Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis

Lys-63-linked multiubiquitin chains play important roles in signal transduction in yeast and in mammals, but the functions for this type of chain in plants remain to be defined. The RING domain protein RGLG2 (for RING domain Ligase2) from Arabidopsis thaliana can be N-terminally myristoylated and localizes to the plasma membrane. It can form Lys-63-linked multiubiquitin chains in an in vitro reaction. RGLG2 has overlapping functions with its closest sequelog, RGLG1, and single mutants in either gene are inconspicuous. rglg1 rglg2 double mutant plants exhibit loss of apical dominance and altered phyllotaxy, two traits critically influenced by the plant hormone auxin. Auxin and cytokinin levels are changed, and the plants show a decreased response to exogenously added auxin. Changes in the abundance of PIN family auxin transport proteins and synthetic lethality with a mutation in the auxin transport regulator BIG suggest that the directional flow of auxin is modulated by RGLG activity. Modification of proteins by Lys-63-linked multiubiquitin chains is thus important for hormone-regulated, basic plant architecture.     

OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice

Recent genomic and genetic analyses based on Arabidopsis suggest that ubiquitination plays crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA). However, few such studies have been reported in rice as a monocotyledonous model plant. Taking advantage of strategies in biochemistry, molecular cell biology and genetics, the RING-finger containing E3 ligase OsSDIR1 (Oryza sativa SALT-AND DROUGHT-INDUCED RING FINGER 1) was found to be a candidate drought tolerance gene for engineering of crop plants. The expression of OsSDIR1 was detected in all tissues of rice and up-regulated by drought and NaCl, but not by ABA. In vitro ubiquitination assays demonstrated that OsSDIR1 is a functional E3 ubiquitin ligase and that the RING finger region is required for its activity. OsSDIR1 could complement the drought sensitive phenotype of the sdir1 mutant and overexpressing transgenic Arabidopsis were more sensitive to ABA, indicating that the OsSDIR1 gene is a functional ortholog of SDIR1. Upon drought treatment, the OsSDIR1-transgenic rice showed strong drought tolerance compared to control plants. Analysis of the stomata aperture revealed that there were more closed stomatal pores in transgenic plants than those of control plants. This result was also confirmed by the water loss assay and leaf related water content (RWC) measurements during drought treatment. Thus, we demonstrated that monocot- and dicot- SDIR1s are conserved yet have diverse functions.     

A poplar DRE-binding protein gene, PeDREB2L, is involved in regulation of defense response against abiotic stress

Dehydration responsive element (DRE)-binding proteins comprise a family of proteins that have important roles in abiotic stress processes. The present study aimed at elucidating the molecular basis of the stress tolerance mechanism in poplar. We isolated a gene, PeDREB2L, from the desert tree Populus euphratica Oliva. PeDREB2L contains a conserved AP2/ERF domain, a nuclear localization signal at the N-terminus and a serine-rich region at the C-terminus. PeDREB2L binds specifically to DRE elements and is expressed ubiquitously in all tissues, including roots, stems, and leaves. Expression of PeDREB2L in leaves increased under dehydration, salt, and abscisic acid stress treatments. Furthermore, PeDREB2L-GFP ectopically expressed in Arabidopsis gave strong green fluorescent protein signals in the nucleus of transgenic Arabidopsis under unstressed conditions and resulted in an improved tolerance against drought and freezing under stress conditions, while the Arabidopsis protein gene DREB2A-GFP used as a control only gave a weak fluorescent. We expected that the PeDREB2L might be useful in improving abiotic stress tolerance in transgenic plants.     

RGLG3 and RGLG4, novel ubiquitin ligases modulating jasmonate signaling

JAs are important hormones for plant development and defense, and JA signaling is regulated by diverse mechanisms. We have recently identified two RING-type ubiquitin ligases, RGLG3 and RGLG4, as essential JA signaling regulators. In this addendum, we discuss some characters of RGLG3 and RGLG4, which further support their important roles in JA pathway. RGLG3 and RGLG4 didn’t interact with known key factors of the core JA pathway, rather, it might target on unknown protein that negatively regulated JA signaling. RGLG3 and RGLG4 expression was suppressed by SA treatment in an NPR1-independent manner, and rglg3 rglg4 moderated SA-inhibited JA-responsive PDF1.2 expression, suggesting RGLG3 and RGLG4 took roles in SA-JA antagonism. RGLG3 and RGLG4 could be important players of a regulatory network and coordinated diverse signals to modulate JA signaling.     

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade‐A PP 2Cs, such as PP 2 CA or ABI 1, is a complementary mechanism to PYR / PYL / RCAR ‐mediated inhibition of PP 2C activity. ABA promotes the degradation of PP 2 CA through the RGLG 1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG 1 with PP 2 CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG 1 and promotes nuclear interaction with PP 2 CA . We found RGLG 1 is myristoylated in vivo , which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG 1 through the downregulation of N‐myristoyltransferase 1 ( NMT 1 ) and promotes nuclear translocation of RGLG 1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP 2 CA protein levels and the formation of RGLG 1–receptor–phosphatase complexes. We show that RGLG 1 ^Gly2Ala mutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG 1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG 1– PP 2 CA – PYL 8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG 1– PP 2 CA interaction and hence PP 2 CA degradation.     

RING finger protein RGLG1 and RGLG2 negatively modulate MAPKKK18 mediated drought stress tolerance in Arabidopsis

Mitogen activated protein kinase kinase kinase 18 (MAPKKK18) mediated signaling cascade plays important roles in Arabidopsis drought stress tolerance. However, the post‐translational modulation patterns of MAPKKK18 are not characterized. In this study, we found that the protein level of MAPKKK18 was tightly controlled by the 26S proteasome. Ubiquitin ligases RGLG1 and RGLG2 ubiquitinated MAPKKK18 at lysine residue K32 and K154, and promoted its degradation. Deletion of RGLG1 and RGLG2 stabilized MAPKKK18 and further enhanced the drought stress tolerance of MAPKKK18‐overexpression plants. Our data demonstrate that RGLG1 and RGLG2 negatively regulate MAPKKK18‐mediated drought stress tolerance in Arabidopsis.     

Phloem-localizing sulfate transporter,Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis. Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots. Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.     

SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1-beta-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.     

The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis

Ascorbic acid (AsA) is an important antioxidant in plants, and its biosynthesis is finely regulated through developmental and environmental cues; however, the regulatory mechanism remains unclear. In this report, the knockout and knockdown mutants of Arabidopsis AtERF98 decreased the AsA level, whereas the overexpression of AtERF98 increased it, which suggests that AtERF98 plays an important role in regulating AsA biosynthesis. AtERF98-overexpressing plants showed enhanced expression of AsA synthesis genes in the d-mannose/l-galactose (d-Man/l-Gal) pathway and the myo-inositol pathway gene MIOX4, as well as of AsA turnover genes. In contrast, AtERF98 mutants showed decreased expression of AsA synthesis genes in the d-Man/l-Gal pathway but not of the myo-inositol pathway gene or AsA turnover genes. In addition, the role of AtERF98 in regulating AsA production was significantly impaired in the d-Man/l-Gal pathway mutant vtc1-1, but the expression of the myo-inositol pathway gene or AsA turnover genes was not affected, which indicates that the regulation of AtERF98 in AsA synthesis is primarily mediated by the d-Man/l-Gal pathway. Transient expression and chromatin immunoprecipitation assays further showed that AtERF98 binds to the promoter of VTC1, which indicates that AtERF98 modulates AsA biosynthesis by directly regulating the expression of the AsA synthesis genes. Moreover, the knockout mutant aterf98-1 displayed decreased salt-induced AsA synthesis and reduced tolerance to salt. The supplementation of exogenous AsA increased the salt tolerance of aterf98-1; coincidently, the enhanced salt tolerance of AtERF98-overexpressing plants was impaired in vtc1-1. Thus, our data provide evidence that the regulation of AtERF98 in AsA biosynthesis contributes to enhanced salt tolerance in Arabidopsis.     

Mutual regulation of Arabidopsis thaliana ethylene-responsive element binding protein and a plant floral homeotic gene, APETALA2

BACKGROUND AND AIMS: It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression. METHODS: Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed. KEY RESULTS: Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens. CONCLUSIONS: AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.     

Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress

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.     

The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses

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.