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. 2022 Aug 9;34(11):4516–4530. doi: 10.1093/plcell/koac245

The deubiquitinating enzymes UBP12 and UBP13 positively regulate recovery after carbon starvation by modulating BES1 stability in Arabidopsis thaliana

Jiawei Xiong 1, Fabin Yang 2, Xiuhong Yao 3, Yuqing Zhao 4, Yu Wen 5, Honghui Lin 6, Hongqing Guo 7, Yanhai Yin 8, Dawei Zhang 9,✉
PMCID: PMC9614486  PMID: 35944221

Abstract

BRI1-EMS-SUPPRESSOR1 (BES1), a core transcription factor in the brassinosteroid (BR) signaling pathway, primarily regulates plant growth and development by influencing BR-regulated gene expression. Several E3 ubiquitin (Ub) ligases regulate BES1 stability, but little is known about BES1 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain BES1 homeostasis. Here, we report that two Arabidopsis thaliana deubiquitinating enzymes, Ub-SPECIFIC PROTEASE (UBP) 12 and UBP13, interact with BES1. UBP12 and UBP13 removed Ub from polyubiquitinated BES1 to stabilize both phosphorylated and dephosphorylated forms of BES1. A double mutant, ubp12-2w ubp13-3, lacking UBP12 and UBP13 function showed both BR-deficient and BR-insensitive phenotypes, whereas transgenic plants overexpressing UBP12 or UBP13 exhibited an increased BR response. Expression of UBP12 and UPB13 was induced during recovery after carbon starvation, which led to BES1 accumulation and quick recovery of stressed plants. Our work thus establishes a mechanism by which UBP12 and UBP13 regulate BES1 protein abundance to enhance BR-regulated growth during recovery after carbon starvation.

Two enzymes deubiquitinate the transcription factor BRI1-EMS-SUPPRESSOR1 to promote its stability and brassinosteroid-regulated growth and thus positively regulate recovery after carbon starvation.

IN A NUTSHELL.

Background: Plants have evolved multiple mechanisms to coordinate growth and stress responses, including global reprogramming of gene expression, RNA processing or sequestration, and post-transcriptional and post-translational modifications. Under stress conditions, plant growth is, in general, placed on hold. During recovery following stress, growth resumes. In contrast to the wealth of studies focusing on stress tolerance, there are relatively few reports on the mechanisms by which plants recover after stress, which is nevertheless crucial as it determines final yield. BRI1-EMS-SUPPRESSOR1 (BES1), a core transcription factor in the brassinosteroid (BR) signaling pathway, as a central regulator for plants in balancing growth and stress response, degradated by several E3 ubiquitin ligases under carbon starvation.

Question: We wanted to know whether and which deubiquitinases antagonize the action of E3 ligases to maintain BES1 homeostasis, and the dynamic regulatory mechanism during carbon starvation and recovery after stress via the regulation of BES1 abundance.

Findings: We found that the deubiquitinases UBIQUITIN-SPECIFIC PROTEASE12 (UBP12) and UBP13 interact with BES1 and enhance its stability. BES1 abundance decreased in a double mutant lacking UBP12 and UBP13 function and conversely increased in transgenic lines overexpressing UBP12 or UBP13. During carbon starvation, BES1 protein was ubiquitinated and degraded, and UBP12 and UBP13 abundance concomitantly decreased. During recovery after carbon starvation, UBP12 and UBP13 transcription was upregulated and their encoded proteins accumulated, leading to the deubiquitination of BES1 by UBP12 and UBP13 and subsequent BES1 accumulation to promote growth.

Next steps: Our work demonstrated a dynamic regulatory mechanism for BES1 during carbon starvation and recovery after carbon starvation. Additional investigations into the transformation of stress to recovery will help us understand the survival strategies of plants.

Introduction

Plants have evolved elaborate mechanisms to regulate protein abundance. One of the most efficient regulatory systems is the ubiquitin (Ub) proteasome system (UPS), in which the small protein Ub is attached to a target protein via the sequential action of three enzymes (Hershko and Ciechanover, 1998). Ub is first activated by an E1 Ub-activating enzyme, transferred to an E2 Ub-conjugating protein, and finally transferred to the substrate itself, a step that is catalyzed by an E3 Ub ligase (Pickart and Eddins, 2004). The E3 Ub ligase specifically binds to its substrate and catalyzes the conjugation of Ub to the substrate protein on specific lysine residues. The UPS is involved in nearly all plant growth and developmental processes, including the cell cycle, embryogenesis, senescence, defense, environmental responses, and phytohormone signaling (Vierstra, 2009).

Deubiquitinating enzymes (DUBs) offer a counterpoint to the UPS by cleaving Ub from UPS-targeted ubiquitinated proteins to regulate their activity and stability (Amerik and Hochstrasser, 2004; Crosas et al., 2006; Hanna et al., 2006). DUBs have two basic biochemical functions: they generate mature Ub from its precursors and they cleave Ub from substrate proteins (Nijman et al., 2005). UBIQUITIN-SPECIFIC PROTEASEs (UBPs) constitute the largest DUB family in Arabidopsis thaliana and are cysteine proteases with two conserved catalytic motifs, the Cys-box and the His-box (Liu et al., 2008).

The Arabidopsis genome encodes 27 UBPs that can be divided into 14 subfamilies (Liu et al., 2008). UBP12 and UBP13, two functionally redundant UBPs, share 91% sequence identity and participate in multiple plant developmental processes (Cui et al., 2013). For instance, UBP12 and UBP13 regulate the circadian clock and the photoperiodic control of flowering through the CONSTANS (CO)-dependent pathway (Cui et al., 2013). In addition, UBP12 and UBP13 work together with the plant-specific Polycomb-group (PcG) protein LIKE HETEROCHROMATIN PROTEIN1 to repress a subset of PcG target genes, and the loss of function of both UBP12 and UBP13 leads to abnormal embryo development (Derkacheva et al., 2016). UBP12 and UBP13 are also involved in phytohormone signals, as they positively regulate jasmonic acid (JA) signaling by enhancing the stability of MYC2, a critical transcription factor in the JA pathway (Jeong et al., 2017). Moreover, UBP12 and UBP13 are critical regulators of root meristem maintenance that act by increasing the stability of ROOT MERISTEM GROWTH FACTOR 1 (RGF1)-perceiving receptor (RGFR1), the cognate receptor for the peptide hormone RGF1 (An et al., 2018). UBP12 and UBP13 also participate in nitrogen deficiency-induced leaf senescence (Park et al., 2019) and regulate leaf size and cell area (Vanhaeren et al., 2020).

The brassinosteroid (BR) steroid phytohormones are widely involved in plant growth, development, and stress responses, such as skotomorphogenesis (seedling development in the dark) (Oh et al., 2012), plant innate immunity (Lozano-Duran et al., 2013; Deng et al., 2016a, 2016b), drought responses (Ye et al., 2017), floral transition (Li et al., 2018), seed germination (Zhao et al., 2019), and chloroplast development (Zhang et al., 2021). The BR signaling transduction pathway is well established (Nolan et al., 2020). BRs are perceived by the membrane receptor BR -INSENSITIVE1 (BRI1), leading to BRI1 activation and its interaction with the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Li and Chory, 1997; Li et al., 2002; Nam and Li, 2002; Hothorn et al., 2011; She et al., 2011; Gou et al., 2012). After BRs are perceived by their receptors, the BR signal is relayed to members of the BRI1-EMS-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1) transcription factor families (Wang et al., 2002; Yin et al., 2002). BES1 and BZR1 activity and abundance are regulated via multiple dynamic pathways that reflect and integrate growth, development, and environmental stimuli (Nolan et al., 2020). Phosphorylation of BES1 and BZR1 by BR-INSENSITIVE2 (BIN2), a glycogen synthase kinase-3-like kinase, leads to their inactivation (He et al., 2002). In contrast, MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) phosphorylates BES1 and promotes its activity in plant immunity (Kang et al., 2015).

In addition to phosphorylation, other posttranslational modifications also regulate BES1 protein accumulation and activity. BES1 and BZR1 are regulated by the UPS and by selective autophagy (Wang et al., 2013, 2021a; Zhang et al., 2016; Nolan et al., 2017; Yang et al., 2017; Kim et al., 2019). BES1 can be ubiquitinated and then degraded via selective autophagy under carbon starvation conditions (Nolan et al., 2017; Wang et al., 2021a). However, little is known about whether and which DUBs antagonize the action of E3 ligases to maintain BES1 homeostasis.

In this study, we show that the DUBs UBP12 and UBP13 interact with BES1 and enhance its stability. BES1 abundance decreased in a double mutant lacking UBP12 and UBP13 function and conversely increased in transgenic lines overexpressing UBP12 or UBP13. During carbon starvation, BES1 protein was ubiquitinated and degraded, and UBP12 and UBP13 abundance concomitantly decreased. During recovery after carbon starvation, UBP12 and UBP13 transcription was upregulated and their encoded proteins accumulated, leading to the deubiquitination of BES1 by UBP12 and UBP13 and subsequent BES1 accumulation to promote growth. Thus, our findings demonstrate a dynamic regulatory mechanism during carbon starvation and recovery after stress via the regulation of BES1 abundance by UBP12 and UPB13.

Results

UBP12 and UBP13 interact with BES1 in vitro and in vivo

BES1 stability is regulated by various mechanisms. In a yeast two-hybrid (Y2H) screen using full-length BES1 as a bait and a normalized Arabidopsis cDNA as a prey library, we showed that BES1 interacted with a partial fragment of UBP13. Since UBP12 and UBP13 are homologs, Y2H assays were performed to further test whether BES1 interacted with UBP12 and UBP13, or other UBPs. The results revealed that BES1 could interact with UBP12 and UBP13, but not with other UBPs (Figure 1A and Supplemental Figure S1). Meanwhile, we found that BES1 homologs BZR1 and BES1/BZR1-HOMOLOG (BEH) 1–4 could also interact with UBP12 and UBP13 in yeast (Supplemental Figure S2). We validated the interaction of BES1 with UBP12 and UBP13 by in vitro His pull-down assays, as recombinant His-UBP12 and His-UBP13 pulled down BES1 fused to maltose-binding protein (MBP-BES1) (Figure 1, B and C).

Figure 1.

Figure 1

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UBP12 and UBP13 interact with BES1. A, Y2H assays show the interaction of BES1 with UBP12 or UBP13. B and C, His pull-down assays detect the interaction between BES1 and UBP12 (B) or UBP13 (C). An anti-MBP antibody was used to detect MBP-BES1. D, BES1 interacts with UBP12 or UBP13, as determined by BiFC assays in N. benthamiana. Scale bars = 50 μm. E, Quantification of the fluorescence intensity in the cytosol and the nucleus from the BiFC assays in (D). Data represent means ± se of 30 cells from three biological replicates. **P < 0.01 based on Student’s t test. F, Co-IP assays confirm the interaction between BES1 and UBP12 or UBP13. BES1 was detected with an anti-BES1 antibody; HA-UBP12 and HA-UBP13 were detected with an anti-HA antibody. G, Co-IP assays show the interaction between different forms of BES1 and UBP13. Ten-day-old UBP13pro:HA-FLAG-UBP13/ubp13-3 seedlings were pretreated with DMSO, 200 nM BL, or 1 μM brassinazole (BRZ) for 2 h to induce BES1 accumulation. BES1 was detected with an anti-BES1 antibody, and HA-UBP13 was detected with an anti-HA antibody.

To expand these observations in planta, we performed bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves. When we co-infiltrated constructs encoding UBP12 or UBP13 fused to the N terminus of yellow fluorescent protein (nYFP) (UBP12-nYFP or UBP13-nYFP, respectively) with a construct encoding BES1 fused to the C terminus of YFP (cYFP) (BES1-cYFP), we detected YFP fluorescence in both the nucleus and the cytoplasm, which supported the interaction of BES1 with UBP12 and UBP13 in planta (Figure 1, D and E). Co-immunoprecipitation (Co-IP) assays using UBP12pro:HA-FLAG-UBP12/ubp12-1 and UBP13pro:HA-FLAG-UBP13/ubp13-3 transgenic seedlings determined that BES1 co-precipitates with UBP12 and UBP13 (Figure 1F).

BES1 can be present in phosphorylated or nonphosphorylated forms in plants. We investigated which BES1 forms interacted with the UBPs by treating UBP13pro:HA-FLAG-UBP13/ubp13-3 transgenic seedlings with brassinolide (BL, the most active BR) or brassinazole (BRZ, a BR biosynthesis inhibitor) to induce the accumulation of dephosphorylated or phosphorylated BES1, respectively, before performing a Co-IP assay. We established that both dephosphorylated and phosphorylated forms of BES1 interact with UBP13 in planta (Figure 1G).

Furthermore, to map the interaction interface between BES1, UBP12, and UBP13, we divided UBP12 and UBP13 into two fragments (Jeong et al., 2017) and BES1 into four regions (Yin et al., 2002; Supplemental Figure S3A). We observed that the N terminus of UBP12 and UBP13 interacts with the BIN2 phosphorylation (Phospho) domain of BES1 in yeast (Supplemental Figure S3, B and C). Taken together, these results demonstrated that UBP12 and UBP13 interact with BES1 in vitro and in vivo.

UBP12 and UBP13 enhance BES1 protein stability

We hypothesized that UBP12 and UBP13 remove Ub from polyubiquitinated BES1. To test this hypothesis, we conducted an in vitro deubiquitination assay, whereby we incubated recombinant His-UBP12 or His-UBP13 with polyubiquitinated BES1, obtained by SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA2 (SINAT2) as an E3 ligase. The results indicated that UBP12 and UBP13 can cleave Ub from polyubiquitinated BES1 (Figure 2A). To assess the deubiquitination effect of UBP12 and UBP13 on BES1 in vivo, we transiently transfected the constructs BES1-GFP and MYC-SINAT2 in Arabidopsis protoplasts isolated from Columbia-0 (Col-0), ubp12-2w ubp13-3, or 35S:HA-FLAG-UBP13 (UBP13OE) (Supplemental Figure S4) and detected the ubiquitination levels of BES1 by immunoblotting. We determined that polyubiquitinated BES1 levels are over three-fold higher in the ubp12-2w ubp13-3 double mutant than in the wild-type Col-0, while BES1 was weakly ubiquitinated in UBP13OE (Figure 2B).

Figure 2.

Figure 2

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UBP12 and UBP13 enhance the protein stability of BES1. A, UBP12 and UBP13 deubiquitinate polyubiquitinated BES1 in vitro. Anti-MBP and anti-Ub antibodies were used to detect the ubiquitination of BES1. B, UBP12 and UBP13 decrease BES1 ubiquitination in vivo. BES1-GFP and MYC-SINAT2 were transiently transfected in Arabidopsis protoplasts prepared from Col-0, ubp12-2w ubp13-3, or UBP13OE plants. BES1 ubiquitination was detected with anti-GFP and anti-Ub antibodies. The BES1-Ub/total BES1 ratio was determined using ImageJ with BES1-Ub or total BES1 abundance in Col-0 set to 1. C, Cell-free degradation assays showing the delayed or accelerated degradation of recombinant MBP-BES1 incubated in extracts prepared from UBP13OE or ubp12-2w ubp13-3, respectively. β-ACTIN served as a loading control in total protein extracts. D, Quantification of relative MBP-BES1 abundance in (C). The initial protein levels before treatment were set to 1. Data represent means ± sd from three independent experiments. Different lowercase letters indicate significant differences (P < 0.05) based on two-way ANOVA followed by Fisher’s least significant difference (LSD) test.

UBP12 and UBP13 regulate the stability of their substrates (Derkacheva et al., 2016; Jeong et al., 2017; An et al., 2018; Park et al., 2019; Vanhaeren et al., 2020). We performed cell-free protein degradation assays using recombinant MBP-BES1 incubated with protein extracts prepared from Col-0, ubp12-2w ubp13-3, or UBP13OE. We observed that MBP-BES1 appears more stable in UBP13OE extracts and less stable in ubp12-2w ubp13-3 extracts compared with Col-0 extracts (Figure 2, C and D). Addition of the proteasome inhibitor MG132 largely inhibited MBP-BES1 degradation, although some degradation still occurred in all genotypes after MG132 treatment (Figure 2D).

To identify the pathway(s) by which UBP12 and UBP13 prevent BES1 degradation, we characterized the degradation rate of BES1 in Col-0, ubp12-2w ubp13-3, or UBP13OE seedlings treated with the protein translation inhibitor cycloheximide (CHX) alone or together with MG132 or the autophagy-related inhibitor E64d or concanamycin A (ConA). E64d blocks vacuolar degradation during autophagy, while ConA inhibits V-type ATPase and prevents the degradation of autophagosomes in the vacuoles or lysosomes (Klionsky et al., 2021). As shown in Figure 3, A and B, the addition of MG132 significantly and equally reduced BES1 degradation in Col-0, ubp12-2w ubp13-3, and UBP13OE, although some BES1 degradation remained even with MG132 treatment. E64d or ConA treatment only moderately decreased BES1 degradation in all genotypes tested, with BES1 being degraded faster in ubp12-2w ubp13-3 and more slowly in UBP13OE relative to Col-0. These results indicated that UBP12 and UBP13 mostly mediate BES1 stability through proteasome-mediated degradation rather than autophagic degradation.

Figure 3.

Figure 3

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UBP12 and UBP13 protect BES1 from proteasomal degradation. A, BES1 protein stability in Arabidopsis seedlings treated with 1 mM CHX) and 50 μM MG132, 1 μM concanamycin A (ConA), or 50 μM E64d for the indicated times. Samples were analyzed by immunoblotting with anti-BES1 antibody. β-ACTIN served as a loading control. B, Quantification of BES1 protein abundance in (A). The initial protein levels before treatment were set to 1. Data represent means ± sd from three independent experiments. C, In vivo lysine 63 (K63)- and lysine 48 (K48)-linked ubiquitination of BES1 in Col-0, ubp12-2w ubp13-3, or UBP13OE seedlings. BES1-GFP and MYC-SINAT2 constructs were transiently transfected in Arabidopsis protoplasts prepared from Col-0, ubp12-2w ubp13-3, or UBP13OE. Proteins were immunoprecipitated with GFP agarose beads, followed by immunoblotting with anti-K63 Ub and anti-K48 Ub antibodies. The BES1-Ub/total BES1 ratio was determined using ImageJ with BES1-Ub or total BES1 abundance in Col-0 set to 1. D and F, Protein stability of phosphorylated BES1 (D) or nonphosphorylated BES1 (F), assessed by treating Col-0, ubp12-2w ubp13-3, or UBP13OE seedlings with 1 mM CHX + 200 nM BL, CHX + BL + 50 μM MG132, CHX + 1 μM BRZ, or CHX + BRZ + MG132. Samples were analyzed by immunoblotting with anti-BES1 antibody. β-ACTIN served as a loading control. E and G, BES1 abundance in (D) and (F), quantified with ImageJ. The initial protein levels before treatment were set to 1. Data represent means ± sd from three independent experiments. Different lowercase letters indicate significant differences (P < 0.05) based on two-way ANOVA followed by Fisher’s LSD test.

The Ub residues lysine 48 (K48) and lysine 63 (K63) are the two most abundant polyubiquitination linkages in Arabidopsis (Kim et al., 2013; Ji and Kwon, 2017). We investigated whether UBP12 and UBP13 target either or both of these residues in Ub by performing a Co-IP assay with GFP agarose beads on protoplasts transfected with the GFP-BES1 and MYC-SINAT2 constructs, followed by an immunoblot with antibodies specific for K63-Ub and K48-Ub (Figure 3C). We detected slightly more K63-linked ubiquitinated BES1 in ubp12-2w ubp13-3 (∼21% more) and slightly less in UBP13OE (9% less) relative to Col-0. K48-linked ubiquitinated BES1 was more abundant (∼2.7-fold) in ubp12-2w ubp13-3 than in Col-0 and less abundant in UBP13OE (only reaching 35% of Col-0 levels) than in Col-0 (Figure 3C). Taken together, these results demonstrated that UBP12 and UBP13 deubiquitinate BES1 by cleaving both K48- and K63-linked ubiquitination, with a preference for K48-linked ubiquitination.

We also assessed the stability of different forms of BES1 in Col-0, ubp12-2w ubp13-3, and UBP13OE by treating seedlings with BRZ (Figure 3, D and E) or BL (Figure 3, F and G). Upon BRZ treatment, phosphorylated BES1 accumulated and was more stable in UBP13OE seedlings but less stable in ubp12-2w ubp13-3 seedlings compared with Col-0 (Figure 3, D and E). Similarly, under BL treatment, dephosphorylated BES1 protein was more stable in UBP13OE and less stable in ubp12-2w ubp13-3 than in Col-0, and the addition of MG132 slowed the degradation of BES1 (Figure 3, F and G). These results indicated that UBP12 and UBP13 protect both the nonphosphorylated and phosphorylated forms of BES1 from proteasomal degradation by cleaving Ub from polyubiquitinated BES1.

UBP12 and UBP13 regulate the deubiquitination of the BR receptor BRI1 (Luo et al., 2022). To clarify whether UBP12 and UBP13 modulate BES1 stability directly or indirectly via the deubiquitination of BRI1, we introduced the UBP13OE transgene into the weak allele bri1-301 by genetic crossing. We established that UBP13 overexpression partially rescues the dwarf stature, shorter hypocotyl, and impaired BR-responsive gene expression of bri1 mutants (Supplemental Figure S5, A and B). We also measured BES1 abundance in bri1-301, seedlings overexpressing BRI1 (BRI1OE) and UBP13OE/bri1-301, which revealed that BRI1 overexpression did not change BES1 levels, while UBP13 overexpression raised BES1 levels in UBP13OE/bri1-301 (Supplemental Figure S5C).

We then explored the effects of blocking BR biosynthesis or BIN2 kinase activity. We grew Col-0, ubp12-2w ubp13-3, and UBP13OE seedlings on half-strength Murashige and Skoog (MS) medium containing the BR biosynthesis inhibitor propiconazole (PCZ) or the BIN2 inhibitor bikinin. Although PCZ inhibited hypocotyl elongation in all genotypes, UBP13OE seedlings appeared to be less sensitive to PCZ than Col-0 (Supplemental Figure S5, D and E). Similar to UBP13OE, hypocotyl elongation of bes1-D seedlings was still repressed by BRZ but was less sensitive than Col-0 (Bernardo-Garcia et al., 2014). Bikinin treatment enhanced hypocotyl elongation, and UBP13OE seedlings were more sensitive and ubp12-2w ubp13-3 seedlings less sensitive than Col-0 (Supplemental Figure S5, D and E).

We also explored BES1 stability in Col-0, ubp12-2w ubp13-3, and UBP13OE seedlings treated with PCZ or bikinin together with CHX. As shown in Supplemental Figure S5, F–H, under PCZ treatment conditions, phosphorylated BES1 was more stable in UBP13OE and less stable in ubp12-2w ubp13-3 than in Col-0; similarly, dephosphorylated BES1 was more stable in UBP13OE and less stable in ubp12-2w ubp13-3 than in Col-0 upon treatment with bikinin. These results indicated that BES1 stability in ubp12-2w ubp13-3 and UBP13OE seedlings is likely under the direct regulation of UBP12- and UBP13-mediated deubiquitination. Moreover, we concluded that UBP12- and UBP13-promoted BES1 accumulation is independent of BRI1 and other upstream BR signaling components such as BIN2.

UBP12 and UBP13 positively regulate BR responses

The results above indicated that UBP12 and UBP13 maintain the stability of BES1, suggesting that UBP12 and UPB13 are involved in regulating BR responses. To substantiate this possibility, we analyzed BR responses of seedlings of various genotypes by measuring their hypocotyl length and elongation with or without BL treatment. Hypocotyl length was shorter in ubp12-2w ubp13-3 seedlings than in Col-0 in normal growth conditions with DMSO as mock treatment or during carbon starvation, and the ubp12-2w ubp13-3 double mutant was less responsive to BL, as were BES1-RNAi seedlings (Figure 4, A–C and Supplemental Figure S6, A–C). The ubp12-2w ubp13-3 mutant also exhibited other typical BR-deficient phenotypes, such as shorter leaf petioles, and a lower ratio between leaf length and leaf width (Supplemental Figure S7, A–C).

Figure 4.

Figure 4

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The ubp12-2w ubp13-3 double mutant shows BR-deficient and BR-insensitive phenotypes. A, BR sensitivity assays of different mutant and transgenic seedlings grown on half-strength MS medium with DMSO (BL carrier) or 20 nM BL at 22°C under long-day conditions for 7 days. Scale bars = 4 mm. B, Hypocotyl length of the genotypes in (A). Hypocotyl lengths were measured in ImageJ. Data represent means ± se of 30 seedlings from three biological replicates. Different lowercase letters indicate significant differences (P < 0.05) based on two-way ANOVA followed by Fisher’s LSD test. C, BL-induced fold change in hypocotyl length of the indicated genotypes in A. BL induction = mean under BL conditions/mean under DMSO conditions. D and E, Relative transcript levels of the BES1-induced gene SAUR-AC1 (D) and the BES1-repressed gene DWF4 (E) under normal or BL-treated conditions. Seven-day-old plants were transferred to liquid half-strength MS medium with DMSO or 200 nM BL for 2 h; ACTIN2 expression was used as an internal reference. Relative transcript levels in DMSO-treated Col-0 seedlings were set to 1. Data represent means ± se from three independent experiments. F, Abundance of endogenous BES1 in different genotypes. Seedlings were grown on half-strength MS medium at 22°C under long-day conditions for 7 days. Samples were analyzed by immunoblotting with anti-BES1 antibody and anti-HSP90 antibody (Solarbio). HSP90 served as a loading control. G, Quantification of BES1 abundance in (F) by ImageJ. The protein levels in Col-0 were set to 1. Data represent means ± sd from three independent experiments. Different lowercase letters indicate significant differences (P < 0.05) based on one-way ANOVA followed by Fisher’s LSD test.

We next measured the relative transcript levels of the BES1-induced gene SMALL AUXIN UPREGULATED RNA-AC1 (SAUR-AC1) and of the BES1-repressed gene DWARF4 (DWF4). We determined that SAUR-AC1 expression is lower and DWF4 expression is higher in ubp12-2w ubp13-3 relative to Col-0 (Figure 4, D and E). Similar to genotypes with higher BR biosynthesis or signaling, 35S:HA-FLAG-UBP12 (UBP12OE), and UBP13OE seedlings had longer hypocotyls and were more sensitive to BL compared with Col-0 (Supplemental Figure S8, A–C). In addition, UBP12OE and UBP13OE seedlings had longer leaf petioles and greater ratios of leaf length to leaf width (Supplemental Figure S7, A–C). SAUR-AC1 expression levels were higher, while those of DWF4 were lower, in UBP12OE and UBP13OE compared with Col-0 (Supplemental Figure S8, D and E).

The Cys domain of UBP12 and UBP13 is essential for their deubiquitinating activity (Ewan et al., 2011; Cui et al., 2013; Jeong et al., 2017). We, therefore, constructed 35S:HA-FLAG-UBP12C208S and 35S:HA-FLAG-UBP13C207S transgenic lines, expressing point mutant variants that abolish the deubiquitinating activity of UBP12 and UBP13, respectively, and investigated the responses of UBP12C208SOE and UBP13C207SOE seedlings to BL treatment. We observed that UBP12C208SOE and UBP13C207SOE seedlings exhibit the same response to BL as Col-0 (Supplemental Figure S9, A–E), indicating that the deubiquitinating activity of UBP12 and UBP13 is essential to the BR response.

To investigate whether the effect of UBP12/UBP13 on BR response was dependent on BES1, we detected BES1 abundance in seedlings of different genotypes. Endogenous BES1 levels were lower in ubp12-2w ubp13-3 and higher in UBP13OE seedlings relative to Col-0 (Figure 4, F and G). We also crossed the BES1-RNAi transgene or the bes1-D mutant to ubp12-2w ubp13-3 or UBP13OE to obtain ubp12-2w ubp13-3 BES1-RNAi, UBP13OE/BES1-RNAi, ubp12-2w ubp13-3 bes1-D, and UBP13OE/bes1-D lines for phenotypic analysis. Seedlings of ubp12-2w ubp13-3 BES1-RNAi had shorter hypocotyls and an impaired BL response compared with ubp12-2w ubp13-3 and BES1-RNAi seedlings (Figure 4, A–C). BES1 accumulated even less in ubp12-2w ubp13-3 BES1-RNAi than BES1-RNAi (Figure 4, F and G).

bes1-D suppressed the short hypocotyl and BL hyposensitivity phenotypes of ubp12-2w ubp13-3, based on the phenotypes displayed by ubp12-2w ubp13-3 bes1-D seedlings (Figure 4, A–C). Moreover, BES1 abundance was significantly higher in ubp12-2w ubp13-3 bes1-D seedlings than in Col-0 (Figure 4, F and G). UBP13 overexpression partially rescued the shorter hypocotyl phenotype of BES1-RNAi in UBP13OE/BES1-RNAi and further enhanced hypocotyl elongation of bes1-D in UBP13OE/bes1-D (Supplemental Figure S8, A–C). Consistent with these phenotypes, BES1 abundance in UBP13OE/BES1-RNAi seedlings was higher than that in BES1-RNAi and slightly lower than that in Col-0; BES1 levels in UBP13OE/bes1-D were even higher than those in bes1-D (Figure 4, F and G). We also established that UBP12 and UBP13 gene expression and protein abundance are not notably regulated by BR (Supplemental Figure S10, A and B). These results demonstrated that UBP12 and UBP13 positively modulate BR responses by increasing BES1 abundance.

UBP12 and UBP13 positively regulate recovery after carbon starvation

BES1 is ubiquitinated by the E3 Ub ligases SINAT2 and BAF1 and degraded through the autophagy pathway dependent on the Ub receptor DOMINANT SUPPRESSOR OF KAR2 (DSK2), which plays an essential role in slowing plant growth under dehydration or fixed-carbon starvation (Nolan et al., 2017; Wang et al., 2021a). To explore the potential involvement of UBP12 and UBP13 in carbon starvation responses, we characterized the expression levels of UBP12, UBP13, and BES1 during carbon starvation. The expression levels of UBP12 and UBP13 did not change much after a 5-day carbon starvation treatment (Figure 5A). The expression levels of UBP12 rose over three-fold after a 4-day recovery period and then decreased by about half after 8 days of recovery. Relative UBP13 transcript levels increased even more, by ∼14-fold after 4 days of recovery and 19-fold after 8 days of recovery (Figure 5A). These results suggested that UBP12 and UBP13 might play an important role in the recovery response after carbon starvation.

Figure 5.

Figure 5

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UBP12 and UBP13 positively regulate recovery after carbon starvation. A, Relative transcript levels of UBP12, UBP13, and BES1 during carbon starvation (S) and recovery (R). Data represent means ± se from three independent experiments. The transcript levels in Col-0 before treatment were set to 1. Different lowercase letters indicate significant differences (P < 0.05) based on one-way ANOVA followed by Fisher’s LSD test. B, BES1 abundance in Col-0 and ubp12-2w ubp13-3 during recovery after carbon starvation. Seedlings were starved for carbon by being placed in the dark for 5 days and were returned to light conditions for recovery. BES1 abundance was detected with an anti-BES1 antibody. β-ACTIN served as a loading control. Relative band intensity of total BES1 was quantified with ImageJ. The initial protein levels before treatment were set to 1. Data represent means ± sd from three independent experiments. *P < 0.05, **P < 0.01 based on Student’s t test. C, BES1 abundance in Col-0, ubp12-2w ubp13-3, and UBP13OE after a 5-day carbon starvation. Data represent means ± sd from three independent experiments. Different lowercase letters indicate significant differences (P < 0.05) based on one-way ANOVA followed by Fisher’s LSD test. D, UBP12 and UBP13 abundance during carbon starvation and recovery. Data represent means ± sd from three independent experiments. *P < 0.05, **P < 0.01 based on Student’s t test. E, Survival of Col-0, ubp12-2w ubp13-3, BES1-RNAi, and bes1-D seedlings after carbon starvation. After an 8-day carbon starvation treatment, seedlings were returned to the light for an 8-day recovery. F, Survival rates in (E). Seedlings remaining alive or with new growth emerging were scored as surviving. Data represent means ± sd from three biological replicates, with each replicate containing at least 30 seedlings. Different lowercase letters indicate significant differences (P < 0.05) based on one-way ANOVA followed by Fisher’s LSD test. G, Seedling phenotypes of different genotypes during recovery. Seedlings after a 5-day starvation period were returned to light for an 8-day recovery; pictures were taken on the indicated days during recovery. Scale bars = 1 mm. H, Weight changes per 30 seedlings after recovery. The initial seedling weights were set to 1. Data represent means ± sd from three biological replicates, with each replicate containing 30 surviving seedlings.

As previously reported, relative BES1 transcript levels decreased ∼80% after 5 days of carbon starvation (Nolan et al., 2017), followed by a slight increase during recovery (Figure 5A). The expression levels of BAF1 and SINAT2 increased after 5 days of starvation, as previously reported (Nolan et al., 2017; Wang et al., 2021a), remained high after a 4-day recovery period, and then decreased slightly after 8 days (Supplemental Figure S11). We then investigated BES1 abundance in Col-0 during recovery after carbon starvation and observed that BES1 protein levels increase with 1 day of recovery and continue to rise as the recovery period is extended (Figure 5B), indicating that BES1 might be required for recovery after carbon starvation. However, BES1 abundance increased only slightly in the ubp12-2w ubp13-3 double mutant during recovery, supporting a role for UBP12 and UBP13 in recovery after stress (Figure 5B).

We tested whether UBP12 and UBP13 can slow BES1 degradation during carbon starvation by measuring BES1 protein levels after a 5-day starvation period in Col-0, ubp12-2w ubp13-3, and UBP13OE seedlings. While we detected lower amounts of BES1 in all genotypes tested after carbon starvation, BES1 abundance was lower in ubp12-2w ubp13-3 seedlings and higher in UBP13OE seedlings compared with Col-0 (Figure 5C). In addition, we observed that UBP12 and UBP13 protein abundance decreases during starvation and increases during recovery (Figure 5D).

We also measured the survival rates of Col-0, BES1-RNAi, bes1-D, ubp12-2w ubp13-3, UBP12OE, UBP13OE, ubp12-2w ubp13-3 bes1-D, and UBP13OE/BES1-RNAi seedlings experiencing carbon starvation. Consistent with previous studies, BES1-RNAi showed higher survival rates and bes1-D showed lower survival rates under carbon starvation than Col-0 (Nolan et al., 2017; Wang et al., 2021a; Figure 5, E and F). Compared with Col-0, the survival rate of ubp12-2w ubp13-3 seedlings was higher (Figure 5, E and F) and that of UBP12OE and UBP13OE seedlings was lower (Supplemental Figure S12, A and B). The hypersensitivity of bes1-D and UBP13OE to carbon starvation was partially rescued in the ubp12-2w ubp13-3 bes1-D triple mutant and UBP13OE/BES1-RNAi lines, respectively (Supplemental Figure S12, A and B), indicating that UBP12 and UBP13 negatively regulate the response to carbon starvation.

We also measured the fresh weight of Col-0, ubp12-2w ubp13-3, UBP13OE, BES1-RNAi, and bes1-D seedlings during recovery after carbon starvation. The weights of all genotypes tested increased during recovery, with BES1-RNAi and ubp12-2w ubp13-3 faring less well than Col-0, while bes1-D and UBP13OE showed a greater increase in fresh weight than did Col-0 (Figure 5, G and H). Together, these results indicate that BES1 accumulates during the recovery period following carbon starvation to support recovery growth, with UBP12 and UBP13 positively regulating BES1 accumulation under these conditions.

Discussion

Plants have evolved multiple mechanisms to coordinate growth and stress responses, including global reprogramming of gene expression, RNA processing or sequestration, and posttranscriptional and posttranslational modifications (Crisp et al., 2016). Under stress conditions, plant growth is, in general, placed on hold. During recovery following stress, growth resumes. In contrast to the wealth of studies focusing on stress tolerance, there are relatively few reports on the mechanisms by which plants recover after stress, which is nevertheless crucial as it determines final yield. In this study, we identified two DUBs, UBP12 and UBP13, that directly regulate the stability of BES1 during recovery after carbon starvation.

UPB12 and UBP13 interact with BES1 and deubiquitinate BES1 to maintain its stability. The ubp12-2w ubp13-3 double mutant displayed lower BES1 accumulation and reduced BR-regulated growth during recovery after carbon starvation, while lines overexpressing UBP13 showed the opposite phenotypes. Furthermore, knockdown of BES1 and its homologs impaired the enhanced BR response in UBP13OE lines, and the ubp12-2w ubp13-3 double mutant largely blocked the enhanced BR responses of bes1-D. We also established that UBP12- and UBP13-mediated BES1 re-accumulation following starvation was critical for the rapid recovery of plant growth. Our results therefore establish a role for the UBP12/UBP13–BES1 signaling module in plant recovery after carbon starvation.

There are more than 1,400 E3 ligases but only 64 DUBs in Arabidopsis (Vierstra, 2009), suggesting that any DUB may be involved in multiple biological functions, each time serving as a hub to connect multiple signals. UBP12 and UBP13 participate in various plant developmental processes, such as plant immunity (Ewan et al., 2011), JA signaling (Jeong et al., 2017), root meristem (An et al., 2018), and leaf senescence (Park et al., 2019). UBP12 and UBP13 positively regulate JA signaling by stabilizing MYC2 (Jeong et al., 2017). Here, we showed that UBP12 and UBP13 regulate BES1 in a similar fashion to positively regulate BR responses. These findings indicate that UBP12 and UBP13 are key components of JA-mediated plant defenses and BR-mediated plant growth.

A living cell must perceive and respond to external nutritional information by quickly adapting its growth and metabolism. These responses determine when to grow, when to assimilate and store nutrients, and when to recycle reserves (Dobrenel et al., 2016). Energy availability is the greatest constraint on plant growth, and stresses such as drought, heat, carbon deficiency, and nitrogen deficiency lead to energy deprivation (Baena-Gonzalez and Sheen, 2008; Izumi et al., 2019). Under energy-constrained conditions, plants may experience transient or long-term carbon starvation, which interrupts plant growth (Baena-Gonzalez and Sheen, 2008). High energy availability increases the carbon costs during carbon starvation and is thus detrimental to plant stress survival, and nutrients are released after stress, which promotes faster recovery (Gessler et al., 2017).

BES1 and BZR1 bind to the promoters of ∼6,600 target genes, as determined by genome-wide chromatin immunoprecipitation studies (Sun et al., 2010; Yu et al., 2011). A large fraction (∼43%) of the genes misregulated under stress conditions are BES1/BZR1 targets (Nolan et al., 2017). It is therefore critical to study the dynamic changes in BES1 abundance under stress and growth conditions. In this study, we determined that ubp12-2w ubp13-3 is insensitive to carbon starvation, similar to BES1-RNAi lines. BES1 protein was degraded during carbon starvation, reflecting changes in UBP12 and UBP13 protein levels. UBP12 and UBP13 transcript and UBP12 and UBP13 protein levels drastically increased during recovery after carbon starvation, increasing the stability of BES1. By contrast, BES1 accumulation was much lower in the ubp12-2w ubp13-3 double mutant during recovery. Together, these observations illustrate how UBP12 and UBP13 play negative roles during carbon starvation responses and are induced during postcarbon starvation recovery to potentiate BES1 accumulation.

K48-linked ubiquitination is the most abundant in plants and serves as a strong proteasomal degron, while K63-linked chains function as general autophagic degrons (Kim et al., 2013; Ji and Kwon, 2017). We determined that UBP12- and UBP13-mediated deubiquitination of BES1 acts primarily through the cleavage of K48-linked ubiquitination. The results were consistent with results from MG132 treatment, which showed that UBP12 and UBP13 promote BES1 stability mainly by inhibiting proteasomal degradation.

The Ub receptor protein DSK2 plays an important role in the autophagy-mediated degradation of BES1 (Nolan et al., 2017). The E3 Ub ligases SINATs and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) target the active dephosphorylated form and phosphorylated form of BES1, respectively, to influence BR-regulated growth in a light-dependent manner (Kim et al., 2014; Yang et al., 2017). SINATs and BAF1 can be induced by starvation stress and target BES1 for degradation under starvation conditions (Nolan et al., 2017; Yang et al., 2017; Wang et al., 2021a). BES1 is therefore a central regulator of balancing growth and stress responses. BES1 abundance decreased during carbon starvation and increased during recovery, indicating that BES1 protein accumulation is necessary during recovery.

Compared with unchanged UBP12/UBP13 gene expression under carbon starvation but rapid upregulation during recovery, expression of BAF1 and SINAT2 was upregulated under carbon starvation but only slight downregulated during recovery. Dynamic changes of UBP12/UBP13 and E3 ligases during carbon starvation and recovery may result in the dynamic change of BES1, which reflects the survival strategies of plants, suppressing growth under energy limit conditions and maximizing growth under favorable conditions. The slight increase of BES1 transcript level and the slight decrease of BAF1/SINAT2 transcript level may not be sufficient for rapid accumulation of BES1 for plant recovery. However, the transcript and protein levels of UBP12/UBP13 were strongly induced during recovery to enhance the stability of BES1, which suggests that enhanced stability of BES1 during recovery is mainly mediated by UBP12/UBP13.

BZR1 protein degradation is promoted by BRs, which recruit cytosolic BZR1 to the nucleus for dephosphorylation (Wang et al., 2021b). A previous study and our study demonstrate that BES1 protein levels show little or no change in bri1-301 and BRI1OE seedlings (Supplemental Figure S5) (Gou et al., 2012). We propose that BR-activated function of BES1 and BZR1 is independent of their protein levels and is dependent on dephosphorylation, which we call a qualitative change. Our results reinforce the notion that environmental signals, such as light and carbon starvation, regulate BES1 function by adjusting its protein level rather than modifying its phosphorylation, which we call a quantitative change.

BES1 protein accumulation enhances the strength of the BR response, such as in bes1-D, UBP12OE, and UBP13OE, in which both phosphorylated and unphosphorylated forms of BES1 accumulate to high levels, resulting in a constitutive BR response phenotype and greater sensitivity to BL treatment when compared with Col-0 (Figure 4 and Supplemental Figure S7). The ubp12-2w ubp13-3 double mutant exhibited a typical BR-deficient phenotype, including shorter hypocotyls and petioles, a smaller ratio between leaf length and leaf width, and reduced BR responses (Figure 4). The ubp12-2w ubp13-3 double mutant phenotypes were similar to those of other BR-deficient mutants, especially BES1-RNAi, though not as strong as those of bri1-701, bin2-1, or the hextuple mutant bzr-h lacking function of BES1, BZR1, and their homologs (Chen et al., 2019). Since UBP12 and UBP13 interacted with BES1 and its five homologs, UBP12 and UBP13 likely also regulate the stability of BES1 homologs.

In summary, this study revealed a dynamic regulatory mechanism for BES1 during carbon starvation and recovery after carbon starvation (Figure 6). Under stress conditions, E3 Ub ligases such as SINATs and BAF1 are activated and BES1 is ubiquitinated for degradation by the proteasome and DSK2-dependent autophagy, which slows plant growth. In the growth or recovery stage, accumulation of UBP12 and UBP13 leads to accumulation of BES1 to enhance plant growth and/or stress recovery.

Figure 6.

Figure 6

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A proposed model illustrating how seedlings regulate BES1 stability via UBP12 and UBP13 in response to carbon starvation and recovery. During carbon starvation stress, the expression of SINAT genes is induced and BES1 is targeted by SINAT E3 Ub ligases to be degraded by ubiquitination. The Ub receptor DSK2 recruits BES1 for degradation by autophagy. Degradation of BES1 leads to growth inhibition. During recovery after carbon starvation, the expression of UBP12 and UBP13 is induced, and BES1 is targeted by UBP12 and UBP13 for deubiquitination, which leads to BES1 accumulation and growth promotion.

Materials and methods

Material and plant growth conditions

The wild-type A. thaliana plants used in this study were the Col-0. The mutants ubp12-1 (GABI_244E11), ubp12-2w (GABI_742C10), ubp13-1 (SALK_128312), and ubp13-3 (SALK_132368) were obtained from Arabidopsis Biological Resource Center and these mutants have been described by Cui et al. (2013). The BR-signaling related mutants and transgenic plants have been used and described in our previous study (Zhang et al., 2021), the bes1-D mutant in Col-0 background has been described previously (Gonzalez-Garcia et al., 2011). Arabidopsis plants used in the study were sterilized using 70% (v/v) ethanol and 0.1% (v/v) Triton X-100 and plated on half-strength MS medium and vernalized at 4°C for 2 days in the dark, and were incubated for 6 h in light (150 µmol/m−2 s−1, 16W T8-type LED) at 22°C for germination and then grown under a long-day condition (22°C, light 16 h/dark 8 h). Nicotiana benthamiana plants were grown on soil under a long-day condition (150 µmol m−2 s−1, 25°C, light 16 h/dark 8 h). Four-week-old soil-grown N. benthamiana plants were used in all experiments. The primers used for genotyping are listed in Supplemental Table S1.

Y2H assays

The plasmid constructions in this study used a Seamless Cloning and Assembly Kit (Vazyme, Nanjing, China). For Y2H assays, full-length or fragments of UBP12 and UBP13 were cloned into pGADT7 (Clontech, Mountain View, California, USA) as prey. Full-length or fragments of BES1 were cloned into pGBKT7 (Clontech) as bait. Each pair of bait and prey constructs was transformed into yeast strain AH109 and grown on Double DO supplement (-Leu/-Trp, -LW) for 3 days, and shifted onto Quadruple DO supplement (-Leu/-Trp/-Ade/-His, -LWHA) to test possible interaction. The procedure conducted is described in detail in the Make Your Own ‘Mate & Plate’ Library System User Manual and Y2H System User Manual (Clontech). The primers used for plasmid construction are listed in Supplemental Table S1.

Plant transformation

Full-length UBP12 and UBP13 were cloned into pCM1307 plasmid to construct 35S:HA-FLAG-UBP12 and 35S:HA-FLAG-UBP13. These all constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Col-0 using the floral dip method as described previously (Zhang et al., 2006). The primers used for plasmid construction are listed in Supplemental Table S1.

In vitro pull-down assays

MBP-BES1 was used in our previous study (Zhang et al., 2014) and purified using amylose resin (NEB, Ipswich, MA, USA). UBP12 and UBP13 were cloned into the pET-28a (+) vector (His tag) and purified using NiNTA agarose (Invitrogen, Carlsbad, California, USA). Purified MBP, MBP-BES1 were incubated with equal amounts of His-UBP12 or His-UBP13 beads in His pull-down binding buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.2% Triton) at 4°C for 2 h. After washing 7 times with His pull-down washing buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% Triton), the beads were collected, boiled in 50 μL 2 × SDS loading buffer for 8 min at 100°C, and the sample examined by immunoblotting using anti-His (1:5,000 dilution, ABclonal, China, AE003) and anti-MBP (1:5,000 dilution, ABclonal, AE016) antibodies. The antibodies source details are listed in Supplemental Table S2. The primers used for plasmid construction are listed in Supplemental Table S1.

BiFC assays and quantification of protein fluorescent signal

Full-length UBP12 and UBP13 were cloned into pXY103 vector carrying N-terminal of YFP and BES1 was cloned into pXY104 vector carrying C-terminal YFP (Yu et al., 2008). Agrobacterium strain GV3101 was transformed with the above vector or control vector. Agrobacterium cultures were grown overnight in LB medium containing 200 mM acetosyringone, washed with infiltration medium (10 mM MgCl2, 10 mM MES, pH 5.7, 200 mM acetosyringone) and resuspended to an OD600 of 1.0. Agrobacterium carrying nYFP and cYFP constructs were mixed in equal ratios, and the Agrobacterium mixtures were infiltrated into the young leaves of N. benthamiana. After 36–48 h, YFP was excited with a 514-nm laser line and detected from 530 to 560 nm. YFP signals were detected using a fluorescence microscope (Leica) (Yu et al., 2008). Oligo primers used for plasmid construction are listed in Supplemental Table S1. Quantification of the fluorescent protein signal involved using ImageJ (http://rsb.info.nih.gov/ij). To measure the nuclear and cytoplasmic signals, small areas were drawn, and measurements of integrated densities were taken from representative areas within the nucleus, cytoplasm, and background (central vacuole) of each cell. Each sample of at least 10 cells was measured three times (Zhang et al., 2012).

Co-IP assays

Ten-day-old UBP12pro:HA-FLAG-UBP12/ubp12-1, UBP12pro:HA-FLAG/ubp12-1, UBP13pro:HA-FLAG-UBP13/ubp13-3, and UBP13pro:HA-FLAG/ubp13-3 transgenic seedlings were used for Co-IP assays. The total proteins were extracted from different plants and then incubated with HA agarose beads (Sigma-Aldrich, St. Louis, Missouri, USA) in IP buffer (10 mM Tris–HCl pH 7.5, 0.5% Nonidet P-40, 2-mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1% plant protease inhibitor cocktail; Amresco). The beads were collected, washed at least five times with IP buffer. Then examined the interaction by immunoblotting using anti-HA (1:5,000 dilution, Sigma-Aldrich, H6908) and anti-BES1 (1:5,000 dilution) antibodies. The anti-BES1 antibody was got from Yanhai Yin’s lab (Yu et al., 2011; Wang et al., 2021a).

In vitro ubiquitination and deubiquitination assays

In vitro ubiquitination assays were performed as described previously (Yang et al., 2018) with some modifications. In brief, an E2-Ub Conjugation Kit (Abcam, Cambridge, UK) was used for in vitro ubiquitination, ubiquitination reaction mixtures (20 μL) contained 1 ×  Ub buffer, 100 mM E1 (wheat E1), 2.5 mM E2 (human E2 UbcH5B), 500 ng GST-SINAT2, 500 ng MBP-BES1, 5 mM Mg-ATP, 2.5 mM biotinylated Ub, and 20 mM ZnCl2. After incubated at 30°C for 6 h, mixtures were purified using amylose resin, purified polyubiquitinated MBP-BES1 was added to a deubiquitination buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, and 2 mM DTT) with His-UBP12, His-UBP13, His-UBP12C208S, and His-UBP13C207S incubated at 30°C. Samples were taken at 4 h, and then examined the ubiquitination by immunoblotting using anti-MBP and anti-Ub (1:5,000 dilution, Abcam, ab7254) antibodies.

In vivo ubiquitination assays

In vivo ubiquitination assays were performed as described previously (Qi et al., 2017) with some modifications. Briefly, the BES1-GFP and MYC-SINAT2 plasmids were transformed into Arabidopsis protoplasts which were isolated from Col-0, ubp12-2w ubp13-3, or UBP13OE. After 16-h expression, added 50 μM MG132 continued to incubate for 2 h. Proteins were extracted and incubated with GFP agarose beads (Chromotek) in IP buffer, then detected the ubiquitination by immunoblotting using anti-GFP (1:5,000 dilution, Transgen, China, HT801Transgen), anti-Ub, anti-K48 Ub (1:5,000 dilution, Abcam, ab140601), and anti-K63 Ub (1:5,000 dilution, Abcam, ab179434) antibodies.

Cell-free protein degradation assays

Cell-free protein degradation assays were described before (Yang et al., 2017). In brief, total proteins were extracted with degradation buffer (25 mM Tris–HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT, and 10 mM ATP), the supernatant was collected and Bio-Rad protein assay was used for determining protein concentration. About 100 ng of recombinant MBP-BES1 proteins were added in 100 μL plant extracts with/without 50 μM MG132 (Sigma-Aldrich) incubated at 30°C for the indicated times, and then detected the protein changes by immunoblotting using anti-MBP and anti-ACTIN (1:10,000 dilution, Cwbio, China, cw0264A) antibodies.

Protein degradation assays and inhibitor treatments

Ten-day-old Col-0, ubp12-2w ubp13-3 and UBP13OE seedlings were transferred to liquid half-strength MS medium with DMSO, 200 nM BL (Wako), 1 μM BRZ (Santa Cruz Biotechnology, Dallas, Texas, USA), 2 μM PCZ (Aladdin), or 50 μM bikinin (Sigma-Aldrich) for 2 h to accumulate different forms of BES1. For experiments using inhibitors, pretreated plants were transferred to liquid half-strength MS medium containing 1 mM CHX (Sigma-Aldrich), 1 mM CHX + 50 μM MG132 (Sigma-Aldrich), 1 mM CHX + 50 μM E64d (Sigma-Aldrich), or 1 mM CHX + 1 μM ConA (Sigma-Aldrich) for indicated times, and then detected the protein changes by immunoblotting using anti-BES1 and anti-ACTIN antibodies. The inhibitor source details are listed in Supplemental Table S2.

Hypocotyl length measurements

For hypocotyl length analysis, seedlings were grown in half-strength MS medium with DMSO, 20 nM BL, 1 μM PCZ, or 10 μM bikinin at 22°C under long-day conditions for 7 days. Hypocotyl lengths were measured using ImageJ software.

RNA extraction and RT-qPCR

Total RNA extracted by Total RNA Extraction Kit (Solarbio, Beijing, China). For reverse transcription-quantitative PCR (RT-qPCR), cDNA was prepared using PrimeScript RT reagent Kit (Takara, Kyoto, Japan). Gene expression was performed using SYBR Green PCR Master Mix (Invitrogen). The CFX Connect Real-Time System (Bio-Rad, Berkeley, California, USA) was used for RT-qPCR analysis. For each sample, three replicates were performed and the expression was normalized to those of ACTIN2. The primers used for RT-qPCR were listed in Supplemental Table S1.

Fixed-carbon starvation

Fixed-carbon starvation assays in seedlings were performed as described previously (Nolan et al., 2017; Wang et al., 2020, 2021a). Four-day-old seedlings from each genotype were transferred to half-strength MS plates without sucrose and grown in darkness for 5- or 8-day starvation, then plates were put back into the light for an 8-day-recovery period.

Statistical analysis

The experimental data were statistically analyzed using three or more averages. The significance of the differences between groups was determined by a two-tailed Student’s t test, *P < 0.05, ** P < 0.01. For multiple comparisons, using one-way or two-way analysis of variance (ANOVA), considered significant when P < 0.05. Statistical data are provided in Supplemental Data Sets S1 and S2.

Accession numbers

The accession numbers of the main genes discussed in this article are: AT1G19350 (BES1), At2g32780 (UBP1), At1g04860 (UBP2), At4g39910 (UBP3), At2g22310 (UBP4), At2g40930 (UBP5), At1g51710 (UBP6), At3g21280 (UBP7), At5g22030 (UBP8), At4g10570 (UBP9), At4g10590 (UBP10), At1g32850 (UBP11), At5g06600 (UBP12), At3g11910 (UBP13), At3g20630 (UBP14), At1g17110 (UBP15), At4g24560 (UBP16), At5g65450 (UBP17), At4g31670 (UBP18), At2g24640 (UBP19), At4g17895 (UBP20), At5g46740 (UBP21), At5g10790 (UBP22), At5g57990 (UBP23), At4g30890 (UBP24), At3g14400 (UBP25), At3g49600 (UBP26), At4g39370 (UBP27), AT3G50660 (DWF4), and At4g38850 (SAUR-AC1).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Interaction tests between BES1 and UBPs.

Supplemental Figure S2. Interaction tests between UBP12 or UBP13 and BES1 and its homologs.

Supplemental Figure S3. Mapping the interaction interface between UBP12 or UBP13 and BES1.

Supplemental Figure S4. Characterization of transgenic lines overexpressing UBP12 or UBP13.

Supplemental Figure S5. UBP12 and UBP13 directly regulate BES1.

Supplemental Figure S6. BR responses of different genotypes during carbon starvation.

Supplemental Figure S7. Other typical BR-related phenotypes of ubp12-2w ubp13-3, UBP12OE, and UBP13OE plants.

Supplemental Figure S8. Seedlings overexpressing UBP12 or UBP13 show BR-enhanced and BR-sensitive phenotypes.

Supplemental Figure S9. The deubiquitinating activity of UBP12 and UBP13 is essential for BR responses.

Supplemental Figure S10. Neither UBP12 and UBP13 expression nor UBP12 and UBP13 protein levels are regulated by BR.

Supplemental Figure S11. Expression levels of BAF1 and SINAT2 during carbon starvation and recovery.

Supplemental Figure S12. Carbon starvation survival of different genotypes.

Supplemental Table S1. The primers used in this study.

Supplemental Table S2 . Key resources.

Supplemental Data Set S1. Data for all statistical analyses performed in this study.

Supplemental Data Set S2. ANOVA results for this study.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32070213); Sichuan Science and Technology Program (2022JDRC0032); Institutional Research Fund of Sichuan University (2020SCUNL212); and Fundamental Research Funds for the Central Universities (SCU2020D003).

Conflict of interest statement. None declared.

Supplementary Material

koac245_Supplementary_Data
Click here for additional data file. (6.2MB, zip)

Contributor Information

Jiawei Xiong, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Fabin Yang, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Xiuhong Yao, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Yuqing Zhao, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Yu Wen, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Honghui Lin, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

Hongqing Guo, Department of Genetics, Development, and Cell Biology, Plant Sciences Institute, Iowa State University, Ames, Iowa 50011, USA.

Yanhai Yin, Department of Genetics, Development, and Cell Biology, Plant Sciences Institute, Iowa State University, Ames, Iowa 50011, USA.

Dawei Zhang, Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China.

 

D.Z. designed the research. J.X. and D.Z. performed most of the experiments with the assistance of F.Y., X.Y., Y.Z., and Y. W. J.X., H.L., H.G., Y.Y., and D.Z. analyzed the data. J.X. and D.Z. wrote the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) is: Dawei Zhang (zhdawei@scu.edu.cn).

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