The Plant Journal (2011) 65, 194–205
doi: 10.1111/j.1365-313X.2010.04416.x
OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment Jong-Jin Park1,2, Jakyung Yi1,2, Jinmi Yoon1,2, Lae-Hyeon Cho1,2, Jin Ping1,2, Hee Joong Jeong1,2, Seok Keun Cho3, Woo Taek Kim3 and Gynheung An1,2,4,* 1 Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea, 2 Crop Biotech Institute, Kyung Hee University, Youngin 446-701, Korea, 3 Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea, and 4 Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Youngin 446-701, Korea Received 2 September 2010; revised 30 September 2010; accepted 18 October 2010; published online 3 December 2010. * For correspondence (fax +82 31 204 3178; e-mail
[email protected]). 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, is Gynheung An (
[email protected]).
SUMMARY The plant U-box (PUB) protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the class-II PUB family. Self-ubiquitination assays showed that bacterially expressed MBPOsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling-lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of H2O2 and oxidized proteins were higher in the knock-out mutant compared with the wild type. OsPUB15 transcript levels were increased upon H2O2, salt and drought stresses; plants overexpressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces reactive oxygen species (ROS) stress and cell death. Keywords: E3 ligase, rice, ROS, seedling lethal, U-box.
INTRODUCTION Reactive oxygen species (ROS) are radical derivatives of molecular oxygen: they include hydrogen peroxide (H2O2), superoxide radical (O2)·), hydroperoxyl radical (HO2·) and hydroxyl radical (·OH), which are produced either by the ionizing radiation of water or through inefficient reduction of oxygen during energy generation (Puntarulo and Cederbaum, 1988). ROS function as signaling molecules that control various cellular processes, including pathogen defense, programmed cell death and stomatal behavior (McAinsh et al., 1996; Maxwell et al., 2002; Tiwari et al., 2002). ROS levels are heightened by various stresses, e.g. increased light and salt, drought, cold, heat, pathogen infection and mechanical damage (Apel and Hirt, 2004). High quantities cause oxidative damage to proteins, DNA and lipids, which eventually leads to cell death (Mittler, 2002). Thus, an excess of ROS must be rapidly removed by various 194
enzymatic and non-enzymatic systems, so that an equilibrium is tightly controlled between their production and scavenging. In addition, the ubiquitin (Ub) proteasome system (UPS) functions during the stress response (Cho et al., 2008). Ubiquitination is accomplished by three enzymes: E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; and E3, Ub-ligase enzyme. Ubiquitin is activated by E1 in an ATP-dependent manner, and this activated Ub is conjugated to a target protein by E2. E3 then mediates covalent conjugation of the target protein to Ub for degradation (Moon et al., 2004; Smalle and Vierstra, 2004; Dreher and Callis, 2007). The ubiquitination system is present in all eukaryotes and is implicated in many cellular processes, such as differentiation, cell division and hormone responses (Zeng et al., 2008; Yee and Goring, 2009). ª 2010 The Authors The Plant Journal ª 2010 Blackwell Publishing Ltd
Abnormal germination in rice seed 195 Plant U-box (PUB) E3 proteins contain a conserved region that resembles the RING-finger domain, except that the zincchelating cystein and histidine residues are absent (Aravind and Koonin, 2000; Ohi et al., 2003). There are 77 members in rice and 63 in Arabidopsis (Zeng et al., 2008). The Spotted leaf 11 (SPL11) is a rice PUB that represses HR-associated cell death and pathogenic defense (Zeng et al., 2004). BnARC1, a PUB member in Brassica, interacts with S-locus kinase, which regulates self-incompatibility (Gu et al., 1998; Stone et al., 1999, 2003). AtPUB17 is an Arabidopsis homolog of BnARC1. Atpub17 knock-out plants display decreased resistance to avirulent Pseudomonas syringae pv. tomato (Yang et al., 2006). PHOR1 functions as a positive regulator during GA signaling: its antisense suppression produces semi-dwarf plants with higher endogenous GA levels and decreased sensitivity to exogenous GA, whereas PHOR1overexpressing lines show greater GA sensitivity (Amador et al., 2001). Under low light, SAUL1 suppresses premature senescence of young seedlings and enhances ABA biosynthesis (Raab et al., 2009). AtPUB22 and AtPUB23 have been proposed as negative regulators of abiotic stresses: the loss of their expression confers drought tolerance, whereas their overexpression results in hypersensitivity to salt and drought (Cho et al., 2008). Finally, the pub22 pub23 pub24 triple knock-out mutants accumulate higher levels of ROS, causing cell death (Trujillo et al., 2008). Here, we report that ospub15 knock-out plants are defective in seedling growth, whereas OsPUB15 overexpressers are tolerant to high salt levels. We propose that OsPUB15 is a negative regulator of cell death and plant responses to abiotic stresses. RESULTS Isolation of a rootless mutant We identified a rootless mutant line, 4A-02107, from the T-DNA insertional population in Oryza sativa ssp. japonica cv. Dongjin rice (Jeon et al., 2000; Jeong et al., 2002; An et al., 2003; Lee et al., 2003; Ryu et al., 2004). Mutant seeds developed normal shoots, but the seedlings did not produce roots (Figure 1a). Longitudinal sections from 3-day-old seedlings showed that radicle growth was hindered, and those tissues eventually turned brown (Figure 1b,c). Although a coleoptile and three leaves appeared, their growth was significantly retarded compared with their segregating wild-type (WT) siblings (Figure 1d). Mutant shoots carried hairs, lamina joints and the fourth leaf primordium, which turned yellow within 2 weeks of germination (Figures 1e and S1). To observe radicle growth in detail, imbibed seeds were cross-sectioned. In the WT, scutellum cells swelled after imbibition, creating an empty space between the radicle and scutellum (Figure 1g). That space continuously enlarged during the first 18 h (Figure 1g,h,o). Radicles were approximately
0.5 mm long at 6 h after imbibition (Figure 1n). They continued to elongate to 1.0 mm, eventually filling the empty space at 24 h (Figure 1i,o). During the first 18 h, radicles of the mutant elongated normally, but scutellum had less swelling, resulting in a smaller space between radicle and scutellum (Figure 1j–l,n,o). At 24 h, the radicles stopped growing because of a lack of space (Figure 1m,o). Magnified images of the seeds at 24 h showed that root-cap cells from the WT were arranged radially, whereas those from the mutant did not elongate and were aligned in straight lines (Figure S2). T-DNA was inserted into OsPUB15 Sequence analysis of the T-DNA flanking region in the mutant revealed that T-DNA was inserted into LOC_Os08g01900 (http://tigrblast.tigr.org/euk-blast) on chromosome 8 (Jeong et al., 2002; An et al., 2003). The gene comprises five exons and four introns (Figure 2a). Its full-length cDNA was identified as AK106557 and AK102080 in the Knowledge-based Oryza Molecular Biological Encyclopedia (KOME) (http:// cdna01.dna.affrc.go.jp/cDNA). T-DNA was inserted 2419 bp downstream from the ATG start codon, in the fifth exon of the gene. The predicted protein encoded by the gene is OsPUB15, a member of the class-II subfamily of U-box proteins (Zeng et al., 2008). Among the members of that subfamily in rice, OsPUB15 is most closely related to OsPUB16 (Figure S3). Functional-domain analysis with PFAM 7.0 (http://www.sanger.ac.uk/Software/Pfam) showed that the region between the 232nd and 295th amino acid residues shares high similarity to the consensus U-box domain sequence, and that the region between the 559th and 812th residues is highly homologous to the armadillo repeat motif (ARM) found in b-catenin of Drosophila (Riggleman et al., 1989). OsPUB15 transcripts were ubiquitous from the young seedling stage through maturity, although levels were higher in shoots than roots during early development (Figure 2b). During seed imbibition, transcripts were rapidly increased, reaching the maximum at 2 h before declining to a steady state at 12 h (Figure 2c). OsPUB15 complementation rescued the mutant phenotypes To examine whether the abnormal seedling phenotypes observed from the T-DNA insertional line resulted from a mutation in OsPUB15, we made the antisense construct using the 381-bp region of the 3¢ untranslated region (3¢-UTR) that started at 17 bp upstream of the stop codon (Figure 3a). The fragment was placed between the maize ubiquitin (ubi) promoter and nopaline synthase (nos) terminator, and the molecule was transferred to embryonic calli via Agrobacterium-mediated transformation (An et al., 1985; Kim et al., 2009). Thirteen independently transformed plants were regenerated and expression was measured for introduced antisense transcripts and those of the
ª 2010 The Authors The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 194–205
196 Jong-Jin Park et al.
(b)
(a)
(e)
(d)
(c)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
Figure 1. Phenotypes of ospub15. (a) Wild-type (left) and ospub15 (right) grown in a closed container for 3 days after germination (DAG); Co, coleoptile; SR, seminal root. (b, c) Hand-sectioned germinating seeds of wild type (WT) (b) and ospub15 (c) at 72 h after imbibition; Co, coleoptile; SR, seminal root; Ra, radicle. Scale bars: 50 lm. (d) Phenotypes of WT (left) and ospub15 (right) at 5 DAG. (e) Phenotype of ospub15 at 14 DAG. (f–m) Safranin O-stained longitudinal sections of radicles from WT (j–m) and ospub15 at 6 (f, j), 12 (g, k), 18 (h, l) and 24 h (i, m); Co, cortex cell; Ep, epidermal cell; Ra, radicle; RC, root cap; VB, vascular bundle. Arrowheads indicate epidermal cells; arrows indicate spaces between radicle and scutellum. Scale bars: 250 lm. (n) Radicle length. (o) Area between radicle and scutellum.
endogenous OsPUB15 (Figure 3b). We selected transgenic plants #4 and #12, in which levels for OsPUB15 were severely reduced because of strong expression of the antisense transcript (Figure 3c). We also chose transgenic plant
#5, in which expression was not significantly reduced. Whereas plant #5 grew almost normally, plants #4 and #12 were semi-dwarf (Figure 3d). Seedlings of the latter two showed retarded development of roots and shoots
ª 2010 The Authors The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2011), 65, 194–205
Abnormal germination in rice seed 197
(a)
B T-DNA
F2
F1
F3
5’
3’ ATG
U-box
ARM repeat
R2 R1
R3
(b)
(OsPUB15/Ubq)
Relative expression
0.09
0.06
OsPUB15 is a member of the U-box E3 ligases
0.03
0
R
Sh 7d
MF
ML
P
70 d
> 90 d
S 5 S 10 > 120 d
(c)
(OsPUB15/Ubq)
0.09
Relative expression
tation of the mutant phenotypes with OsPUB15. Among the transformants in the WT background, those from one group weakly expressed the introduced OsPUB15 (#11 and #16), whereas the others (#2, #6, #27 and #35) had high expression (Figure S4d). However, both groups had normal and similar patterns of growth (Figure S4c). Therefore, it appears that overexpression of OsPUB15 does not affect plant development under standard growing conditions.
0.06
0.03
0 0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h) Figure 2. Expression patterns of OsPUB15. (a) Schematic diagram of OsPUB15 and insertional position of T-DNA. Closed boxes represent five exons; connecting black lines represent four introns. Dark closed boxes indicate conserved domains. Arrowheads show primers used for genotyping progeny and measuring expression levels. (b) OsPUB15 expression pattern in various organs: MF, mature flower; ML, mature leaf at 70 DAG; P, panicle
