Lijing Liu

COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability

Seasonal changes in day length are perceived by plant photoreceptors and transmitted to the circadian clock to modulate developmental responses such as flowering time. Blue-light-sensing cryptochromes, the E3 ubiquitin-ligase COP1, and clock-associated proteins ELF3 and GI regulate this process, although the regulatory link between them is unclear. Here we present data showing that COP1 acts with ELF3 to mediate day length signaling from CRY2 to GI within the photoperiod flowering pathway. We found that COP1 and ELF3 interact in vivo and show that ELF3 allows COP1 to interact with GI in vivo, leading to GI degradation in planta. Accordingly, mutation of COP1 or ELF3 disturbs the pattern of GI cyclic accumulation. We propose a model in which ELF3 acts as a substrate adaptor, enabling COP1 to modulate light input signal to the circadian clock through targeted destabilization of GI.

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana.

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate-specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase-substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well-known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene-silencing suppressor p19 and by time-courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.

Arabidopsis Ubiquitin Conjugase UBC32 Is an ERAD Component That Functions in Brassinosteroid-Mediated Salt Stress Tolerance

This work demonstrates that the Arabidopsis thaliana ubiquitin conjugation enzyme UBC32 is a component of the plant endoplasmic reticulum (ER)-associated protein degradation pathway. Biochemical and genetic studies demonstrate the functional connection between ER-associated protein degradation and brassinosteroid-mediated salt stress signaling. Plants modify their growth and development to protect themselves from detrimental conditions by triggering a variety of signaling pathways, including the activation of the ubiquitin-mediated protein degradation pathway. Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important aspect of the ubiquitin-proteasome system, but only a few of the active ERAD components have been reported in plants. Here, we report that the Arabidopsis thaliana ubiquitin-conjugating enzyme, UBC32, a stress-induced functional ubiquitin conjugation enzyme (E2) localized to the ER membrane, connects the ERAD process and brassinosteroid (BR)-mediated growth promotion and salt stress tolerance. In vivo data showed that UBC32 was a functional ERAD component that affected the stability of a known ERAD substrate, the barley (Hordeum vulgare) powdery mildew O (MLO) mutant MLO-12. UBC32 mutation caused the accumulation of bri1-5 and bri1-9, the mutant forms of the BR receptor, BRI1, and these mutant forms subsequently activated BR signal transduction. Further genetic and physiological data supported the contention that UBC32 plays a role in the BR-mediated salt stress response and that BR signaling is necessary for the plant to tolerate salt. Our data indicates a possible mechanism by which an ERAD component regulates the growth and stress response of plants.

Integration of Light- and Brassinosteroid-Signaling Pathways by a GATA Transcription Factor in Arabidopsis

Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here, we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant cop1. Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses GATA2 transcription through the BR-activated transcription factor BZR1, whereas light causes accumulation of GATA2 protein and feedback inhibition of GATA2 transcription. Dark-induced proteasomal degradation of GATA2 is dependent on the COP1 E3 ubiquitin ligase, and COP1 can ubiquitinate GATA2 in vitro. This study illustrates a molecular framework for antagonistic regulation of gene expression and seedling photomorphogenesis by BR and light.

The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca2+ release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.

The SINA E3 Ligase OsDIS1 Negatively Regulates Drought Response in Rice

Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice.

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.

The RING Finger Ubiquitin E3 Ligase SDIR1 Targets SDIR1-INTERACTING PROTEIN1 for Degradation to Modulate the Salt Stress Response and ABA Signaling in Arabidopsis

An endoplasmic reticulum-bound ubiquitin E3 ligase targets its chloroplast/nucleus-bound substrate for degradation to promote abscisic acid signaling and the salt stress response. The plant hormone abscisic acid (ABA) regulates many aspects of plant development and the stress response. The intracellular E3 ligase SDIR1 (SALT- AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1) plays a key role in ABA signaling, regulating ABA-related seed germination and the stress response. In this study, we found that SDIR1 is localized on the endoplasmic reticulum membrane in Arabidopsis thaliana. Using cell biology, molecular biology, and biochemistry approaches, we demonstrated that SDIR1 interacts with and ubiquitinates its substrate, SDIRIP1 (SDIR1-INTERACTING PROTEIN1), to modulate SDIRIP1 stability through the 26S proteasome pathway. SDIRIP1 acts genetically downstream of SDIR1 in ABA and salt stress signaling. In detail, SDIRIP1 selectively regulates the expression of the downstream basic region/leucine zipper motif transcription factor gene ABA-INSENSITIVE5, rather than ABA-RESPONSIVE ELEMENTS BINDING FACTOR3 (ABF3) or ABF4, to regulate ABA-mediated seed germination and the plant salt response. Overall, the SDIR1/SDIRIP1 complex plays a vital role in ABA signaling through the ubiquitination pathway.

Arabidopsis SDIR1 Enhances Drought Tolerance in Crop Plants

Arabidopsis E3 ligase salt- and drought-induced RING-finger 1 (SDIR1) has been found to be involved in abscisic acid (ABA)-related stress signaling. SDIR1-overexpressing Arabidopsis plants exhibit improved tolerance to drought. Tobacco (Nicotiana tabacum) and rice (Oryza sativa) are two important agronomic crop plants. To determine whether SDIR1 enhances drought resistance in crop plants, SDIR1 transgenic tobacco and rice plants were generated. Ectopic expression of SDIR1 in both plants conferred improved drought tolerance ability. These results suggest that SDIR1 can function as a drought-tolerance gene in both dicotyledons and monocotyledons, and that it can serve as a drought-tolerance engineering candidate gene in crop plants.

A Ubiquitin Ligase of Symbiosis Receptor Kinase Involved in Nodule Organogenesis

The symbiosis receptor kinase (SymRK) is required for morphological changes of legume root hairs triggered by rhizobial infection. How protein turnover of SymRK is regulated and how the nodulation factor signals are transduced downstream of SymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase (SIE3) was shown to bind and ubiquitinate SymRK. The SIE3-SymRK interaction and the ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3 represents a new class of plant-specific E3 ligases that contain a unique pattern of the conserved CTLH (for C-terminal to LisH), CRA (for CT11-RanBPM), and RING (for Really Interesting New Gene) domains. Expression of SIE3 was detected in all tested tissues of Lotus japonicus plants, and its transcript level in roots was enhanced by rhizobial infection. The SIE3 protein was localized to multiple subcellular locations including the nuclei and plasma membrane, where the SIE3-SymRK interaction took place. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas downregulation of SIE3 transcripts by RNA interference inhibited infection thread development and nodule organogenesis. These results suggest that SIE3 represents a new class of E3 ubiquitin ligase, acts as a regulator of SymRK, and is involved in rhizobial infection and nodulation in L. japonicus.