Cosuppression of AtGELP22 and AtGELP23, two ubiquitinated target proteins of RING E3 ligase AtAIRP5, increases tolerance to drought stress in Arabidopsis.

AtAIRP5 RING E3 ubiquitin ligase was recently identified as a positive regulator of the abscisic acid (ABA)-mediated drought stress response by stimulating the degradation of serine carboxypeptidase-like 1. Here, we identified GDSL-type esterase/lipase 22 (AtGELP22) and AtGELP23 as additional interacting partners of AtAIRP5. Yeast two-hybrid, pull-down, co-immunoprecipitation, and ubiquitination analyses verified that AtGELP22 and AtGELP23 are ubiquitinated target proteins of AtAIRP5. AtGELP22 and AtGELP23 were colocalized with AtAIRP5 to punctate-like structures in the cytosolic fraction, in which PYK10 and NAI2, two ER body marker proteins, are localized. T-DNA insertion atgelp22 and atgelp23 single knockout mutant plants showed phenotypes indistinguishable from those of wild-type plants under ABA treatment. In contrast, RNAi-mediated cosuppression of AtGELP22 and AtGELP23 resulted in hypersensitive ABA-mediated stomatal movements and higher tolerance to drought stress than that of the single mutant and wild-type plants. Taken together, our results suggest that the putative GDSL-type esterases/lipases AtGELP22 and AtGELP23 act as redundant negative regulators of the ABA-mediated drought stress response in Arabidopsis.

E3 ligase AtAIRP5/GARU regulates drought stress response by stimulating SERINE CARBOXYPEPTIDASE-LIKE1 turnover.

Ubiquitination is a major mechanism of eukaryotic posttranslational protein turnover that has been implicated in abscisic acid (ABA)-mediated drought stress response. Here, we isolated T-DNA insertion mutant lines in which ABA-insensitive RING protein 5 (AtAIRP5) was suppressed, resulting in hyposensitive ABA-mediated germination compared to wild-type Arabidopsis (Arabidopsis thaliana) plants. A homology search revealed that AtAIRP5 is identical to gibberellin (GA) receptor RING E3 ubiquitin (Ub) ligase (GARU), which downregulates GA signaling by degrading the GA receptor GID1, and thus AtAIRP5 was renamed AtAIRP5/GARU. The atairp5/garu knockout progeny were impaired in ABA-dependent stomatal closure and were markedly more susceptible to drought stress than wild-type plants, indicating a positive role for AtAIRP5/GARU in the ABA-mediated drought stress response. Yeast two-hybrid, pull-down, target ubiquitination, and in vitro and in planta degradation assays identified serine carboxypeptidase-like1 (AtSCPL1), which belongs to the clade 1A AtSCPL family, as a ubiquitinated target protein of AtAIRP5/GARU. atscpl1 single and atairp5/garu-1 atscpl1-2 double mutant plants were more tolerant to drought stress than wild-type plants in an ABA-dependent manner, suggesting that AtSCPL1 is genetically downstream of AtAIRP5/GARU. After drought treatment, the endogenous ABA levels in atscpl1 and atairp5/garu-1 atscpl1-2 mutant leaves were higher than those in wild-type and atairp5/garu leaves. Overall, our results suggest that AtAIRP5/GARU RING E3 Ub ligase functions as a positive regulator of the ABA-mediated drought response by promoting the degradation of AtSCPL1.

OsATL38 mediates mono-ubiquitination of the 14-3-3 protein OsGF14d and negatively regulates the cold stress response in rice.

One of the major regulatory pathways that permits plants to convert an external stimulus into an internal cellular response within a short period of time is the ubiquitination pathway. In this study, OsATL38 was identified as a low temperature-induced gene that encodes a rice homolog of Arabidopsis Tóxicos en Levadura RING-type E3 ubiquitin (Ub) ligase, which was predominantly localized to the plasma membrane. OsATL38-overexpressing transgenic rice plants exhibited decreased tolerance to cold stress as compared with wild-type rice plants. In contrast, RNAi-mediated OsATL38 knockdown transgenic progeny exhibited markedly increased tolerance to cold stress relative to that of wild-type plants, which indicated a negative role of OsATL38 in response to cold stress. Yeast two-hybrid, in vitro pull-down, and co-immunoprecipitation assays revealed that OsATL38 physically interacted with OsGF14d, a rice 14-3-3 protein. An in vivo target ubiquitination assay indicated that OsGF14d was mono-ubiquitinated by OsATL38. osgf14d knockout mutant plants were more sensitive to cold stress than wild-type rice plants, indicating that OsGF14d is a positive factor in the response to cold stress. These results provide evidence that the RING E3 Ub ligase OsATL38 negatively regulates the cold stress response in rice via mono-ubiquitination of OsGF14d 14-3-3 protein.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

PUB22 and PUB23 U-box E3 ubiquitin ligases negatively regulate 26S proteasome activity under proteotoxic stress conditions.

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo-proteasome complex, resulting in intracellular accumulation of UbG76V -GFP, an artificial substrate of the proteasome complex, and insoluble poly-ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26S proteasome integrity.

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA-mediated microtubule depolymerization, stomatal closure, and tolerance response to drought stress.

Ubiquitination is a critical post-translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T-DNA insertion mutant in the At5g10650 locus. Compared to wild-type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C-terminal C3HC4-type Really Interesting New Gene (RING) motif, which was essential for ABA-mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule-associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA-promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild-type levels of H2 O2 after ABA treatment. These findings indicated that JUL1 acts downstream of H2 O2 and calcium in the ABA-mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H2 O2 , and calcium, which in turn resulted in ABA-hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild-type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA-mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.

OsPUB41, a U-box E3 ubiquitin ligase, acts as a negative regulator of drought stress response in rice (Oryza Sativa L.).

KEY MESSAGE: OsPUB41 plays a negative role in drought stress response through the mediation of OsUBC25 and interacts with OsCLC6, suggesting a putative substrate. The notable expansion of Plant U-Box E3 ligases (PUB), compared with those in mammals, implies that PUB proteins have evolved to perform plant-specific functions. OsPUB41, a potential ortholog of CMPG1, was recently reported to regulate the cell wall degrading enzyme (CWDE)-induced innate immune response in rice. Here, we characterized the OsPUB41 gene, which encodes a dual-localized cytosolic and nuclear U-box E3 ligase in rice. OsPUB41 expression was specifically induced by dehydration among various abiotic stresses and abscisic acid (ABA) treatments. Furthermore, we revealed that the core U-box motif of OsPUB41 possesses the E3 ligase activity that can be activated by OsUBC25 in rice. The Ubi:RNAi-OsPUB41 knock-down and ospub41 suppression mutant plants exhibited enhanced tolerance to drought stress compared with the wild-type rice plants in terms of transpirational water loss, long-term dehydration response, and chlorophyll content. Moreover, the knock-down or suppression of the OsPUB41 gene did not cause adverse effect on rice yield-related traits. Yeast two-hybrid and an in vitro pull-down analyses revealed that OsCLC6, a chloride channel, is a putative substrate of OsPUB41. Overall, these results suggest that OsPUB41 acts as a negative regulator of dehydration conditions and interacts with OsCLC6, implying that it is a substrate of OsPUB41.

Suppression of DRR1 results in the accumulation of insoluble ubiquitinated proteins, which impairs drought stress tolerance.

Drought stress has detrimental effects on plants. Although the abscisic acid (ABA)-mediated drought response is well established, defensive mechanisms to cope with dehydration-induced proteotoxicity have been rarely studied. DRR1 was identified as an Arabidopsis drought-induced gene encoding an ER-localized RING-type E3 Ub ligase. Suppression of DRR1 markedly reduced tolerance to drought and proteotoxic stress without altering ABA-mediated germination and stomatal movement. Proteotoxicity- and dehydration-induced insoluble ubiquitinated protein accumulation was more obvious in DRR1 loss-of-function plants than in wild-type plants. These results suggest that DRR1 is involved in an ABA-independent drought stress response possibly through the mitigation of dehydration-induced proteotoxic stress.

OsBZR1 turnover mediated by OsSK22-regulated U-box E3 ligase OsPUB24 in rice BR response.

Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1-EMS-SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U-box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome-dependent degradation of OsBZR1. The ospub24 T-DNA knock-out mutant and Ubi:RNAi-OsPUB24 knock-down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild-type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3-like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine-tuning the BR response in rice.

Poaceae Type II Galactinol Synthase 2 from Antarctic Flowering Plant Deschampsia antarctica and Rice Improves Cold and Drought Tolerance by Accumulation of Raffinose Family Oligosaccharides in Transgenic Rice Plants.

Deschampsia antarctica is a Poaceae grass that has adapted to and colonized Antarctica. When D. antarctica plants were subjected to cold and dehydration stress both in the Antarctic field and in laboratory experiments, galactinol, a precursor of raffinose family oligosaccharides (RFOs) and raffinose were highly accumulated, which was accompanied by upregulation of galactinol synthase (GolS). The Poaceae monocots have a small family of GolS genes, which are divided into two distinct groups called types I and II. Type II GolSs are highly expanded in cold-adapted monocot plants. Transgenic rice plants, in which type II D. antarctica GolS2 (DaGolS2) and rice GolS2 (OsGolS2) were constitutively expressed, were markedly tolerant to cold and drought stress as compared to the wild-type rice plants. The RFO contents and GolS enzyme activities were higher in the DaGolS2- and OsGolS2-overexpressing progeny than in the wild-type plants under both normal and stress conditions. DaGolS2 and OsGolS2 overexpressors contained reduced levels of reactive oxygen species (ROS) relative to the wild-type plants after cold and drought treatments. Overall, these results suggest that Poaceae type II GolS2s play a conserved role in D. antarctica and rice in response to drought and cold stress by inducing the accumulation of RFO and decreasing ROS levels.

AtKPNB1, an Arabidopsis importin-ß protein, is downstream of the RING E3 ubiquitin ligase AtAIRP1 in the ABA-mediated drought stress response.

MAIN CONCLUSION: AtKPNB1, an Arabidopsis importin-ß protein, was regulated by AtAIRP1 E3 ubiquitin ligase, which intensified the ABA-mediated drought stress response. As an early step in the abscisic acid (ABA)-mediated drought response, the ABA signal is transduced into the nucleus, and thus the nuclear transport system is crucially involved in the drought stress response. AtKPNB1, an importin-ß protein, which is a core component of nuclear transport, was previously reported to be a negative factor in the ABA-mediated drought stress response (Luo et al. Luo et al., Plant J 75:377-389, 2013). Here, we report that AtAIPR1, an Arabidopsis RING-type E3 ubiquitin (Ub) ligase, interacted with and ubiquitinated AtKPNB1. A null mutation of AtKPNB1 suppressed the ABA-insensitive germination phenotype of atairp1 mutant seedlings as compared to that of the wild-type plants. Furthermore, the ABA-insensitive stomatal closure and drought-susceptible phenotypes of atairp1 were rescued in atairp1atkpnb1 double mutant progeny, indicating that AtKPNB1 functions downstream of AtAIRP1. These data suggest that AtAIRP1 regulates the ABA-mediated drought response in Arabidopsis via ubiquitination of AtKPNB1.

Inverse Correlation Between MPSR1 E3 Ubiquitin Ligase and HSP90.1 Balances Cytoplasmic Protein Quality Control.

MISFOLDED PROTEIN SENSING RING1 (MPSR1) is a chaperone-independent E3 ubiquitin ligase that participates in protein quality control by eliminating misfolded proteins in Arabidopsis (Arabidopsis thaliana). Here, we report that in the early stages of proteotoxic stress, cellular levels of MPSR1 increased immediately, whereas levels of HEAT SHOCK PROTEIN90.1 (AtHSP90.1) were unaltered despite massively upregulated transcription. At this stage, the gene-silencing pathway mediated by microRNA 414 (miR414) suppressed AtHSP90.1 translation. By contrast, under prolonged stress, AtHSP90.1 was not suppressed, and instead competed with MPSR1 to act on misfolded proteins, promoting the destruction of MPSR1. Deficiency or excess of MPSR1 significantly abolished or intensified the suppression of AtHSP90.1, respectively. Similar to the MPSR1-overexpressing transgenic plants, the miR414-overexpressing plants showed an increased tolerance to proteotoxic stress as compared to the wild-type plants. Although the functional relationship between MPSR1 and miR414 remains unclear, both MPSR1 and miR414 demonstrated negative modulation of the expression of AtHSP90.1. The inverse correlation between MPSR1 and AtHSP90.1 via miR414 may adjust the set-point of the HSP90-mediated protein quality control process in response to increasing stress intensity in Arabidopsis.

ROS1-Dependent DNA Demethylation Is Required for ABA-Inducible NIC3 Expression.

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD+ salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.

MPSR1 is a cytoplasmic PQC E3 ligase for eliminating emergent misfolded proteins in Arabidopsis thaliana.

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.

Classification of barley U-box E3 ligases and their expression patterns in response to drought and pathogen stresses.

BACKGROUND: Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. RESULTS: In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. CONCLUSION: This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley.

The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination Specificity for Exocyst Subunit Exo70B1.

The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stress responses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereas PUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecular mechanism that mediates the functional difference between PUB18 and PUB22 and found that the UNDPUB18 was critically involved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, was identified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ∆UND-PUB18 derivative failed to ubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UNDPUB18-PUB22 chimeric protein ubiquitinated Exo70B1 instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, respectively, are dependent on the presence or absence of the UNDPUB18 motif. The ABA-insensitive phenotypes of the pub18 pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1 functions downstream of PUB18. Overall, our results suggest that the UNDPUB18 motif is crucial for the negative regulation of ABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.

Low binding affinity and reduced complement-dependent cell death efficacy of ofatumumab produced using a plant system (Nicotiana benthamiana L.).

The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the capping of CD20 after ofatumumab binding was also confirmed using Ramos B cells. In the functional equivalence tests, the binding affinities and complement-dependent cell cytotoxicity efficacy of plant-ofatumumab-HDEL and plant-ofatumumab without HDEL were significantly reduced compared to those of CHO-derived ofatumumab. Therefore, we suggest that although ofatumumab is not a good candidate as a template for plant-derived monoclonal antibodies because of its decreased affinity when produced in plants, it is an interesting target to study the differences between post-translational modifications in mammals and plants.

The proper localization of RESPONSIVE TO DESICCATION 20 in lipid droplets depends on their biogenesis induced by STRESS-RELATED PROTEINS in vegetative tissues.

Arabidopsis LD surface proteins, SRPs are found only in higher plants and are important for LD biogenesis and abiotic stress signaling. However, the cellular mechanism of SRPs is still unclear. To investigate molecular functions of SRPs, we used tobacco transient expression system. Transient expression of SRPs was sufficient and synergistic for LD biogenesis, and SRPs participated in the formation step of LD in tobacco leaves. RESPONSIVE TO DESICCATION 20 (RD20), a known LD-localizing peroxygenase, localized to LD in the presence of an SRP, and its peroxygenase activity correlated with proper localization of RD20 to LD. Our data suggest that Arabidopsis SRPs play roles as positive factors for LD biogenesis to provide a proper localization of LD-localizing proteins in vegetative tissues.

AtAIRP2 E3 Ligase Affects ABA and High-Salinity Responses by Stimulating Its ATP1/SDIRIP1 Substrate Turnover.

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.