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The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1 K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

AtAIRP2 is a cytosolic RING-type E3 ubiquitin ligase that positively regulates an abscisic acid (ABA) response in Arabidopsis ( Arabidopsis thaliana ). Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein1) as a substrate of AtAIRP2. ATP1 was found to be identical to SDIRIP1, which was reported recently to be a negative factor in ABA signaling and a target protein of the RING E3 ligase SDIR1. Accordingly, ATP1 was renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast cotransfection assays. The ABA-mediated germination assay of 35S : ATP1/SDIRIP1-RNAi/atairp2 double mutant progeny revealed that ATP1/SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA- and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and bimolecular fluorescence complementation experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were colocalized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco ( Nicotiana benthamiana ) leaf cells. The 26S proteasome subunits RPN12a and RPT1 and the molecular chaperones HSP70 and HSP101 were colocalized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that the down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 ubiquitin ligases is critical for ABA and high-salinity responses during germination in Arabidopsis., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H(2)O(2)) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H(2)O(2) and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H(2)O(2), and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.

Among approximately 480 RING domain-containing E3 Ub ligases in Arabidopsis, three, At3g46620, At5g59550, and At2g39720, have a domain-of-unknown-function (DUF) 1117 motif in their C-terminal regions. At3g46620 and At5g59550 were identified as homologous ABA- and drought-induced RING-DUF1117 genes and were designated AtRDUF1 and AtRDUF2, respectively. Single and double knock-out mutations of AtRDUFs resulted in hyposensitive phenotypes toward ABA in terms of germination rate and stomatal closure and markedly reduced tolerance to drought stress relative to wild-type plants. These results are discussed in the context that AtRDUF1 and AtRDUF2 play combinatorial, but still distinguishable, roles in ABA-mediated dehydration stress responses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABA-mediated drought stress responses.

Ubiquitination is a eukaryotic posttranslational protein modification that is mediated by the cascade of E1, E2, and E3 ubiquitin (Ub) ligases and is involved in regulating numerous cellular functions. In this study, we obtained 100 different Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant plants in which RING E3 Ub ligase genes were suppressed and monitored their phenotypes in the presence of exogenous abscisic acid (ABA), a plant stress hormone. One of these loss-of-function mutants displayed ABA-insensitive phenotypes at the germination stage and was named atairp1 (for Arabidopsis ABA-insensitive RING protein 1). AtAIRP1 encodes a cytosolic protein containing a single C3H2C3-type RING motif with in vitro E3 Ub ligase activity. AtAIRP1 was significantly induced by ABA and drought stress. In contrast to atairp1 mutant plants, AtAIRP1-overexpressing transgenic plants (35S:AtAIRP1-sGFP) were hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure. Ectopic expression of AtAIRP1-sGFP in atairp1 effectively rescued the loss-of-function ABA-insensitive phenotype. Both 35S:AtAIRP1-sGFP and atairp1/35S:AtAIRP1-sGFP plants accumulated higher amounts of hydrogen peroxide in response to exogenous ABA than did wild-type and atairp1 mutant plants. AtAIRP1 overexpressors were markedly tolerant to severe drought stress, as opposed to atairp1, which was highly susceptible. The levels of drought stress-related genes and basic leucine zipper transcription factor genes were up-regulated in the 35S:AtAIRP1-sGFP lines relative to wild-type and atairp1 mutant plants in response to ABA. Overall, these results suggest that AtAIRP1, a C3H2C3-type RING E3 Ub ligase, is a positive regulator in the Arabidopsis ABA-dependent drought response.

Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box-containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.