PUB13, a U-box/ARM E3 ligase, regulates plant defense, cell death, and flowering time

Abstract

The ubiquitination pathway is involved in a variety of cellular processes in plant growth, development, and immune responses. However, the function of this pathway in connecting plant development and innate immunity is still largely unknown. Recently, we characterized the U-box/ARM E3 ubiquitin ligase PUB13, which regulates both immune responses and flowering time in Arabidopsis. Here, we show that the rice Spl11 gene can complement the cell death and flowering functions of PUB13 in the pub13 mutant. In addition, HFR1, which functions mainly in photomorphogenesis, was identified as one of the PUB13-interacting proteins through yeast two-hybrid screening and pull-down assays. Because the flowering phenotype of pub13 depends on photoperiod, we propose that PUB13 may regulate HFR1 to fine-tune photomorphogenesis and flowering time in Arabidopsis.

The U-box/ARM E3 Ligase PUB13 Negatively Regulates Cell Death, Disease Resistance, and Flowering Time

The ubiquitination proteasome system (UPS) in plants regulates diverse cellular processes, such as defense, cell death, floral transition, circadian rhythm, and photomorphogenesis.^1-4 Although these cellular functions cross talk and affect each other in plants, few studies have demonstrated the role of the UPS in interconnecting these cellular functions.

The Arabidopsis PUB13, a highly conserved ortholog of rice SPL11, is a U-box/ARM protein possessing E3 ubiquitin ligase activity.^5-7 We recently showed that PUB13 negatively regulates cell death and H₂O₂ accumulation, especially under high humidity and long-day conditions.⁵ Moreover, PUB13 negatively regulates broad-spectrum disease resistance to biotrophic pathogens, whereas it positively regulates disease resistance against necrotrophic pathogens after high humidity treatment.⁵ Another recent study showed that PUB13 is involved in regulating the FLS2-mediated PAMP-triggered immunity (PTI).⁸ In addition, we found that PUB13 negatively regulates flowering time in a photoperiod-dependent manner.⁵ Our genetic data further proved that salicylic acid (SA) is required for PUB13 regulation of cell death, defense, and flowering time. Taken together, these results suggest that PUB13, through the SA signaling pathway, acts as a junction between plant innate immunity and development.

PUB13 and SPL11 Are Highly Conserved in Protein Structure and Biological Functions

PUB13 is the closest ortholog of the rice SPL11 in Arabidopsis. PUB13 displays 73% identity of amino acids with SPL11, and both contain a U-box/ARM structure with E3 ligase activity.^5,7,9 Furthermore, like SPL11, PUB13 also regulates cell death, defense, and flowering time.^5,9,10 The high similarity of protein structure and biological functions between PUB13 and SPL11 prompted us to test whether the rice Spl11 gene can functionally complement the pub13 null mutant. Interestingly, overexpressing Spl11 with the 35S promoter in the pub13 mutant restored the flowering phenotype of pub13 to wild-type level under long-day conditions (Fig. 1A). This result indicates that Spl11 can functionally complement PUB13 in regulating flowering time, although SPL11 is a positive regulator in the short-day rice, while PUB13 is a negative regulator in the long-day Arabidopsis.^5,9

Figure 1. Flowering and cell death phenotypes of Spl11/pub13 transgenic plants. The plants were grown under long-day conditions (day/light: 16 h/8 h). The rice Spl11 gene under the control of the 35S promoter was transformed into pub13, and the T3 transgenic lines were used in (A), (B), and (C). (A) Flowering phenotypes of pub13 and Spl11/pub13 transgenic plants. Plants were 6 weeks old. (B) Lesion mimic phenotypes of 4 weeks old pub13 and Spl11/pub13 treated with high humidity (95% RH) for 48 h. (C) Cell death in pub13 and Spl11/pub13. Plants were treated with high humidity for 48 h, then the seventh or eighth leaves were harvested for trypan blue cell-death staining.

We further detected whether the cell death phenotype of pub13 could be complemented by Spl11. As expected, we found that the lesion mimic and cell death phenotypes of pub13 were partially rescued to the wild-type level after the overexpression of Spl11 under high humidity and long-day conditions (Fig. 1B, C). Taken together, the results demonstrate that PUB13 and SPL11 are functionally conserved in regulating flowering time and cell death. Identification and characterization of PUB13 and SPL11 have provided the first example of a U-box/ARM E3 ubiquitin ligase that has multiple functions in cell death, innate immunity and flowering in plants.

PUB13 Interacts with HFR1, a Positive Regulator of Photomorphogenesis

To identify the targets of PUB13 in the regulation of flowering and defense, we performed a yeast two-hybrid screen using PUB13^V273R, an E3 ligase activity-compromised mutant, as the bait. One of the isolated interactors was HFR1 (long hypocotyl in far-red light 1), which encodes a bHLH-type transcription factor.¹¹ We confirmed the screening result by transforming both plasmids into yeast (Fig. 2A). The interaction between PUB13 and HFR1 was further detected in E. coli in the GST pull-down assay. As shown in Figure 2B, PUB13 and HFR1, fused with the GST and MBP tags, respectively, interact specifically in E. coli. However, whether PUB13 ubiquitinates HFR1 remains unknown.

Figure 2. The interaction between PUB13 and HFR1 in vitro. (A) The interaction between PUB13 and HFR1 in yeast. The full-length CDS of PUB13V273R was cloned into pDBleu, and the full-length CDS of HFR1 was cloned into pPC86. PUB13V273R and/or HFR1 or the corresponding empty vector (control) were transformed into yeast strain MaV203. The transformed yeast cells were plated on YPDA medium for X-gal assay. Control A-E indicate the spectrum of interaction strengths. (B) The GST pull-down assay of PUB13 and HFR1. PUB13 and HFR1 were fused with GST and MBP, respectively. The two E. coli-expressed proteins were incubated together with glutathione-agarose beads. The complexes recovered from the beads were resolved by SDS-PAGE and analyzed by western blotting with the anti-MBP antibody to confirm the interaction. The GST protein was used as the control.

HFR1 promotes photomorphogenesis and is ubiquitinated and degraded by the E3 ligase COP1 in darkness.¹² COP1 is involved in photomorphogenesis, plant growth, flower shape, and flowering time.^13-15 pub13 displays early flowering under middle-day or long-day conditions but not under short-day conditions, suggesting that the flowering time regulated by PUB13 depends on photoperiod.^5,16,17 Although little is known about the role of HFR1 in the regulation of flowering time, transcriptional levels of HFR1 are 4- to 6-times higher in floral organs than in other tissues based on TAIR microarray data (jsp.weigelworld.org/expviz/expviz.jsp?experiment = development&normalization = absolute&probesetcsv = At1g02340&action = Run). These results suggest that PUB13 may regulate HFR1 in a COP1-dependent or -independent pathway to fine-tune photomorphogenesis and flowering time in Arabidopsis.