Journal of Integrative Plant Biology

Protein-protein interactions (PPIs) play a crucial role in enabling proteins to carry out their functions within various biological processes (Hui et al., 2003). Since the introduction of the yeast two-hybrid (Y2H) method for PPI detection in 1989 (Fields and Song, 1989), the identification of PPIs has become a significant focus in modern biological research. PPI goes beyond examining individual proteins, allowing researchers to establish a comprehensive network that regulates biological processes. Rice, as a key model organism in plant biological studies, has been at the forefront of PPI research. In 2008, prominent rice scientists in China called for concerted efforts to define a comprehensive protein-protein interaction network experimentally, which aimed to facilitate the prediction of the functional mechanisms operating throughout a plants lifecycle (Zhang et al., 2008). With efforts for 2 decades, the experimentally identified rice PPIs have reached over ten thousand. Several public databases have been established to systematically collate and store PPIs, including STRING (Szklarczyk et al., 2019), BioGRID (Oughtred et al., 2020), IntAct (del Toro et al., 2022), PRIN (Gu et al., 2011), RicePPINet (Liu et al., 2017) and RiceNet v2 (Lee et al., 2015). However, most PPI datasets in rice stem from computational predictions, while experiment-based rice PPI datasets are fragmented due to the lack of systematic profiling at the rice PPIome level, which largely hinders information sharing in the rice research community. To bridge this gap, we constructed the Port of Protein-Protein Interactomes (POPPIN; https://riceome.hzau.edu.cn/poppin/), an integrated database dedicated to sharing experimentally verified PPIs and functional clues in rice. Empowered by high-throughput PPIome profiling technologies and text mining assisted by a large language model (Huang et al., 2025; Liu et al., 2025), POPPIN currently has deposited over 150,451 pieces of rice PPI-related information. Additionally, POPPIN provides detailed protein information, including GO annotations, subcellular localizations, domains, trait ontology (TO) information, and hyperlinks to external biological databases. Through offering a user-friendly web interface for search and dynamic network visualization, POPPIN serves as the first large-scale, experiment-based database for searchable PPIs in rice, and has the potential to be extended to other species under this structural framework.

The Arabidopsis thaliana nonhost resistance proteins 2A (AtNHR2A) and 2B (AtNHR2B) play crucial roles in plant immunity as the single mutants Atnhr2a and Atnhr2b and the double mutant Atnhr2bAtnhr2a are susceptible to the non-adapted pathogen Pseudomonas syringae pv. tabaci that is unable to infect wild-type Col-0 plants. The localization of fluorescent versions of AtNHR2A and AtNHR2B to compartments of the endomembrane system together with their interaction with secreted proteins suggested a function in endomembrane-mediated secretory processes participating in plant immunity. Comparative apoplastic proteomics analysis between wild type Col-0 and the double mutant Atnhr2bAtnhr2a after treatment P. syringae pv. tabaci, revealed that AtNHR2A and AtNHR2B are indeed required for the secretion of proteins containing N-terminal signal peptides that occurs through the conventional protein secretion pathway. In this work, we leveraged these apoplastic proteomics datasets to identify proteins lacking N-terminal signal peptide and expected to be secreted through unconventional secretion pathway(s). We discovered that AtNHR2A and AtNHR2B are also required for the secretion of proteins through an unconventional secretion pathway that, intriguingly, included proteins previously associated with abiotic stress. These findings led us to define the subcellular dynamics of AtNHR2A and AtNHR2B, and through co-localization analyses and the use of vesicle trafficking inhibitors, we uncovered their trafficking pathways transitioning through Golgi-dependent and Golgi-independent pathways to ultimately reach the central vacuole. Our findings suggest that AtNHR2A and AtNHR2B participate in a multivesicular bodies-vacuole-mediated unconventional secretion pathway that results in the release of proteins involved in plant responses to environmental stresses.

Cellulose synthase complexes (CSCs) synthesize cellulose at the plasma membrane, and their activity and trafficking are critical for maintaining cell wall integrity during plant growth. Clathrin-mediated endocytosis (CME) regulates CSC internalization and has been implicated in their rapid stress-induced removal from the plasma membrane. Stress adaptation, instead, requires the maintenance of a subset of CSCs at the plasma membrane, yet the mechanisms underlying this homeostasis remain poorly understood. The Arabidopsis Bcl-2-associated athanogene4 (BAG4) was identified as an interactor of the adaptor protein 2 complex (AP-2) and the TPLATE complex (TPC), two key components of plant CME. Here, we show that AP-2 and the TPC associated with four closely related BAG proteins, BAG1-BAG4. A quadruple mutant exhibited abnormal growth, increased sensitivity to salt stress, and reduced endocytic flux. However, the abundance, localization and dynamics of CME machinery was largely unaffected, suggesting that BAG proteins are not core regulators of CME. Instead, BAG1-BAG4 deficiency caused hypersensitivity to cellulose biosynthesis inhibitors and impaired hypocotyl elongation in darkness, consistent with defective cellulose-dependent growth. BAG1-BAG4 also interacted with CESA6, and salt-induced CESA6 degradation and ubiquitination was enhanced in the quadruple mutant. Together, these findings identify BAG1-BAG4 as redundant proteostasis factors that safeguard CESA6 stability during salt stress, thereby maintaining cellulose synthesis, cell wall integrity, and plant stress tolerance.

The transport protein particle II (TRAPPII) complex is a conserved regulator of post-Golgi membrane trafficking. In Arabidopsis, phosphorylation of the TRAPPII-specific subunit TRS120 by SHAGGY-like kinases modulates adaptive growth responses, but the phosphatases that reverse this phosphorylation remain unknown. Here, proteomic analyses identified subunits of Protein Phosphatase 2A (PP2A) in the TRAPPII interactome. PP2A subunits physically associated with TRAPPII, and double-mutant analyses revealed genetic interactions between PP2A and TRAPPII. Loss of TRAPPII function reduced the relative membrane association of PP2A scaffolding subunits. We established an in vitro assay for Arabidopsis PP2A holoenzyme activity using complexes transiently co-expressed and affinity-purified from Nicotiana benthamiana. Structural modelling and interface analysis predicted binding of a phosphorylated peptide encompassing a TRS120 phosphosite cluster at the PP2A catalytic interface, while biochemical assays showed that a PP2A holoenzyme containing the B2 regulatory subunit dephosphorylated this peptide. Together, these findings identify a B2-containing PP2A holoenzyme as a candidate phosphatase for TRS120 and support a model in which antagonistic SHAGGY-like kinase and PP2A activities couple signalling to membrane trafficking during plant development and environmental adaptation.

Canonical ribonucleases such as Argonaute (AGO) participate in RNA-based gene regulatory processes. They are important for small RNA (sRNA) association and the formation of RNA-induced RNA silencing complexes. Potato (Solanum tuberosum) has 14 AGO-encoding genes, including the Solanum-specific StAGO15, which is activated upon infection by filamentous pathogens. The resistant phenotype conferred by ectopic expression of StAGO15 was abolished in plants in which a 21-nt sequence in the PAZ-encoding region of StAGO15 was targeted using an artificial miRNA. We then analyzed the sRNAs associated with StAGO15 in Phytophthora infestans leaf infection. Small RNAs were coimmunoprecipitated, subjected to deep sequencing, and analyzed against the P. infestans and potato genomes, in which cleavage sites were predicted by applying the R package, smartPARE. The most notable difference between pathogen- and water-inoculated StAGO15 samples was observed in the transposon (TE) category. Here, two categories of nonautonomous TE dominated: the miniature inverted-repeat transposable elements (MITEs) and short interspersed nuclear elements. The StAGO15-associated sRNAs bind and target complementary endogenous potato RNAs. One example is siRNA122, which acts on a cleavage site in the resistance gene PGSC0003DMG400015681 (StRES1). siRNA122 is derived from a MITE; upon deletion of this source sequence, resistance is restored. Our findings provide new insights into the plant immune surveillance network and demonstrate that deleting a 21 nt RNA sequence from a MITE transposon released a defense response, against two important filamentous pathogens.

Under nitrogen limiting conditions, legume plants interact with nitrogen fixing bacteria known as rhizobia, resulting in the formation of a new organ, the nodule. This process is accompanied by dramatic changes in gene expression, which operate at different levels. A previous study revealed that histone methylation is differentially modulated during nodulation. However, the histone methyl transferases and demethylases involved in this modulation have not been characterized. In this study we report the identification of the Medicago truncatula putative histone lysine demethylase MtPKDM9B, which is subject to alternative splicing (AS), and the differential modulation of AS variants at translational level during nodule symbiosis. Knockdown of MtPKDM9B impaired infection by rhizobia, nodule development, bacterial viability and the expression of the leghemoglobin coding gene MtLHB1. MtPKDM9B is the putative ortholog of Arabidopsis EARLY FLOWERING 6 (ELF6/AtPKDM9B) gene involved in the removal of the repressive mark H2K27me3. A combination of ChIP-seq and RNA-seq experiments revealed that MtPKDM9B is required for demethylation of H3K27me3 in regions nearby or contained within gene bodies of symbiotic genes and the upregulation of the cognate mRNAs in response to rhizobia, including those encoding the putative ubiquitin ligase MtPUB2, the MYB transcription factor MtMYB040 and the auxin conjugating enzyme MtGH3 (Gretchen Hagen 3). Our findings illustrate how AS and translational regulation of this plant specific histone lysine demethylase contributes to the removal of the repressive mark H3K27me3, promoting transcriptional activation of symbiotic genes required for the formation of functional nitrogen fixing nodules.

Plants perceive pathogenic threats through receptors localized at the plasma membrane and inside cells to initiate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. PTI and ETI pathways are interconnected and partially converge on the EDS1-PAD4-ADR1 branch which stimulates anti-pathogen defence. Besides these canonical immune pathways, proteolysis plays a pivotal role in immunity by regulating the abundance and activities of diverse components. We previously identified Arabidopsis papain-like cysteine protease RD19 as a PAD4 interactor and showed that RD19 promotes resistance to host-adapted pathogens. Here, we investigated the contribution of RD19 to bacterial flg22 and nlp20 elicited PTI responses which exhibit, respectively, partial and high PAD4 dependence. We show that although PAD4 and RD19 both promote PTI, they have separate functions. In resting (non-triggered) and nlp20-triggered tissues, RD19 limits RBOHD protein accumulation and consequently ROS production. Using N-terminomic enrichment and protease cleavage site identification methods we identify candidate RD19 substrates in resting and nlp20-responding tissues. Our analysis shows that two immune-related aspartyl proteases AED3 and AED2/ASPG1 are likely direct RD19 targets. This study reveals a role of RD19 in ROS regulation and advances our understanding of proteolysis and dynamic RD19 substrate targeting in plant immunity.

Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.

Plants rely on an innate immune system to defend against pathogens through various molecular responses. In addition to classical damage- and pathogen-associated molecular patterns (DAMPs and PAMPs), plants produce endogenous signaling peptides termed phytocytokines that amplify and regulate immune responses following stress. Although most characterized phytocytokines originate from dedicated precursor proteins, the contribution of multifunctional proteins to phytocytokine generation remains poorly understood. Here, we show that salicylic acid (SA) rapidly remodels the maize apoplastic peptidome through an early, transient proteolytic program driven by apoplastic serine hydrolases. Time course peptidomics identified fourteen candidate phytocytokines, including two cryptic peptides, PC13 and PC14, released from the stress-associated zinc-finger protein ZmSAP7 and the migration inhibitory factor-like protein ZmMDL1, respectively. Both peptides activated immune-associated gene expression but triggered distinct transcriptional responses and exerted opposing effects on Ustilago maydis infection, with PC13 enhancing resistance and PC14 promoting susceptibility. Biochemical analysis demonstrated that PMSF-sensitive apoplastic serine proteases directly process ZmMDL1 to release PC14. Together, our findings uncover a SA-responsive proteolytic pathway that generates functionally distinct phytocytokines from multifunctional proteins, expanding the repertoire of immune signaling peptides and revealing an additional layer of regulation in plant defense.

Aphids deplete their host plants of amino acids and other essential nutrients by feeding on phloem sap. Autophagy, a conserved process across eukaryotes, maintains cellular homeostasis during starvation and environmental stress by recycling damaged or excess cellular components. Here, we identified and characterized a previously uninvestigated aphid salivary protein, termed Aphid Autophagy Related Factor 1 (AAF1), that is secreted into plant cells and manipulates plant physiology in a manner that benefits the aphid. We show that AAF1 interacts with four known or putative host autophagy related proteins: RABD1 and RABD2a (plant homologs of Ypt1/RAB1), ATG8, a ubiquitin like protein required for autophagosome formation, and PI3PT, a previously uncharacterized ER localized putative lipid binding or lipid transfer protein annotated as a phosphoinositide 3 phosphate transfer protein. We further demonstrate that the RAB binding domain of AAF1 and its interaction with RABD2a may be specifically required for AAF1 function. In addition, we identified unique, highly mobile compartments containing interacting AAF1 and RABD2a that are associated with the endoplasmic reticulum (ER) and dynamically traffic with autophagosomes. We propose that these compartments may be derived from endosomes or endosome to ER contact sites (EERCSs) and serve as platforms for autophagosome trafficking and/or biogenesis. We further propose that AAF1, RABD2a, and PI3PT function together to regulate the formation or function of these compartments, thereby supporting autophagosome formation and trafficking. Finally, we show that stable expression of AAF1 in plants enhances growth and biomass in an autophagy dependent manner.

The Arabidopsis PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) and Telomere Repeat-Binding Proteins 1-3 (TRB1-3, TRBs) associate with distinct and shared protein complexes involved in epigenetic regulation, yet their cooperative roles in chromatin control and plant development remain largely unexplored. Here, we show that the interaction between PWO1 and TRBs is evolutionarily conserved. Both PWO1 and TRBs associate with plant telomeres, interact at these regions, and are co-enriched at subsets of interspersed telo-box motifs across regulatory regions genome-wide. TRBs facilitate PWO1 binding at shared genomic regions, including telo-box motifs. PWO1 and TRBs share a substantial number of genomic targets and preferentially bind chromatin regions associated with transcriptionally active states, whereas TRBs alone associate with repressive marks at thousands of loci. Genetic analyses show that the pwo1 trb1 trb3 triple mutant displays severe developmental defects, including main stem arrest and early maturation associated with aberrant lignin deposition in interfascicular tissues. In the triple mutant, key enzymes in the lignin biosynthesis pathway are upregulated, indicating that PWO1, TRB1, and TRB3 cooperatively regulate secondary cell wall formation. Together, our findings provide new insights into how PWO1 and TRBs cooperate to regulate chromatin states and orchestrate plant development, highlighting their central role in controlling gene expression programs. Significance statementThis study shows that PWO1 and TRB proteins co-occupy telomeres, including interspersed telo-box motifs, to regulate chromatin organization and plant development, particularly ectopic lignin deposition. Our findings reveal how these nuclear protein factors coordinate epigenetic states in Arabidopsis thaliana, providing a framework for understanding the control of developmental programs. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740627v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1370171org.highwire.dtl.DTLVardef@3faea5org.highwire.dtl.DTLVardef@e30921org.highwire.dtl.DTLVardef@16c948e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Evolutionarily conserved PWO-TRB interactions and their shared roles in chromatin regulation and plant development. Created with BioRender.com. C_FIG

Topologically associating domains (TADs) are essential structural and functional modules of the genome that play a crucial role in regulating gene expression. In this study, we systematically investigated the conservation and evolution of TADs in five closely related crops, including maize, sorghum, coix, foxtail millet and broomcorn millet. Our results show that 74% of TAD boundaries are conserved between two inbred maize lines, B73 and Mo17, and that approximately 50% or more of TAD boundaries are conserved across different crop species. TAD number remains relatively stable in the face of changes in genome size. However, the length of TADs varies depending on genome size. Furthermore, we found that large-scale transposable element expansion leads to TAD expansion, while chromosomal inversions lead to TAD fusion and the formation of new TAD boundaries. Frequent chromatin interactions between subgenome chromosomes occur after whole-genome duplication. Moreover, we also found that crossovers are enriched at TAD boundaries in maize, indicating the importance of TADs as a fundamental unit during species evolution. Overall, our study provides insights into the conservation and evolution of TADs in crop genomes and their roles in genome organization and function.

Brown spot (BS), caused by the fungal pathogen Bipolaris oryzae, is a major disease threatening global rice production. However, the genetic basis of host BS resistance remains unclear. Here, we identified brown spot resistance 1 (bsr1), a quantitative trait locus conferring BS resistance, by map-based cloning. We show that bsr1 encodes a sucrose transporter and that a near-isogenic line carrying bsr1 (bsr1-NIL) in the susceptible Koshihikari genetic background exhibited resistance to BS by suppressing sucrose efflux into the apoplast after pathogen attack. Furthermore, bsr1-NIL also showed strain-specific resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae through the same mechanism. These findings demonstrate that bsr1 confers dual resistance to fungal and bacterial diseases by regulating sucrose efflux. Our study identifies a previously unrecognized mechanism underlying resistance to both BS and bacterial blight and highlights bsr1 as a promising target for breeding disease-resistance rice cultivars. Rice (Oryza sativa L.) is a staple food for more than half of the worlds population1. Brown spot (BS), caused by the fungus Bipolaris oryzae, is one of the most prevalent fungal diseases of rice, and its incidence has increased under global warming2. BS infects coleoptiles, leaves, leaf sheaths, panicle branches, glumes, and spikelets, and severe infection can substantially reduce grain yield.

DNA methylation dynamics regulate diverse plant stress responses, but the mechanisms governing arsenic (As) tolerance through epigenetic pathways remain largely unknown. Here we report that Arabidopsis mutants lacking the DNA demethylase, REPRESSOR OF SILENCING (ROS1), show substantially enhanced As resistance. Integrating transcriptome and methylome data, we found that PIN-FORMED 2 (PIN2), encoding an auxin efflux carrier that also transports As out of cells, represents a key ROS1 target. In ros1, we observed increased methylation in the last exon of PIN2, a region functioning as the promoter for an antisense long non-coding RNA (lncRNA). Hypermethylation of the promoter suppresses the antisense lncRNA, which follows reduction of sense PIN2 transcription. As a result, PIN2 expression increases in ros1 mutants during As exposure, enhancing As tolerance. Analysis of ros1 pin2 double mutants confirmed that PIN2 acts downstream of ROS1 in As tolerance. Our study demonstrates an epigenetic pathway in which ROS1 regulates As tolerance by controlling PIN2 expression through antisense lncRNA-mediated transcriptional interference, offering new epigenetic strategies using plants in heavy metal contaminated environments.

The Unfolded Protein Response (UPR) maintains endoplasmic reticulum (ER) homeostasis during proteotoxic stress while also contributing to plant immunity. However, the functional relationship between UPR signaling, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA) remains poorly understood. Here, we investigated this interplay using Arabidopsis mutants impaired in the two major UPR branches. Mutants defective in the INOSITOL-REQUIRING ENZYME 1 (IRE1)-bZIP60 branch displayed enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and to the type III secretion-deficient strain hrcC-, indicating potentiation of PTI. Consistently, the ire1a ire1b double mutant exhibited increased flg22-induced callose deposition, whereas reactive oxygen species production was unaffected. Despite enhanced basal resistance, all UPR mutants retained full responsiveness to benzothiadiazole, demonstrating that downstream SA signaling remains functional independently of UPR integrity. Conversely, acute ER stress induced by tunicamycin promoted SA accumulation and extensive leaf necrosis. Reverse genetics revealed that SA-deficient mutants were largely protected from ER stress-induced cell death, whereas mutants over-accumulating SA exhibited enhanced susceptibility, demonstrating that endogenous SA is required for the full execution of this response. Analysis of an independent transcriptomic dataset further showed that loss of IRE1 de-represses genes involved in SA biosynthesis, PTI and defense under ER stress. Together, our results identify IRE1 as a key regulator coordinating ER homeostasis with immune signaling by limiting SA-dependent defense activation during ER stress, while simultaneously preventing excessive cell death.

Plants produce diverse small interfering RNA (siRNA) molecules that modulate development, environmental responses, and immunity. Although transgene-derived siRNAs are traditionally viewed as mediators of gene silencing, whether they can actively regulate endogenous host pathways remains largely unexplored. Previously obtained Arabidopsis, wheat, and soybean plants expressing the sunflower gene encoding the transcription factor HaHB4 exhibited water deficit tolerance. Here, we show that expressing inverted-repeat constructs that generate HaHB4-derived siRNAs without producing the HaHB4 protein bypasses transgenic growth penalties and instead enhances vegetative vigor and reproductive performance. In Arabidopsis, these DCL-dependent siRNA-producing lines exhibited enhanced root growth, increased stem and pith areas, increased cauline branching, and higher seed yield under both optimal and water-limiting conditions. Transcriptomic analysis revealed convergent repression of biotic stress-related genes, accompanied by increased bacterial susceptibility and reduced sensitivity to salicylic acid-mediated growth inhibition, suggesting an altered balance between immunity and growth. Functional characterization of candidate endogenous HD-Zip I targets further showed that athb20 and athb53 mutants recapitulated the increased stem expansion and cauline branching of the RNAi lines, respectively, pointing to endogenous HD-Zip I genes as candidate mediators of these traits. Remarkably, these effects were observed in newly obtained transgenic soybean plants, where expression of HaHB4-derived siRNAs enhanced vegetative vigor under controlled growth conditions. Overall, these findings show that transgene-derived siRNAs act independently of protein function to rewire endogenous regulatory networks, providing a potential strategy to optimize crop architecture and yield.

- The ascomycete fungal pathogen Blumeria hordei (Bh) is predicted to secrete over 530 effector proteins to infect barley and cause powdery mildew disease, yet their individual and collective functions are largely unexplored. We used yeast two-hybrid next-generation interaction screening (Y2H-NGIS) to probe the host targets of 48 Bh candidate secreted effectors (CSEPs), including multiple AVRA effectors that are recognized by MLA nucleotide-binding leucine-rich repeat (NLR) receptors. - Y2H-NGIS and subsequent binary retesting identified 356 high-confidence protein-protein interactions (PPI) among Bh CSEPs, MLA NLR fragments, and barley proteins. We uncovered unique and shared host targets, including numerous proteins involved in ubiquitin-associated processes, gene expression, immune and kinase signaling, metabolism, and vesicle trafficking. - We identified a host protein (HvCAP2) that interacts with MLA and at least five Bh effectors, as well as additional effector-host PPI that correlate with loss of recognition of AVRA13 and AVRA6 by MLA13 and MLA6, respectively. - Results were integrated with an interolog-based barley interactome (HvInt) to assemble a host-pathogen network of 1248 proteins and 1701 interactions. This PPI resource can serve as a platform for functional investigations into the molecular basis of powdery mildew pathogenesis and host recognition.

In angiosperms, ovule development requires the activity of the C, D and E classes of MADS genes, which encode key transcriptional regulators of reproductive development. The SEPALLATA (SEP) MADS transcription factors (MTFs), which belong to the E class, act as organizing hubs of MADS heterotetrameric complexes and play an essential role in the development of flower organs. However, the role of the SEP genes in ovule and seed development has been difficult to determine due to redundancy in the subclade, the lack of observable phenotypes in single and double sep1 sep2 mutants and the homeotic conversion of the carpel into sepal or leaf in higher order sep mutants. Here, we engineered a version of SEP3 (SEP3{Delta}M) that encodes a protein lacking the DNA-binding MADS-domain but retains the oligomerization domains needed for MADS protein heterotetramerization. In vitro experiments demonstrated the ability of SEP3{Delta}M to interact with the C and D classes of MTF, reducing the capability of such MADS complex to efficiently bind DNA. sep3{Delta}M plants showed a delay in flower opening and organ maturation and a reduced fertility. The ovules exhibited reduced outer integument growth, and the few seeds that developed showed impaired mucilage secretion upon imbibition. RNA-seq analysis of sep3{Delta}M demonstrated misregulation of genes involved in outer integument and seed coat development. Taken together, these data indicate the key role of SEP3-containing MADS complexes in proper ovule outer integument growth and seed coat development.

Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.

The ubiquitin-proteasome system (UPS) serves as the primary proteolytic machinery in eukaryotes, governing intracellular protein turnover to maintain proteome homeostasis. In plants, the HECT-type UPL3/4 ubiquitin ligases play vital roles in developmental and immune signaling. After ubiquitination by pathway-specific E3 ligases, substrates are physically relayed to proteasome-associated UPL3/4 ligases for further modification, which is necessary for their proteasome-mediated degradation. In this study, we investigated if the cellular influence of UPL3/4 extends beyond their direct role in substrate degradation. We discovered that UPL3/4 govern the ubiquitination not only of a broad array of immune-related substrates, but also of many UPS components, including E3 ligases. UPL3 physically interacts with PUB22, a pathway-specific U-box E3 ligase that negatively regulates immunity. PUB22 is controlled by a phospho-switch that converts it from an instable autoubiquitinated state to a stable phosphorylated E3 ligase that marks substrates for degradation. Remarkably, UPL3 only interacted with unphosphorylated PUB22 and facilitated its autoubiquitination-mediated degradation, thereby promoting the accumulation of PUB22 substrates. Moreover, the compromised immune phenotypes of upl3 upl4 mutant plants were largely dependent on PUB22 and its close paralogues. Thus, UPL3/4 control the stability of immune-related substrates not only through direct ubiquitination, but also indirectly by promoting autoubiquitination of PUB22 ligase and its paralogues. Controlling the stability of autoubiquitinating E3 ligases may be a universal mechanism whereby HECT-type ligases and the proteasomes they associated with, orchestrate cellular proteostasis in eukaryotes. Significance StatementThe ubiquitin-proteasome system (UPS) governs intracellular protein turnover to maintain proteome homeostasis in eukaryotes. Proteasome-associate HECT-type ubiquitin ligases play an important role in processing and degrading substrates delivered to the proteasome by pathway-specific E3 ligases. Here, we discover that in plants, HECT-type ligases not only promote the degradation of substrates, they also modify the E3 ligases that target these substrates to the proteasome. Specifically, HECT-type ligases facilitated or expanded the autoubiquitination of immune-suppressive E3 ligases, resulting in their proteasome-mediated degradation and onset of immunity. Our discoveries suggest that during plant immunity, HECT-type ligases and the proteasomes they associate with, control cellular proteostasis by governing the stabilities of both E3 ligases and their substrates.