Ubiquitin-specific proteases UBP12 and UBP13 act in circadian clock and photoperiodic flowering regulation in Arabidopsis

Abstract

Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.

Figure 1.

UBP12 and UBP13 have deubiquitination activities. A, Schematic diagram of the UBP12 and UBP13 protein structures. The MATH domain, conserved Cys-box, and His-box are shown as squares or rectangles. The star indicates the Cys residue required for enzymatic activity. B, UBP12 and UBP13 are active DUBs. In vivo cleavage of hexameric polyubiquitin (UBQ10, left) and ubiquitin extension protein (UBQ1, right). UBP12, UBP13, and their mutants UBP12C208S and UBP13C207S were coexpressed with the substrates UBQ1 and UBQ10 in E. coli; the cleavage products were detected by immunoblot analyses with anti-ubiquitin antibodies. The positions of the substrates and cleaved products are indicated by arrows.

Figure 2.

UBP12 and UBP13 have similar expression pattern and protein localization. A to D, GUS staining of dark-grown seedlings (A), 4-d-old seedlings under LD condition (B), 14-d-old seedlings under LD condition (C), and inflorescences (D) of the transformants containing UBP12pro:GUS. E to H, GUS staining of dark-grown seedlings (E), 4-d-old seedlings under LD condition (F), 14-d-old seedlings under LD condition (G), and inflorescences (H) of the transformants containing UBP13pro:GUS. I to L, UBP12 fused to GFP (K) and UBP13 fused to CFP (L) localize to the nuclei and cytoplasm; 35Spro:GFP (I) and 35Spro:CFP (J) were used as control. Bar = 200 µm. UBP12/UBP13 protein was detected in both cytoplasmic and nuclear fractions. Histone H3 and phosphoenolpyruvate carboxylase (PEPC) were used as control for nuclear or cytoplasmic fraction, respectively.

Figure 3.

Mutations of UBP12 and UBP13 affect plant development and flowering time. A, Schematic diagrams of the UBP12 and UBP13 gene structures, with the T-DNA insertion sites indicated. Black boxes indicate exons, white boxes indicate untranslated regions, and lines indicate introns. B, Northern blots showing the expression levels of UBP12 and UBP13 in the T-DNA insertion mutants (top). Ribosomal RNA stained with methylene blue was used as loading control (bottom). C to L, Phenotypes of 24-d-old seedlings of the wild type (C), ubp12-1 (D), ubp12-2w (E), ubp13-1 (F), ubp13-2 (G), ubp13-3 (H), ubp12-2w ubp13-1 (I), ubp12-2w ubp13-2 (J), ubp12-2w ubp13-3 (K), and ubp12-1 ubp13-3 (L). Bar = 1 cm. M to R, Phenotypes of the wild type (M), ubp12-2w (N), ubp12-2w ubp13-1 (O), ubp12-2w ubp13-2 (P), ubp12-2w ubp13-3 (Q), and ubp12-1 ubp13-3 (R) after bolting. Bar = 1 cm. S and T, Statistical analysis of leaf numbers of ubp12-2w, ubp13-3, ubp12-2w ubp13-3 double mutants, and ubp12-1 under LD (S) and SD (T) conditions compared with wild-type plants. Values are means ± sd of at least 20 plants. WT, Wild type.

Figure 4.

UBP12 and UBP13 regulate photoperiodic flowering. A, Expression patterns and transcript levels of CO (left) and FT (right) under LD condition in the wild type, ubp12-2w, ubp13-3, and ubp12-2w ubp13-3 double mutant. Values are means ± sd of three independent experiments. B, Expression patterns and transcript levels of CO (left) and FT (right) under SD condition in the wild type, ubp12-2w, ubp13-3, and ubp12-2w ubp13-3 double mutant. Values are means ± sd of three independent experiments. C, Statistical analysis of leaf numbers of ubp12-2w co and ubp12-2w gi-4 double mutants under LD condition. Values are means ± sd of at least 20 plants. D, Statistical analysis of leaf numbers of ubp12-2w svp32 and ubp12-2w ubp13-3 svp32 plants under LD condition. Values are means ± sd of at least 20 plants. WT, Wild type.

Figure 5.

Mutation of UBP12 and UBP13 shortens the circadian period under LL. The expression patterns of LHY (A) and TOC1 (B) under LL condition were analyzed by qRT-PCR. Wild-type, ubp12-2w, and ubp12-2w ubp13-3 plants were grown for 10 d in 12-h-light/12-h-dark cycles before released to LL and sampled every 4 h from ZT0. Circadian rhythm of pCCA1:LUC was detected in transgenic seedlings in ubp12-2w (n = 23; C) or ubp12-2w ubp13-3 (n = 7; D). The pCCA1:LUC transgenic plants in ubp12-2w and ubp12-2w ubp13-3 were grown for 7 or 15 d, respectively, in 12-h-light/12-h-dark cycles and transferred to LL at ZT 0. Relative amplitude error is a measure of the strength of the oscillation. Statistical analysis of period length showed that in each case, the periods of ubp12-2w and ubp12-2w ubp13-3 were shorter than that of the wild type (P < 0.001). UBP12 and UBP13 expression levels were tested from ZT 24 to 48 by qRT-PCR in wild-type plants (E). Values are means ± sd of three independent experiments. The protein level of UBP12/UBP13 under LL was tested by immunoblot using UBP12/UBP13 antibodies from ZT 24 to 48 (F). The arrow indicates the UBP protein position. The asterisk indicates the nonspecific band. H3 was used as the loading control. WT, Wild type.