsis (Arabidopsis thaliana) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GSyellow tag, along the growth zone of the maize (Zea mays) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GSyellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research.

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf.

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.

A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls.

Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM.

Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA double-strand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases--the Arabidopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs--as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.

Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.

Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway.

Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17 Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions.

The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis.

In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita-induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment.

ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development.

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues.

Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.

The Arabidopsis Iron-Sulfur Protein GRXS17 is a Target of the Ubiquitin E3 Ligases RGLG3 and RGLG4.

The stability of signaling proteins in eukaryotes is often controlled by post-translational modifiers. For polyubiquitination, specificity is assured by E3 ubiquitin ligases. Although plant genomes encode hundreds of E3 ligases, only few targets are known, even in the model Arabidopsis thaliana. Here, we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 using a substrate trapping approach involving tandem affinity purification of RING-dead versions. Simultaneously, we used a ubiquitin-conjugating enzym (UBC) panel screen to pinpoint UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 and mediating auto-ubiquitination of RGLG3 and ubiquitination of GRXS17 in vitro. Accordingly, GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta. The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. Our results suggest that the RGLG family is intimately linked to the essential element iron.

The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability.

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCF(COI1)) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCF(COI1) components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability.