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 H₂O₂ after ABA treatment. These findings indicated that JUL1 acts downstream of H₂O₂ and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H₂O₂, 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.     

The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis

Recent work has established a core ABA signaling pathway in which A‐type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA‐receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA‐hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1‐1/pir2 double mutant became more insensitive to ABA than the wild‐type or pir1‐1 and pir2 single mutants. Using a cell‐free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1‐1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation.     

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.     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

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.     

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.