RING-type E3 Research Articles

The Arabidopsis RING-Type E3 Ligase TEAR4 Controls Seed Germination by Targeting RGA for Degradation.

Light and DELLA proteins are central factors controlling seed germination which is critical for seed plant survival and agricultural production. However, the mechanisms underlying DELLA degradation under different light conditions during seed germination remain to be clarified. Here, it is reported that TIE1-ASSOCIATED RING-TYPE E3 LIGASE4 (TEAR4) and other TEARs redundantly promote DELLA degradation to positively regulate seed germination in Arabidopsis. The tear1/2/3/4/5/6 sextuple mutant displayed delayed seed germination under the white or PhyB-dependent light condition, and nearly no seed germination under the PhyA-dependent light condition. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in tear1/2/3/4/5/6, and disruption of RGA and GA-INSENSITIVE (GAI) in tear1/2/3/4/5/6 rescued defective seed germination. Far-red (FR) light rapidly induced TEARs, and TEAR4 is shown to act as an E3 ligase. It is showed that both GA-dependent and TEAR-mediated DELLA degradation pathways are indispensable for PhyA-dependent germination. It is found that TEAR homologs PpTEAR1 and PpTEAR2 from the moss Physcomitrium patens interacted with Arabidopsis DELLAs to promote their degradation, and overexpression of PpTEAR1 or PpTEAR2 completely rescued defective PhyA-dependent seed germination in phya-211. This findings demonstrate that TEARs act as critical players in fine-tuning seed germination, and TEAR-mediated DELLA degradation might be an ancient pathway conserved in plant kingdom.

Characterization of tripartite motif containing 59 (TRIM59) in Epinephelus akaara: Insights into its immune involvement and functional properties in viral pathogenesis, macrophage polarization, and apoptosis regulation

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84 kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H2O2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara.

Genome-wide association study and transcriptome analysis reveal the genetic basis underlying the environmental adaptation of plant height in a woody plant

Studies on plant height have been conducted in several crops. However, the underlying genetic mechanisms in woody plants remain unclear. To improve the genetic understanding of plant height, the genome-wide association study (GWAS) was conducted on the 298 individuals of paper mulberry (Broussonetia papyrifera), and the individuals with the highest and lowest plant heights were selected for comparative transcriptome analysis. The analysis of phenotypic data showed that plant height decreased from low latitude (N: 24°30′) to high latitude (N: 41°00′), ranging from 372 to 150 cm. Furthermore, the plant height of paper mulberry was significantly correlated with environmental factors, such as latitude, frost-free period, hours of sunshine and so on, indicating adaptive phenotypic divergence across environmental gradients. A total of 228 candidate genes were identified through the GWAS, including three genes (Bp10g0547, Bp10g0551 and Bp10g0817) that contained nonsynonymous SNP variations significantly affecting plant height. A total of 2554 differentially expressed genes (DEGs) were identified through RNA sequencing (RNA-seq) analysis, including 28, 5, 3, 20 and 138 DEGs involved in auxin, gibberellin, cytokinin, ubiquitylation and transcription factors, respectively. Besides, there were 13 common genes identified by integrating GWAS and RNA-seq analysis, including Bp10g0817, which encodes COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1) and belongs to the RING type E3 ubiquitin ligase gene family. Collectively, this study provides valuable insights into the genetic mechanisms underlying plant height and adaptation of woody plants to diverse environments.

Transcription factors PuNAC37/PuWRKY74 and E3 ubiquitin ligase PuRDUF2 inhibit volatile ester synthesis in 'Nanguo' pear.

As major contributors to flavor in many fruit species, volatile esters are useful for investigating the regulation of the biosynthesis pathways of volatile aroma compounds in plants. Ethylene promotes the biosynthesis of volatile esters during fruit storage while accelerating fruit ripening; thus, the ethylene perception inhibitor 1-Methylcyclopropene (1-MCP) is employed to prolong the shelf life of fruits. However, the mechanisms by which 1-MCP regulates volatiles synthesis remain unclear. In this study, we analyzed the pathway of 1-MCP-mediated volatile ester synthesis in 'Nanguo' pear (Pyrus ussuriensis). 1-MCP significantly decreased volatile ester synthesis during storage. Comparative transcriptome analysis showed that the genes encoding two transcription factors (PuNAC37, PuWRKY74) and a RING-type E3 ubiquitin ligase which have a domain of unknown function (PuRDUF2) were expressed at high levels, whereas ALCOHOL ACYLTRANSFERASE 1 (PuAAT1), encoding an enzyme responsible for volatile ester synthesis, was expressed at low levels in 1-MCP-treated fruit. Moreover, PuNAC37 inhibited the expression of PuWRKY74 via transcriptional regulation, whereas PuNAC37 and PuWRKY74, after directly binding to the promoter of PuAAT1, synergistically inhibited its expression in 1-MCP-treated fruit. In addition, in vitro and in vivo ubiquitination experiments revealed that PuRDUF2 functions as an E3 ubiquitin ligase that ubiquitinates PuAAT1, causing its degradation via the 26S proteasome pathway following 1-MCP treatment. Subsequent PuAAT1 degradation resulted in a reduction of volatile esters during fruit storage. Our findings provide insights into the complex transcriptional regulation of volatile ester formation in 'Nanguo' pears and contribute to future research on AAT protein ubiquitination in other species.

The RING-type E3 ligase RIE1 sustains leaf longevity by specifically targeting AtACS7 to fine-tune ethylene production in Arabidopsis

Ethylene is widely recognized as a positive regulator of leaf senescence. However, how plants coordinate the biosynthesis of ethylene to meet the requirements of senescence progression has not been determined. The rate-limiting enzyme in the ethylene biosynthesis pathway is ACC synthase. AtACS7 was previously considered one of the major contributors to the synthesis of "senescence ethylene" in Arabidopsis. However, the "brake signal" that fine-tunes the expression of AtACS7 to ensure optimal ethylene production during leaf development has yet to be identified. In the present study, the RING-H2 zinc-finger protein RIE1 was found to specifically interact with and ubiquitinate AtACS7, among all functional ACSs in Arabidopsis, to promote its degradation. Overexpression of RIE1 markedly decreased ethylene biosynthesis and delayed leaf senescence, whereas loss of function of RIE1 significantly increased ethylene emission and accelerated leaf senescence. The ethylene-related phenotypes of RIE1 overexpressing or knockout mutants were effectively rescued by the ethylene precursor ACC or the competitive inhibitor of ACS, respectively. In particular, AtACS7-induced precocious leaf senescence was strongly enhanced by the loss of RIE1 but was significantly attenuated by the overexpression of RIE1. The specific regions of interaction between AtACS7 and RIE1, as well as the major ubiquitination sites of AtACS7, were further investigated. All results demonstrated that RIE1 functions as an important modulator of ethylene biosynthesis during leaf development by specifically targeting AtACS7 for degradation, thereby enabling plants to produce the optimal levels of ethylene needed.

The E3 ligase TaE3V-B1 ubiquitinates proteins encoded by the vernalization gene TaVRN1 and regulates developmental processes in wheat.

In wheat (Triticum aestivum), early maturity is desired to avoid the hot and dry summer season, especially in view of climate change. Here, we report that TaE3V1, a C3H2C3 RING-type E3 ligase that interacts with TaVRN1, is associated with early development. Aside from its RING domain, TaE3V1 does not harbor any domains that are conserved in other RING-type or other E3 ligase proteins. TaE3V-B1b, encoded by the functional TaE3V1 allele, interacts with and ubiquitinates TaVRN1. In contrast, TaE3V-B1a, encoded by a natural nonfunctional TaE3V1 allele, neither interacts with TaVRN1 nor has E3 ligase activity. TaE3V-B1b activity decreases with plant age under warmer temperatures, but not under the low temperatures required for vernalization. We employed a gene editing method to simultaneously inactivate the three homoeologous TaE3V1 genes to validate their functions. Overall, our results suggest that the naturally mutated and edited TaE3V1 alleles can accelerate wheat development and aid adaptation to warming climates.

The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.

The endoplasmic reticulum-associated degradation (ERAD) system eliminates misfolded and short-lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain-of-function mutant, doa10a-1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono-ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING-type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono-ubiquitination.

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2~Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48-specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48-specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1~Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.

Interaction of the cyclin-dependent kinase inhibitor GhKRP6 with RING-Type E3 ligase influences the fiber length in Upland cotton

Upland cotton (Gossypium hirsutum L.) is the major crop producing renewable fiber for textiles. Cotton fiber length is an economically important trait that affects the quality of the yarn. The Upland cotton multi-parent advanced generation intercross (MAGIC) population was developed for genetics research to understand how fiber traits can be improved without reducing fiber yield. A genome-wide association study of the MAGIC population identified a significant fiber length QTL on Chromosome D11 (qFL-D11–1). To understand how the qFL-D11–1 regulates fiber length, we developed F5 lines by crossing the longest and the shortest fiber recombinant inbred lines (RILs) of the MAGIC population to establish lines with parental haplotypes at the qFL-D11–1 but segregating for other loci. The F5 lines and parental RILs were sequenced to detect exchanges from parents' genomic regions in the progeny. One segregating genomic region in the F5 lines coincided with qFL-D12–1, which additively affected fiber length. Correlation analysis of the expression patterns of genes from the two QTLs with the fiber lengths in the MAGIC population showed the highest correlation of the RING-type ubiquitin E3 ligase (GhUbE3) from qFL-D12–1. Kip-related protein-6 (KRP6) is the candidate gene from the qFL-D11–1. A yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated a physical interaction between GhKRP6 (Ghir_D11G020340) and GhUbE3 (Ghir_D12G006080). RNAseq showed that the GhUbE3 expression is reciprocal to the interacting GhKRP6 during fiber development. Virus-induced gene silencing (VIGS) of GhKRP6 increased fiber length. The data indicates that GhUbE3 controls the dose of GhKRP6's inhibitory activity and, therefore, the duration of fiber elongation.

Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

TRAF7 determines circadian period through ubiquitination and degradation of DBP.

D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP.

RLIM-specific activity reporters define variant pathogenicity in Tonne-Kalscheuer syndrome

Tonne-Kalscheuer syndrome (TOKAS, OMIM # 300978) is an X-linked recessive disorder with devastating consequences for patients such as intellectual disability, developmental delay, and multiple congenital abnormalities. TOKAS is associated with hemizygous variants in the RLIM gene that encodes a RING-type E3 ubiquitin ligase. The current sustained increase in reported RLIM variants of uncertain significance creates an urgent need to develop assays that can screen these variants and experimentally determine their pathogenicity and disease association. Here, we engineered flow cytometry-based RLIM-specific reporters to measure RLIM activity in TOKAS. This paper describes the design and use of RLIM-specific reporters to determine the pathogenicity of a TOKAS RLIM gene variant. Our data demonstrates that RLIM-specific flow cytometry reporters based on either the full length or a degron region of the substrate REX1 measure RLIM activity in cells. Further, we describe the TOKAS variant RLIM p.Asn581Lys and using reporter assays, determine that it disrupts RLIM catalytic activity. This data reveals how the p.Asn581Lys variant impairs RLIM function and suggests pathogenic mechanisms. The use of RLIM-specific reporters will greatly accelerate the resolution of variants of uncertain significance and disease association in TOKAS.

The ubiquitin E3 ligase RZFP1 affects drought tolerance in poplar by mediating the degradation of the protein phosphatase PP2C-9.

Abscisic acid signaling has been implicated in plant responses to water deficit-induced osmotic stress. However, the underlying molecular mechanism remains unelucidated. This study identified the RING-type E3 ubiquitin ligase RING ZINC FINGER PROTEIN1 (PtrRZFP1) in poplar (Populus trichocarpa), a woody model plant. PtrRZFP1 encodes a ubiquitin E3 ligase that participates in protein ubiquitination. PtrRZFP1 mainly functions in the nucleus and endoplasmic reticulum and is activated by drought and abscisic acid. PtrRZFP1-overexpressing transgenic poplars (35S:PtrRZFP1) showed greater tolerance to drought, whereas PtrRZFP1-knockdown lines (KD-PtrRZFP1) showed greater sensitivity to drought. Under treatment with polyethylene glycol and abscisic acid, PtrRZFP1 promoted the production of NO and H2O2 in stomatal guard cells, ultimately enhancing stomatal closure and improving drought tolerance. Additionally, PtrRZFP1 physically interacted with the clade A Protein Phosphatase 2C protein PtrPP2C-9, a core regulator of abscisic acid signaling, and mediated its ubiquitination and eventual degradation through the ubiquitination-26S proteasome system, indicating that PtrRZFP1 positively regulates the abscisic acid signaling pathway. Furthermore, the PtrPP2C-9-overexpression line was insensitive to abscisic acid and more sensitive to drought than the wild-type plants, whereas the opposite phenotype was observed in 35S:PtrRZFP1 plants. In general, PtrRZFP1 negatively regulates the stability of PtrPP2C-9 to mediate poplar drought tolerance. The results of this study provide a theoretical framework for the targeted breeding of drought-tolerant traits in perennial woody plants.

The RING-Type E3 Ligase BOI Interacts with EXO70E2 and Mediates Its Ubiquitination in Arabidopsis.

The exocyst is a hetero-octameric complex that exhibits significant functional diversity in regulating biological processes and defense responses. In plants, the EXO70 proteins are important components of the exocyst complex and are involved in membrane trafficking, biotic and abiotic interactions, as well as cell wall formation. A previous study has indicated that a member of the EXO subfamily, EXO70E2, interacts with RIN4 to mediate plant immunity. In this study, we found that EXO70E2 interacts with the RING-type E3 ligase Botrytis susceptible1 interactor (BOI), and the C-terminal domain of BOI is necessary for its interaction with EXO70E2. Moreover, the protein level of EXO70E2 was degraded and ubiquitinated by BOI in vitro. Collectively, our study reveals a mechanism for regulating the stability of EXO70E2 by a RING-type E3 ligase BOI-mediated ubiquitination.

Genome-wide identification of SINA gene family in Medicago truncatula and functional analysis of MtSINAL7.

Ubiquitination is an essential biological process that is vital for maintaining cellular activity and plays a critical role in precisely regulating protein levels within cells. The SINA (seven in absentia) protein belongs to the RING-type E3 ubiquitin ligase, which is one of the key enzymes involved in the process of ubiquitination. However, there have been few reports on the genome-wide identification of SINA gene family and the functional analysis of its specific genes, particularly in leguminous plants. In this study, a total of 20 MtSINA genes were identified from the genomes of Medicago truncatula, and classified into three subfamilies. These genes are distributed on 7 of 8 chromosomes with chromosome preference. The gene structures of most MtSINA genes are quite similar, and all MtSINA proteins contain conserved RING and SINA functional domains. Moreover, various cis-regulatory elements related to abiotic stress and hormone signals were found in the promoters of MtSINA genes. The expression profile indicates that a majority of MtSINA genes exhibit a significant response to abiotic stress. Furthermore, the study characterized the function of MtSINAL7 in plants and discovered its pivotal role in improving plant stress resistance. In summary, this study provides a new insight into the potential functions of MtSINA genes in Medicago truncatula.

Pepper RING-Type E3 Ligase CaFIRF1 Negatively Regulates the Protein Stability of Pepper Stress-Associated Protein, CaSAP14, in the Dehydration Stress Response.

As part of the cellular stress response in plants, the ubiquitin-proteasome system (UPS) plays a crucial role in regulating the protein stability of stress-related transcription factors. Previous study has indicated that CaSAP14 is functionally involved in enhancing pepper plant tolerance to dehydration stress by modulating the expression of downstream genes. However, the comprehensive regulatory mechanism underlying CaSAP14 remains incompletely understood. Here, we identified a RING-type E3 ligase, CaFIRF1, which interacts with and ubiquitinates CaSAP14. Pepper plants with silenced CaFIRF1 exhibited a dehydration-tolerant phenotype when subjected to dehydration stress, while overexpression of CaFIRF1 in pepper and Arabidopsis resulted in reduced dehydration tolerance. Co-silencing of CaFIRF1 and CaSAP14 in pepper increased sensitivity to dehydration, suggesting that CaFIRF1 acts upstream of CaSAP14. A cell-free degradation analysis demonstrated that silencing of CaFIRF1 led to decreased CaSAP14 protein degradation, implicating CaFIRF1 in the regulation of CaSAP14 protein via the 26S proteasomal degradation pathway. Our findings suggest a mechanism by which CaFIRF1 mediates the ubiquitin-dependent proteasomal degradation of CaSAP14, thereby influencing the response of pepper plants to dehydration stress.

GL5.2, a Quantitative Trait Locus for Rice Grain Shape, Encodes a RING-Type E3 Ubiquitin Ligase.

Grain weight and grain shape are important traits that determine rice grain yield and quality. Mining more quantitative trait loci (QTLs) that control grain weight and shape will help to further improve the molecular regulatory network of rice grain development and provide gene resources for high-yield and high-quality rice varieties. In the present study, a QTL for grain length (GL) and grain width (GW), qGL5.2, was firstly fine-mapped into a 21.4 kb region using two sets of near-isogenic lines (NILs) derived from the indica rice cross Teqing (TQ) and IRBB52. In the NIL populations, the GL and ratio of grain length to grain width (RLW) of the IRBB52 homozygous lines increased by 0.16-0.20% and 0.27-0.39% compared with the TQ homozygous lines, but GW decreased by 0.19-0.75%. Then, by analyzing the grain weight and grain shape of the knock-out mutant, it was determined that the annotation gene Os05g0551000 encoded a RING-type E3 ubiquitin ligase, which was the cause gene of qGL5.2. The results show that GL and RLW increased by 2.44-5.48% and 4.19-10.70%, but GW decreased by 1.69-4.70% compared with the recipient. Based on the parental sequence analysis and haplotype analysis, one InDel variation located at -1489 in the promoter region was likely to be the functional site of qGL5.2. In addition, we also found that the Hap 5 (IRBB52-type) increased significantly in grain length and grain weight compared with other haplotypes, indicating that the Hap 5 can potentially be used in rice breeding to improve grain yield and quality.

The RING-type E3 ligase, TaFRFP, regulates flowering by controlling a salicylic acid-mediated floral promotion

The initiation of transition to flowering is carefully managed by endogenous and environmental cues, which is critical for flowering plant reproductive success. Here, we found that wheat RING-type E3 ligase TaFRFP was highly expressed from the double ridge to degeneration stage (WS2.5-WS9). TaFRFP is localized in the nucleus and has E3 ligase activity in vitro. TaFRFP overexpression in Arabidopsis resulted in an early flowering phenotype, but to a lesser extent, under short-day conditions. Under the SA-treated condition, overexpression of TaFRFP shows higher root growth and has more accumulation of SA contents. A proteomic comparison revealed that the amount of FRL4A protein, a FRIGIDA LIKE 4 A, was considerably lower in SA-treated TaFRFP seedlings compared to normal condition. We further found that TaFRFP directly interacts with FRL4A in the nucleus and recruits it to the FLC locus in Arabidopsis. Moreover, an ubiquitination assay showed that TaFRPF physically interact and ubiquitinates TaFRL as a substrate. Our findings support the concept that the TaFRFP E3 ligase works as a positive regulator, and that the ubiquitination of its substrate proteins plays a significant role in controlling flowering time via an SA-dependent pathway.

Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

The RING-Type E3 Ubiquitin Ligase Gene GhDIRP1 Negatively Regulates Verticillium dahliae Resistance in Cotton (Gossypium hirsutum).

Cotton is one of the world's most important economic crops. Verticillium wilt is a devastating cotton disease caused by Verticillium dahliae, significantly impacting cotton yield and quality. E3 ubiquitin ligases are essential components of the ubiquitin-mediated 26S proteasome system, responsible for recognizing ubiquitinated target proteins and promoting their degradation, which play a crucial regulatory role in plant immune responses. In this study, on the basis of the confirmation of differential expression of GhDIRP1, a RING-type E3 ubiquitin ligase encoding gene, in two cotton varieties resistant (Zhongzhimian 2) or susceptible (Jimian 11) to V. dahliae, we demonstrated that GhDIRP1 is a negative regulator of V. dahliae resistance because silencing GhDIRP1 in cotton and heterogeneously overexpressing the gene in Arabidopsis enhanced and compromised resistance to V. dahliae, respectively. The GhDIRP1-mediated immune response seemed to be realized through multiple physiological pathways, including hormone signaling, reactive oxygen species, and lignin biosynthesis. Based on the sequences of GhDIRP1 isolated from Zhongzhimian 2 and Jimian 11, we found that GhDIRP1 had identical coding but different promoter sequences in the two varieties, with the promoter of Zhongzhimian 2 being more active than that of Jimian 11 because the former drove a stronger expression of GUS and LUC reporter genes. The results link the ubiquitination pathway to multiple physiological pathways acting in the cotton immune response and provide a candidate gene for breeding cotton varieties resistant to V. dahliae.