The Plant Journal (2009) 60, 666–678
doi: 10.1111/j.1365-313X.2009.03992.x
The Arabidopsis SUMO E3 ligase AtMMS21, a homologue of NSE2/MMS21, regulates cell proliferation in the root Lixia Huang1,†, Songguang Yang1,†, Shengchun Zhang1, Ming Liu1, Jianbin Lai2, Yanli Qi1, Songfeng Shi1, Jinxiang Wang3, Yaqin Wang4, Qi Xie2 and Chengwei Yang1,5,* 1 Guangdong Key Lab of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, China, 2 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China, 3 College of Natural Resources and Environment, Root Biology Center, South China Agricultural University, Guangzhou 510642, China, 4 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China, and 5 MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510631, China Received 29 March 2009; revised 20 July 2009; accepted 24 July 2009; published online 1 September 2009. *For correspondence (fax +86 20 85210855; e-mail
[email protected]). † These authors contributed equally to this work.
SUMMARY hMMS21 is a SUMO E3 ligase required for the prevention of DNA damage-induced apoptosis, and acts by facilitating DNA repair in human cells. The Arabidopsis genome contains a putative MMS21 homologue capable of interacting with the SUMO E2 conjugating enzyme AtSCE1a, as indicated by a yeast two-hybrid screen and bimolecular fluorescence complementation experiments. In vitro and in vivo data demonstrated that AtMMS21 was a SUMO E3 ligase. We identified the Arabidopsis AtMMS21 null T-DNA insertion mutant mms21-1, which had a short-root phenotype, and affected cell proliferation in the apical root meristem, as indicated by impaired expression of the cell division marker CYCB1:GUS in mms21-1 roots. The mms21-1 roots had reduced responses to exogenous cytokinins, and decreased expression of the cytokinin-induced genes ARR3, ARR4, ARR5 and ARR7, compared with the wild type. Thus, our findings suggest that the AtMMS21 gene is involved in root development via cell-cycle regulation and cytokinin signalling. Keywords: Arabidopsis thaliana, AtMMS21, cytokinin, root development, cell cycle.
INTRODUCTION All organisms use a variety of chemical modifiers for posttranslational control of proteins that affect development, growth and homoeostasis. Ubiquitin (Ub) is one such polypeptide that was first described to attach covalently to other proteins upon completion of their synthesis. Conjugation of Ub (termed ubiquitination) has a well-established role in earmarking proteins for degradation by the 26S proteosome. In addition to Ub, a panoply of proteins termed ‘ubiquitin-like modifiers’ (UBLs) that function in a manner analogous to Ub have been discovered in eukaryotes; this group includes proteins like SUMO (small ubiquitin-like modifier), Rub1 (also named Nedd8), Apg8 and Apg12. SUMO conjugation to protein substrates (sumoylation) is a reversible post-translational modification that is regulated by environmental stimuli in animals and yeast (Johnson, 666
2004). Similar to ubiquitination, sumoylation of substrates is catalysed by a cascade of enzymes: the E1 SUMO-activating enzyme (AOS1-UBA2), the E2 conjugating enzyme (UBC9), and the E3 SUMO ligases (Hay, 2005). Sumoylation of target proteins in yeast and metazoans has been implicated in the regulation of innate immunity, cell-cycle progression and mitosis, DNA repair, chromatin stability, cell division, nucleo-cytoplasmic trafficking, subnuclear targeting, ubiquitination antagonism, and transcriptional regulation (Johnson, 2004; Gill, 2005). PIAS/Siz proteins are SUMO E3 ligases that mediate the final step of SUMO conjugation (Hay, 2005). Transcription factors are direct targets of SUMO conjugation mediated by PIAS/Siz proteins (Gill, 2005). The yeast SUMO E3 ligases Siz1 and Siz2 facilitate cell division at low temperatures (Johnson and Gupta, 2001). The yeast ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd
AtMMS21 regulates cell proliferation 667 MMS21 and its human homologue of MMS21 contain an SP-RING domain that is related to the PIAS family of SUMO E3 ligases that autosumoylate, and are required for the prevention of DNA damage-induced apoptosis by facilitating DNA repair (Andrews et al., 2005; Potts and Yu, 2005). SUMO conjugation/deconjugation in plants has been demonstrated in responses to abiotic and biotic stresses. AtSUMO1/2-overexpression increased sumoylation levels, attenuated ABA-mediated growth inhibition, and induced the expression of ABA-responsive genes RD29A and AtPLC1 (Lois et al., 2003). AtSIZ1 is a SUMO E3 ligase that is involved in phosphate starvation signalling by facilitating the sumoylation of PHR1, regulates the freezing response by stabilizing ICE1 activity, and also plays important roles in ABA signalling, and in heat and drought stress responses (Miura et al., 2005, 2009; Yoo et al., 2006; Catala et al., 2007). Two SUMO proteases, OVERLY TOLERANT TO SALT1 (OTS1) and OTS2, act redundantly to regulate salt stress responses in Arabidopsis thaliana (Conti et al., 2008). However, SUMO conjugation is also essential for normal development, as demonstrated by the embryonic-lethal phenotype associated with mutations in either SAE1/2 encoding activating (E1) or SCE1 encoding conjugating (E2) enzymes, or in both the SUMO1 and SUMO2 genes (Saracco et al., 2007). Recently, some Arabidopsis mutants with altered sumoylation, which regulates plant development, have been described (esd4, Murtas et al., 2003; siz1, Miura et al., 2005; ost1/ost2, Conti et al., 2008). The SUMO protease mutants esd4 and ost1/ost2 display early-flowering phenotypes (Murtas et al., 2003; Conti et al., 2008), demonstrating that SUMO protein conjugates in these developmental processes. The SUMO E3 ligase SIZ1 has been implicated in the regulation of flowering via both control of a salicylic acid-mediated floral promotion pathway and effects on FLC chromatin structure (Miura et al., 2005; Yoo et al., 2006; Lee et al., 2007; Jin et al., 2008). A small rice SUMO E3 ligase-like protein, SaM, has been suggested to play an important role in hybrid male sterility (Long et al., 2008). Although sumoylation has been implicated in the regulation of plant development (e.g. flowering time or shoot development; Miura et al., 2007), there is currently no information regarding the role of sumoylation in root development. In this report, we functionally characterized the Arabidopsis homologue gene of human SUMO E3 ligase MMS21:AtMMS2. AtMMS21 encodes a functional SUMO E3 ligase, and its mutant mms21-1 exhibits a short-root phenotype and altered responses to exogenous cytokinins. Phenotypic analysis, genetic complementation studies, and cellular and molecular characterization of AtMMS21 T-DNA mutants demonstrated that AtMMS21 is an important regulator of cell proliferation and cytokinin signalling during root development.
RESULTS Identification of an hMMS21 homologue in Arabidopsis To identify putative homologues of human and yeast MMS21 in Arabidopsis, the protein sequences of human MMS21 were used as queries to search against the TIGR A. thaliana annotation database using the BLASTP program. The search resulted in a significant hit: at3g15150 (Figure 1), which was previously classified as the hMMS21 homologue of Arabidopsis (Miura et al., 2007). AtMMS21 has additional homology with hMMS21 outside of the SP-RING domains, and a total amino acid identity of 23.5% (Figure 1a). Both Arabidopsis and human MMS21 proteins contain an SPRING domain, which is present in human PIAS1, as well as a known binding motif that contains five cysteine/histidine residues as Zn2+-coordinating ligands (Gill, 2004). Sequence alignment between Arabidopsis MMS21 and other MMS21 proteins from various species revealed the five conserved cysteine/histidine residues thought to be responsible for Zn2+ coordination in the SP-RING domain (Figure 1b). Phylogenetic analysis of AtMMS21 and the SP-RING domain proteins from Arabidopsis and rice indicated that AtMMS21 is evolutionarily most closely related to yeast SUMO E3 ligase ScNSE2/MMS21 (Figure 1c). The mms21-1 mutant displays defective primary root growth AtMMS21 is composed of seven exons and six introns (Figure 2a). The gene is located on chromosome III of the Arabidopsis genome. To elucidate the biological role of the AtMMS21 homologue in plants, two AtMMS21 T-DNA insertion mutant lines were obtained from the ABRC (Salk Institute Genomic Analysis Laboratory, http://www.biosci. ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm). The respective plants were obtained and propagated, and homozygous individuals of the respective insertions were identified. The insertion sites were determined in detail by PCR (Figure S1), and the T-DNA insertion positions are illustrated in Figure 2(a). The first insertion, mms21-1 (CS848340), occurs in the sixth intron. The second insertion, mms21-2 (CS803668), is located in the three prime untranslated region (3¢-UTR) (Figure 2a). Reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to assess the expression level of AtMMS21 in the homozygous T-DNA lines. No expression was detected in mms21-1 (Figure 2b), indicating that this allele is most likely a ‘true’ null AtMMS21 mutation. When grown on MS medium, there were no obvious differences between wild-type plants and two AtMMS21 mutants at the germination stage. However, mms21-1 had short primary roots compared with wild-type plants when grown on vertical MS medium (Figure 2b). In addition to abnormal root growth, the mms21-1 plant was relatively normal when grown on nutrient agar (Figure 2b,c), but
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
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Figure 1. Gene structure of AtMMS21, and sequence comparison between AtMMS21 and SP-RING proteins from other species. (a) A schematic representation of the AtMMS21 intron-exon structure and sequence identities among MMS21 proteins of Arabidopsis thaliana, Homo sapiens and Saccharomyces cerevisiae. Exons are represented by grey boxes; introns are represented by black bars. In total, the AtMMS21 gene counts seven exons encoding a protein of 249 amino acids. The identities of the full-length, the N-terminal domain and the C-terminal SP-RING domain (indicated by dashed lines) of MMS21 proteins are shown. (b) Sequence alignment of the SP-RING domain of AtMMS21 and the MMS21 proteins from other species. The putative Zn-coordinating cysteine and histidine residues are highlighted and conserved residues are shaded: AtSIZ1 (GI:145362697) from Arabidopsis thaliana; OsSIZ1 (GI:115461835), OsSIZ2 (GI:115454983) and OsSaM (GI:169261412) from Oryza sativa, hMMS21(GI:27734761) from Homo sapiens, ScNSE2(GI:7276232) from Saccharomyces cerevisiae. The species identifying codes of each protein are as follows: Arabidopsis thaliana (At), Oryza sativa (Os), Homo sapiens (Hs) and Saccharomyces cerevisiae (Sc). Alignment was performed using CLUSTALW. (c) Phylogenetic relationship between AtMMS21 and SP-RING proteins of other species. The phylogenies were generated by neighbour joining with 400 bootstrap replicates, and were rooted at the midpoint. The bootstrap values are shown as percentages: AtPIAs1 (GI:42561833) and AtPIAs2 (GI:79527303) from A. thaliana; OsPIAs (GI:115466602), LOC_Os05g47780 (GI:115465291) and LOC_Os07g25890 (GI:115454983) from O. sativa; the other proteins are as shown in (b).
showed a stunted phenotype when grown in soil (Figure 2f). In contrast, mms21-2 and the heterozygous mms21-1 plants exhibited a phenotype similar to the wild type, suggesting that AtMMS21 is a recessive gene involved in controlling root growth. Seedlings were germinated and grown on vertical agar plates to further investigate the root phenotype of the mms21-1 mutant. Primary root length was then measured over a time course. The root growth rate for mms21-1 homozygous seedlings was less than that of wild-type plants (Figure 2d). By 13 days after germination, the length of the
mutant primary roots was one-third that of the wild-type roots. To confirm whether the short-root phenotype of the mms21-1 mutant was indeed caused by the loss of AtMMS21 function, 35S-AtMMS21-GFP was overexpressed in the mms21-1 mutant, and the short-root mutant phenotype was completely rescued (Figure 2e). For example, 8 days after germination, mms21-1 mutant roots had a mean length of 8.2 mm; the mean root lengths of transgenic lines were a little longer than that of the wild-type plants. RT-PCR analysis confirmed that each of these lines expressed the
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
AtMMS21 regulates cell proliferation 669 Figure 2. Phenotype analysis of mms21 mutants. (a) T-DNA insertion sites of mms21 mutants. One insertion is located in the sixth intron, named mms21-1, whereas another insertion is located in the 3¢-UTR, named mms21-2. (b) Root phenotype of wild-type (WT; left), mms21-1 mutant (middle) and mms21-2 mutant (right) seedlings grown on vertical MS plates for 14 days (top panel). RT-PCR analysis of the AtMMS21 transcripts in wild-type and mms21 mutant seedlings (bottom panel). Actin was used as an internal control. (c) An enlarged shoot picture of representative wild-type and mms21-1 seedlings shown in (b). (d) Primary root growth of wild-type and mms211 mutant plants on MS medium in vertical growth conditions. The values are means SDs (n = 30). (e) AtMMS21 cDNA complements the mms21-1 phenotype. Eight-day-old seedlings of the wild type, mms21-1 mutant and the complementation line 35S-AtMMS21 in mms21-1 showing the restoration of the wild-type root phenotype to the complemented mutant (top panel). RT-PCR analysis of the AtMMS21 transcripts in wild-type, mms21-1 and 35S-AtMMS21 in mms21-1 seedlings (bottom panel). Actin was used as an internal control. (f) The phenotypes of 5-week-old wild type, mms21-1 and 35S::MMS21 in mms21-1 plants grown on soil. (g) Transverse section through the primary root elongation (top panel) and meristem (bottom panel) parts of 7-day-old wild-type and mms21-1 mutant seedlings. (h) Whole-mount preparations of the root apical meristem (RAM) in PI-stained root tips of 36-hold seedlings. Scale bars: 10 lm.
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AtMMS21 transcript, whereas the control (untransformed) mms21-1 seedlings demonstrated no amplification product (Figure 2e). Transverse sections of elongation and division zones of mms21-1 roots indicated that the cortex and endodermis cell layers were present (Figure 3f), suggesting that the function of AtMMS21 differs from that of the SCARECROW (SCR) and SHORT ROOT (SHR), which have important roles in the maintenance of the quiescent centre (QC) in root development (Sabatini et al., 2003). We then examined whether the mms21-1 mutant exhibited any structural or functional defects in the root apical
meristem. At 36 h after germination, the mms21-1 mutant showed a typical cell arrangement (Figure 2g). The QC was identified morphologically, which suggests that the mms21-1 mutant had a normal QC identity, and regulates the cell division pattern in the post-embryonic root apical meristem. mms21-1 plants display shorter and fewer root cells We analysed the primary roots of mms21-1 seedlings at the cellular level to characterize the mms21-1 phenotype in further detail. In principle, the short-root phenotype of mms21-1 could be caused by either reduced cell length or
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decreased cell numbers. To distinguish between these two possibilities, we microscopically analysed the cell files of the mature and elongation zones. Analysis of the size and number of epidermal cells revealed that mms21-1 roots had fewer (Figure 3a,c,d,f) and shorter (Figure 3b,c,e,f) cells in the mature and elongation zones. To quantify our observations, we measured the size and number of root mature and elongation zones of mms21-1 and wild-type seedlings grown for 6 days after germination by analysing epidermal cell files. This measurement showed that mms21-1 root mature zones were comprised of roughly 60% of the number of cells present in wild-type mature zones, and the average cell length was only 80% that of wild-type mature zone cells (Figure 3a,b). We also counted the number of epidermal cells in the elongation zone, from the first rapidly elongating cell up to the first cell of mature size; which provided an indicator of the elongation zone size (Mouchel et al., 2004). The mms21-1 root elongation zones consisted of roughly 27% of the number of cells present in wild-type elongation zones, and the average cell length was only 25% that of wild-type elongation zone cells (Figure 3d,e). Thus, although the sizes of both the mature and elongation zones of the mms21-1 root decreased, the elongation zone was more severely affected. The MMS21 gene affects cell proliferation in the apical root meristem The organization of mms21-1 root meristems appeared normal in 6-day-old seedlings when investigated by confo-
Figure 3. Cell size and number in the mature and elongation zones of primary roots of wild-type (WT) and mms21-1 mutant seedlings. (a) Epidermal cell length of the root mature zone at 6 days after germination (6 dag). For each genotype, three seedlings were measured. The number of cells measured in each seedling was 20 in the WT and 25 in the mms21-1 mutant. (b) Epidermal cell numbers of the root mature zone at 6 dag. For each genotype, five seedlings were counted. (c) Confocal microscopy images of the root mature region of WT and mms21-1 mutant seedlings. (d) Epidermal cell length of the root elongation zone at 6 dag. For each genotype, three seedlings were measured. The number of cells measured in each seedling was 46 for the WT and 50 for the mms21-1 mutant. (e) Epidermal cell numbers of the root elongation zone at 6 dag. For each genotype, five seedlings were counted. (f) Confocal microscopy images of the root elongation region of wild-type and mms21-1 mutant seedlings.
cal microscopy (Figure 4a,b). However, the length of root meristems and the number of cells in the meristematic zone of mms21-1 mutants undergoing division appeared to be less than in wild-type meristems (Figure 4a,c). To quantify our observations, we measured from the root cap to the first rapidly elongating cell, as an indicator of root meristem size: the meristem length of the mutant primary root was 40% of that of the wild type (Figure 4a). To further quantify root meristem size, we counted the number of cortical cells from the initial cell up to the first rapidly elongating cell in the meristematic zone of mms21-1 and wild-type seedlings (Casamitjana-Martinez et al., 2003). The mms21-1 root meristems consisted of roughly 46% of the number of cells of the wild-type plants (Figure 4d). We also counted the number of cortical cells from the first rapidly elongating cell to the first cell of mature size, as an indicator of elongation-zone size (Mouchel et al., 2004). The mms21-1 root meristems consisted of roughly 20–25% of the number of cells in wild-type elongation zones (Figure 4d). These results suggest that cell proliferation in mms21-1 mutants was severely reduced. Impaired expression of the cell division marker CYCB1:GUS in the mms21-1 mutant To analyse the role of AtMMS21 in cell proliferation in the meristematic region of the root, we crossed the mms21-1 mutant with a transgenic reporter plant for cell proliferation expressing CYCB1:GUS (Colon-Carmona et al., 1999), and obtained a CYCB1:GUS-containing wild type and mms21-1 mutant lines. The CYCB1:GUS gene contains the GUS reporter gene fused to the mitotic destruction sequence
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
AtMMS21 regulates cell proliferation 671
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(b) Figure 4. Root meristem morphology and size in the primary roots of wildtype (WT) and mms21-1 seedlings. (a) The meristem lengths of WT and mms21-1 roots, and images of 6-day-old root tips. The vertical lines indicate the meristem lengths in the root tips. (b) Confocal images of root meristems grown for 6 days on half-strength MS medium on vertical plates. Scale bars: 10 lm. (c) Magnification of cortical cell files (marked by white asterisks), starting from the initial cell, shown in B. Scale bars: 10 lm. (d) Number of cells in cortical cell files of the root meristematic zones, as defined in the text, grown on half-strength MS medium containing 1.5% sucrose, and scored at day 6. The values are means SDs (n > 30).
(D-box) and the promoter of the cyclin CYCB:1. These fusion genes are expressed upon entry into G2 (via the CYCB1:1 promoter), and their protein product is degraded upon exit from the metaphase (via the D-box) (Criqui et al., 2001). Subsequently, GUS activity marks cells in G2 and early M phase (Colon-Carmona et al., 1999). GUS-positive cells were detected in the root tips and at the initiation sites of lateral root formation in wild-type plants (Figure 5). However, the size of the zone of cells expressing the GUS gene was significantly reduced in root tips of mms21-1 mutants (Figure 5a,b). There were no differences in GUS activity in lateral root formation initiation sites (Figure 5c). Examination of 3- and 7-day-old seedlings revealed similar results (Figure 5d,e). This finding indicates severely decreased mitotic activity in the primary root meristem of mms21-1 mutants. The mms21-1 mutant showed reduced sensitivity to cytokinin The reduced longitudinal cell expansion and cell number in mms21-1 mutant roots could be caused by increased accumulation or sensitivity to several hormones, including ethylene, auxin (Ljung et al., 2001) and cytokinin (Beemster and Baskin, 2000). To investigate the sensitivity of
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(e) Figure 5. Cell cycle arrest and lateral root initiation in the mms21-1 mutant. Wild-type and mms21-1 mutant plants at 3- and 7-days old, containing the G2/ M marker CYCB1:GUS levels were analyzed by histochemical staining for GUS activity. Scale bars: 200 lm. (a) Primary root tips of 3-day-old wild-type and mms21-1 seedlings. (b) Primary root tips of 7-day-old wild-type and mms21-1 seedlings. (c) Lateral root formation of 3-day-old wild-type and mms21-1 seedlings. (d) Early stages of lateral root formation of 7-day-old wild-type and mms21-1 seedlings. (e) Late stages of lateral root formation of 7-day-old wild-type and mms21-1 seedlings.
mms21-1 root growth to exogenous hormones, homozygous mutant and wild-type seedlings were grown in the medium in the presence of different concentrations of exogenous auxin [1–100 nM 2,4-dichlorophenoxyacetic acid (2,4-D); 1–250 nM 1-naphthaleneacetic acid (NAA)], cytokinin [1 nM–1 lM benzyladenine (BA)], AgNO3 (inhibitor of ethylene biosynthesis, 10 nM–1 lM) and gibberellic acid (GA, 1–100 nM). For auxin and GA treatment, 3-day-old seedlings were transferred from hormone-free medium to plates containing auxin or GA for another 4 days in the light. For cytokinin and AgNO3 treatments, seedlings were grown continuously in the presence of either BA or AgNO3 for 7 days after germination in darkness, as used previously to
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
672 Lixia Huang et al. identify mutants defective in cytokinin and ethylene signalling (Vogel et al., 1998). Changes in primary root lengths of mutant and wild-type seedlings were determined (Table 1). Cytokinin treatment resulted in reduced primary root growth in both mutant and wild-type seedlings. Interestingly, the mms21-1 roots were proportionally longer than wild-type roots in the presence of exogenous 6-BA, over a range of concentrations. At the highest 6-BA concentration tested (1 lM), there was a 4% reduction of root length in the mms21-1 mutant, but a 70% reduction in wild types. Also, the growth inhibitory effects of auxin on the mms211 root compared with the wild-type root in the presence of exogenous 2,4-D and NAA was less pronounced. However, there was no difference between the relative growth enhancement of mms21-1 and wild-type roots in the presence of GA and AgNO3 over the concentration range tested. These results suggest that the growth of the mms21-1 primary root has reduced responses to cytokinin. Alterations in the gene expression of the mms21-1 mutant To determine whether transcription of the native AtMMS21 gene is regulated by cytokinin, 1-week-old Arabidopsis seed-
WT (n = 10)
MS 6-BA (1 nM) 6-BA (10 nM) 6-BA (100 nM) 6-BA (1 lM) GA (1 nM) GA (10 nM) GA (100 nM) MS 2,4-D (1 nM) 2,4-D (10 nM) 2,4-D (100 nM) NAA (1 nM) NAA (10 nM) NAA (100 nM) NAA (250 nM) ABA (10 nM) ABA (100 nM) ABA (250 nM) ABA (500 nM) ABA (1 lM) ABA (5 lM) ABA (10 lM) AgNO3 (10 nM) AgNO3(100 nM) AgNO3 (1 lM)
lings were subjected to different concentrations of cytokinin. RNA was prepared for RT-PCR analysis. The AtMMS21 transcript levels were downregulated by cytokinin (Figure 6a). The mms21-1 mutation had reduced sensitivity to cytokinin and was downregulated by cytokinin, and we next sought to uncover the molecular events responsible for this change in mutant plants. The observation that mms21-1 mutants had reduced cytokinin responsiveness suggests that the expression of cytokinin-regulated genes might be changed in mms21-1 seedlings. To test this, the expression levels of several cytokinin response genes were analysed by quantitative PCR in the absence of exogenous cytokinin. The cytokinin primary-response genes ARR3, ARR4, ARR5, ARR6 and ARR7 (Brandstatter and Kieber, 1998; D’Agostino et al., 2000), which are transcriptionally upregulated by cytokinin, were investigated in mms21-1 and wild-type roots in the absence of exogenous cytokinin by quantitative and semi-quantitative PCR. ARR3, ARR4, ARR5 and ARR7 were downregulated in the mms21-1 mutant (Figure 6a,b), whereas there was no difference in ARR6 expression between mms21-1 and wild-type plants. This result indicated that ARR3, ARR4, ARR5 and ARR7 genes, which are specifically
mms21-1 (n = 10)
Mean root length (mm SE)
Percentage change from control
Mean root length (mm SE)
Percentage change from control
23.2 2.1 21.9 3.5 21.8 2.3 17.3 1.3 7.0 2.0 40.8 2.1 42.0 1.6 28.0 1.4 32.3 2.7 34.3 2.2 29.3 1.9 9.8 0.8 29.6 2.2 34.6 1.9 26.0 1.7 20.2 1.0 30.0 1.9 34.6 2.8 34.9 2.0 35.2 2.2 32.1 3.1 29.8 0.9 29.7 1.1 35.3 1.8 33.0 2.1 37.4 2.3
100 94.4 94 74.6 30.2 175.9 181 120.7 100 106.2 90.7 30.3 91.6 107.1 80.5 62.5 92.9 107.1 108 109 99.4 92.3 92.0 109.3 102.2 115.8
5.6 0.8 5.3 0.9 5.8 0.4 8.1 0.9 5.4 0.7 7.1 0.9 9.8 1.5 7.5 0.7 7.4 1.3 9.7 0.5 8.4 0.6 4.8 0.4 9.0 0.7 8.1 1.8 7.0 1.0 6.5 0.9 8.9 2.0 10.0 1.0 8.5 1.3 8.7 0.6 8.9 1.6 7.7 1.5 7.5 0.7 7.4 1.0 7.4 0.9 9.3 2.0
100 94.6 103.6 144.6 96.4 126.8 175 133.9 100 131.1 113.5 64.9 121.6 109.5 94.6 87.8 120.3 135.1 114.9 117.6 120.3 104.1 101.4 100 100 125.7
Table 1 Changes in primary root lengths of mms21-1 and wild-type seedlings after 7 days of continuous growth on cytokinin (6-BA), and after transfer at 3 days after germination from hormone-free to hormone-containing (2,4-D, 1-NAA and GA) medium for another 4 days
The relative change in primary root length is shown as a percentage change compared with control seedlings grown on hormone-free medium (MS). ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
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The AtMMS21 protein is localized to the nucleus and cytoplasm To determine the subcellular localization of the AtMMS21 protein, we made a fusion protein of AtMMS21-EGFP expressed under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. The 35S-AtMMS21-GFP can complement the phenotype of mms21-1 mutants compared with 35S-GFP plants (Figure 2e), demonstrating that AtMMS21 fused to GFP functions in a similar manner to AtMMS21 alone. When the construct was permanently expressed in wild-type Arabidopsis (Figure 7), its subcellular localization was monitored by fluorescence microscopy. Similar to GFP alone, the AtMMS21-GFP fusion protein was localized to the nucleus and cytoplasm in this assay.
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AtMMS21 is a SUMO ligase in vitro and in vivo
Figure 6. AtMMS21 expression is downregulated by cytokinin and the expression analysis of cytokinin response genes. (a) Expression of AtMMS21 transcript levels after treatment with cytokinin (10 lM kinetin). Seedlings were grown for 7 days on hormone-free medium and were then transferred to the hormones for the times indicated. RT-PCR was performed with either AtMMS21-specific primers (top gel) or Actinspecific primers (bottom gel). (b) The expression analysis of the cytokinin response gene in wild-type and mms21-1 seedlings by semiquantitative RT-PCR (left) and real-time PCR (right). DR5-GUS gene fusion activities in the root tips of wild-type and mms21-1 mutant seedlings 7 days after germination.
Thus far, three types of SUMO ligases have been identified in animals and fungi. These three ligases have been termed SIZ/PIAS, RanBP2 and Pc2, and they all interact with ubc9 (SUMO E2 conjugating enzyme) and enhance sumoylation both in vivo and in vitro (Potts and Yu, 2005; Sharrocks, 2006). AtSCE1a is the only SUMO E2-conjugating enzyme, and plays an indispensable role in the plant sumoylation process. A yeast two-hybrid screen was used to identify any interaction between AtMMS21 and AtSCE1a (SUMO E2 conjugating enzyme; Figure 8a). To further validate the yeast two-hybrid data, we used the bimolecular fluorescence complementation (BiFC) approach. BiFC detects protein interactions in living cells by relying on the fluorescence from split YFP peptide fragments brought into close proximity by the two interacting proteins to which they are fused (Hu et al., 2002; Walter et al., 2004). If AtMMS21 physically interacts with AtSCE1a, YFP fluorescence would be restored. YFP fluorescence was indeed observed in the nucleus and
regulated by cytokinins, were downregulated in the mms21-1 mutant. Considering that auxins and cytokinins interact and often have apparently antagonistic effects in root development (Casson et al., 2002; Muller and Sheen, 2008), the expression levels of the IAA1 and IAA2 genes (Abel and Theologis, 1996), which are transcriptionally upregulated by auxin, were also investigated in mms21-1 and wild-type seedlings. The expression of IAA1/IAA2 did not differ between mms21-1 mutant and wild-type plants (Figure S2). Consistent with this finding, the expression of the GUS gene driven by the DR5 gene promoter and introduced into the mms21-1 mutant background by crossing (to avoid positional effects on the level of expression) was unchanged in the root tips of wild-type and mutant seedlings (Figure 6c). These results suggest that the AtMMS21 gene might be specifically involved in cytokinin signalling in root development.
(a)
(b)
Figure 7. Subcellular localization of AtMMS21. 35S-AtMMS21-EGFP transgenic Arabidopsis root cells (a) and control 35S-EGFP root cells (b). Left, GFP fluorescence image; middle, bright-field image; right, merged GFP fluorescence image. Scale bars: 40 lm.
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674 Lixia Huang et al.
(a)
(b)
(c)
(d)
An immunoblot of total root protein by anti-AtSUMO1 demonstrated no difference in the SUMO conjugates of wild-type, mms21-1 and 35S-AtMMS21-GFP in mms21-1 seedlings that were grown at 22C; however, the extent of sumoylation was substantially less in mms21-1 plants after being treated by 6-BA, and a similar pattern was observed between wild-type and 35S-AtMMS21-GFP in mms21-1 plants (Figure 8c). These results suggest that AtMMS21 is involved in the sumoylation of Arabidopsis root protein. We also performed in vitro sumoylation reactions using bacterially purified GST-AtMMS21. In the presence of ATP, Aos1-Uba2 and SCE1a, these high-molecular weight species of GST-AtMMS21 were present in reaction mixtures (Figure 8d). Together, the in vitro and in vivo analyses established that AtMMS21 functions as a SUMO E3 ligase in Arabidopsis. DISCUSSION
Figure 8. In vitro and in vivo assays indicate that AtMMS21 is a SUMO E3 ligase. (a) AtMMS21 interacts with AtSCE1a in yeast cells. Yeast cells were cotransformed with: pBD53 and pADT (+) (sector 1, positive control); pBDLam and pADT ()) (sector 2, negative control); pBD and pADAtMMS21 (sector 3); pAD and pBDSCE1a (sector 4); and pBDAtSCE1 and pADAtMMS21 (sectors 5 and 6). Transformants were streaked on yeast synthetic drop-out selection medium that lacked Leu and Trp or that lacked Leu, Trp and His, supplemented with 3-AT (1 mM). After spotting, plates were kept at 28C. Blue colonies in an X-Gal assay indicate the interaction in yeast. (b) BiFC visualization of the AtMMS21 interaction with AtSCE1a in the nuclei and cytoplasm of onion cells. Left, YFP fluorescence image of onion epidermal cells co-transfected with AtMMS21-YFP and AtSCE1a-YFP; middle, bright-field image of the onion cells; right, merged image showing YFP fluorescence in the nucleus and cytoplasm. (c) In planta sumoylation profiles of wild-type, mms21-1 and 35S-AtMMS21GFP in mms21-1 seedlings that were grown for 7 days on full-strength MS medium at 23C, and then treated with 6-BA for 3 h. Ponceau staining of total protein was used as the loading control. Lanes 1 and 4, wild type; lanes 2 and 5, mms21-1 mutant; and lanes 3 and 6, 35S-AtMMS21-GFP in mms21-1. Analysis of in vitro sumoylation profiles indicates that AtMMS21 facilitates the production of SUMO conjugates. GST-AtMMS21 was expressed and purified from Escherichia coli, and then tested for sumoyaltion activity in the presence of E1 (His-Aos1/Uba1), E2 (His-SCE1a) and His-SUMO1. The indicated quantity of GST-AtMMS21 (2 or 6 lg) was added to the reaction mixture. The immunoblots were probed with anti-SUMO1 antibodies. The arrow indicates the sumoylated GST-AtMMS21 band.
cytoplasm of onion epidermal cells co-bombarded with pSAT6-nEYFPAtMMS21 and pSAT6-cEYFPAtSCE1a (Figure 8b). In contrast, YFP fluorescence was not detected in onion epidermal cells when either pSAT6-cEYFPAtSCE1a or pSAT6-cEYFPN1 was in combination with pSAT6-nEYFPAtMMS21. These data demonstrate that AtMMS21 directly interacts with AtSCE1a, suggesting that AtMMS21 may be a putative SUMO E3 ligase.
Sumoylation plays important roles in floral initiation, ABA signalling, and in plant responses to various stresses like heat and cold shock, phosphate deficiency and pathogen resistance (Kurepa et al., 2003; Lois et al., 2003; Murtas et al., 2003; Miura et al., 2005, 2007; Yoo et al., 2006). In this report we showed that AtMMS21 encodes a functional SUMO E3 ligase, and that the null mms21-1 mutant displayed a short-root phenotype and altered responses to exogenous cytokinins. Our genetic, phenotypic, cellular and molecular characterization of mutants demonstrates that AtMMS21 plays a crucial role in cell proliferation and cytokinin signalling in roots. AtMMS21 encodes an SP-RING protein with SUMO E3 ligase activity The SP-RING is a Zn-finger domain-containing C2HC3 in SIZ/ PIAS of C2HC2 in NSE2/MMS21 (Sharrocks, 2006). The SPRING domains are necessary for the SUMO E3 ligase activity of SIZ/PIAS and NSE2/MMS21 proteins, and the SP-RING facilitates the binding of SIZ/PIAS and NSE2/MMS21 to SCE1 (Potts and Yu, 2005; Sharrocks, 2006). Alignment of AtMMS21 with protein sequences of AtSIZ1, OsSIZ1, OsSIZ2, human MMS21, yeast ScNSE2 and rice MMS21 showed it was most closely related to OsMMS21 (Figure 1). Previous studies have identified human MMS21 and yeast ScNSE2 as SUMO E3 ligases that autosumoylate (Potts and Yu, 2005). Although AtMMS21 contains all of the canonical amino acids (Figure 1C) present in other SP-RING proteins, we needed to establish whether AtMMS21 was a SUMO E3 ligase. Our results concur with the previous results, and demonstrated that AtMMS21 was a SUMO ligase that undergoes autosumoylation in vitro and in vivo (Figure 8c,d). Furthermore, SUMO E3 ligases are SCE1-interacting proteins that are necessary for the transfer of SUMO from SCE1 to substrate proteins in vivo (Johnson, 2004). To confirm this, a yeast two-hybrid screen and BiFC analysis
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AtMMS21 regulates cell proliferation 675 showed that AtMMS21 interacts with SCE1a (Figure 8a,b). These results indicated that AtMMS21 was a SUMO E3 ligase. Role of AtMMS21 in root cell proliferation AtMMS21 is expressed in almost all plant tissues (Figure S1), and so it is reasonable to assume that this SUMO E3 ligase plays a role in plant development. Morphological analysis showed that mms21-1 has a short-root phenotype with shorter and fewer root cells, especially in elongation zones (Figure 2). These results suggest that AtMMS21 is involved in cell proliferation and cell expansion. This phenotype can be attributed to AtMMS21 deficiency because wild-type morphology was restored in the complementation line (Figure 2). The involvement of sumoylation in cell cycle progression is best understood in yeast and animal cells (Johnson, 2004; Hay, 2005). In Saccharomyces cerevisiae, mutations in the single genes that encode SUMO (SMT3), either the E1 subunits (UBA2 and AOS1) or the E2 ligase (UBC9), are not viable: the mutants have strong cell-cycle defects and arrest at the G2/M boundary (see review in Hay, 2005). Mutations in equivalent subunits in Schizosaccharomyces pombe are viable, but exhibit severely impaired growth, mitotic defects and greater genomic instability (see review in Hay, 2005). The Arabidopsis SUMO E3 ligase siz1-3 mutant has reduced plant height and leaf size (Catala et al., 2007). Similar to the mms21-1 short-root phenotype, knockout mutants for some of these genes have a short-root phenotype caused by defects in cell elongation (OsCyt-invl, Jia et al., 2008; brx, Mouchel et al., 2004) and cell proliferation (nrp1-1/nrp2-1, Zhu et al., 2006; brx, Mouchel et al., 2004; hlr, Ueda et al., 2004; stp1, Beemster and Baskin, 2000). Interestingly, the short-root phenotype in mms21-1 plants showed significant similarity to the root tip phenotype of seedlings in which cell proliferation has been slowed by cytokinin treatment (Beemster and Baskin, 2000) or by overexpression of cell-cycle progression inhibitors (De Veylder et al., 2001). Furthermore, previous analyses suggest that root growth rate is primarily controlled at the cell proliferation step (Beemster et al., 2002, 2003; Mouchel et al., 2004). Thus, the primary cause of the mms21-1 shortroot phenotype might be reduced cell proliferation in the root meristem. B-type cyclins are good markers of cell proliferation, as their expression patterns are specific to the G2/M phase of the cell cycle (Hemerly et al., 1992; Donnelly et al., 1999). CYCB1 overexpression increases root elongation (Doerner et al., 1996). Our observation that AtMMS21 is involved in cell proliferation implies that the SUMO E3 ligase AtMMS21 is also actively involved in cell cycle regulation. CYCB1:GUS expression was reduced in salt-stressed roots as a result of both decreased cell production and mature cell length (West et al., 2004), indicating severe disruption of mitotic activity. In the present report, we analysed a transgenic line contain-
ing a CYCB1:GUS construct as a reporter of mitotic activity. There was reduced CYCB1:GUS expression in mms21-1 mutants compared with wild types (Figure 5). Together with the decreased meristem size, this finding is consistent with a model in which the cells become blocked at the G2/M transition. Thus, our results suggest that AtMMS21 is actively involved in cell-cycle regulation during root meristem development. AtMMS21 negatively mediates cytokinin signalling to control primary root development Cytokinins are purine derivatives that promote and maintain plant cell division in culture, and are also involved in various differentiation processes, including shoot formation, primary root growth and callus formation (Catterou et al., 2002; Werner et al., 2003; Higuchi et al., 2004). Three sensor hiskinases, CRE1/AHK4/WOL, AHK2 and AHK3, have been shown to act as cytokinin receptors (Kakimoto, 2003). These receptors activate the expression of several response regulators (e.g. ARR4, ARR5 and ARR6) in a cytokinin-dependent manner (Brandstatter and Kieber, 1998). Further downstream, cytokinin signalling stimulates the G1/S transition of the cell cycle. It is very interesting that expression levels of the cytokinin primary-response genes ARR3, ARR4, ARR5 and ARR7 were downregulated in mms21-1 plants (Figure 6b), which is consistent with the idea that the AtMMS21 mutant is less sensitive to exogenous cytokinin (Table 1). These results suggest that cytokinin signalling is likely to be involved in the alterations in root cell proliferation of AtMMS21 mutants. These findings agree well with the conclusion that cytokinins negatively regulate root meristem activity (Werner et al., 2003). Thus, we speculated on the relationship between cytokinin signalling and AtMMS21 in the regulation of root meristem activity in mms21-1 mutants. AtMMS21 may encode a component that negatively regulates cytokinin signalling in controlling root development. The STUNTED PLANT1 gene has been suggested to be a negative regulator of root development mediated by cytokinin signalling (Baskin et al., 1995). The following observations support this hypothesis: (i) mms21-1 mutant roots phenocopied cytokinin-treated wild-type roots; (ii) the expression of the AtMMS21 gene was strongly downregulated in root tips in the presence of exogenous BA; (iii) the mms21-1 mutant had less inhibited root growth in the presence of the cytokinin BA; (iv) the mms21-1 mutant exhibited decreased expression of the cytokinin-induced genes ARR3, ARR4, ARR5 and ARR7, which are expressed in the root tip (Brandstatter and Kieber, 1998); and (v) there were significant differences when leaf explants were cultured on a callus induction medium for a 35-day period without being transferred to fresh medium (Figure S3). Therefore, the mutant exhibits altered responses to cytokinins. AtMMS21 might thus be an important component that regulates cytokinin signalling.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
676 Lixia Huang et al. Model for the role of AtMMS21 in the control of root development AtSIZ1-mediated sumoylation of the MYC-like transcription factor ICE1 and the MYB transcription factor PHR1 regulate both cold and phosphate starvation signalling (Miura et al., 2005, 2007). Our results suggest that AtMMS21 represents a new class of negative regulators of root development involved in cell-cycle regulation. Thus, we hypothesize that AtMMS21 functions as a SUMO E3 ligase that mediates the sumoylation of targets. The sumoylated target protein regulates cytokinin signalling, the cell cycle and the development of the root meristem. Thus, further functional dissection of AtMMS21, as well as the identification of AtMMS21 target proteins, and their interactions in cytokinin signalling and the cell cycle, are necessary for the complete understanding of the cytokinin signalling networks and cell-cycle regulation that function during primary root development.
RT-PCR indicates the transcript levels of marker genes (including ARR5, ARR6, IAA1 and IAA2) in wild-type and mms21-1 after treatment with cytokinin (10 lM kinetin or 6-BA; Casson et al., 2002). Seedlings were grown for 7 days on hormone-free medium, and were then transferred to the hormones for the times indicated.
Yeast two-hybrid The full-length coding sequence of AtMMS21 was PCR-amplified and cloned in vector pGADT7 (Clontech, http://www.clontech.com), expressed as pAD-AtMMS21, to produce a protein fusion with the GAL4 DNA AD. The AtSCE1a PCR fragment was cloned into pGBKT7, expressed as pBD-AtSCE1a, to produce a protein fusion with the GAL4 DNA BD. The GAL4-based interaction between the pBD-AtSCE1a protein and pAD-AtMMS21 fusion was tested in the yeast strain AH109 (Clontech). Co-transformed yeast strains were selected on synthetic defined (SD)/–Leu/–Trp medium. Protein–protein interactions were tested using stringent (SD/–Leu/–Trp/–His) selection. The interaction between pAD-T and pBD-53 was used as the positive control, and that between pAD-T and pBD-Lam was used as the negative control. GUS activity was measured to assay for the interaction between two known proteins AtMMS21 and AtSCE1a in a GAL4 two-hybrid system.
EXPERIMENTAL PROCEDURES
Confocal microscopic observation
Plant materials and growth conditions
To prepare the 35S-AtMMS21-GFP fusion construct, the entire coding region of AtMMS21 was inserted directly upstream of the EGFP coding region in pBEGFP (pBEGFP is reconstructed based on pBin19). The construct was introduced into Arabidopsis by transformation, and T2 plants were used for GFP subcellular localization analysis. Roots of 7-day-old transgenic seedlings were used for the green fluorescence analysis (GFP localization) by a Carl Zeiss laser scanning system LSM 510 (http://www.zeiss.com). For the primary root tip cell morphorous analysis, seedlings of wild type and mms21-1 were grown vertically on MS agar plates, 2day-old seedlings were stained with a propidium iodide (PI) solution (10 lg mL)1) for 1 min, followed by washing with sterilized water, and were placed on slides in a drop of water and then imaged under confocal microscopy. Then, the 7-day-old seedlings were also stained with PI and imaged under confocal microscopy, and the lengths of elongating and mature epidermal cells were measured by the tool DIGIMIZER 3.2.1.0.
Arabidopsis plants were grown in a controlled growth room at 22 2C under long-day conditions (16-h light/8-h dark). For in vitro experiments, seeds were surface-sterilized for 2 min in 75% ethanol, followed by 5 min in 1% NaClO solution and washed five times in sterile distilled water, plated on growth medium (MS medium, 1.5% sucrose and 0.8% agar), vernalized at 4C for 2 days in the dark and then exposed to white light. The constructs in binary vectors were transformed into Agrobacterium EHA105, which were then used to transform A. thaliana (Columbia ecotype) following the floral-dip method (Clough and Bent, 1998). Transgenic seedlings were selected on MS agar plates with 50 mg L)1 kanamycin.
Hormone treatments and primary root length measurements For hormone application experiments, seeds were sown on MS agar plates containing various concentrations of hormones, and were then cultured vertically. For cytokinin treatment, seedlings were grown continuously in the presence of 6-BA for 7 days after germination in the dark (Casson et al., 2002). For other hormone (2,4-D, 1-NAA, GA and ABA) and AgNO3 treatment, 3-day-old seedlings were transferred from hormone-free medium to hormone- or AgNO3-containing medium for another 4 days in the light (Casson et al., 2002; Chilley et al., 2006). The plates were scanned by an Epson perfection V200 photo scanner, and the primary root length of the 7-day-old seedlings was measured by the tool DIGIMIZER 3.2.1.0 (http://www.digimizer.com).
Histochemical analysis and microscopy The plant lines that include CYCB1:GUS and DR5:GUS in the mms21-1 mutant background were obtained by crossing the homozygous CYCB1:GUS and DR5:GUS lines with the homozygous mms21-1 line. The progeny was followed to the second generation, and then genotyped for the presence and absence of the wild-type AtMMS21 allele. The T3 generation was analysed by histochemical GUS staining. The stained materials were cleared for 10 min in an 8:3:1 mixture of chloral hydrate:distilled water:glycerol (Willemsen et al., 1998), then visualized and imaged under a light microscope (BX51; Olympus, http://www.olympus-global.com).
Gene expression analysis The total RNA was extracted from Arabidopsis seedlings or organs with TRIzol Reagent (Invitrogen, http://www.invitrogen.com), according to the manufacturer’s instructions. Reverse transcription (RT) was performed with an oligo (dT) primer. RNA (1 lg) was heated at 70C for 10 min and then immediately chilled on ice. RNA was then subjected to RT with reverse-transcriptase MMLV-RT SPCL (Invitrogen) at 42C for 1 h, following the manufacturer’s protocol. Synthesized cDNA was used as the PCR template.
BiFC assay We performed BiFC assays in vivo as described by Walter et al. (2004). The coding sequence of AtMMS21 was amplified by PCR and cloned into the plasmid pSAT6-nEYFPN1, thus resulting in the plasmid pSAT6-nEYFPN1-AtMMS21 expressed as the nEYFPN1AtMMS21 fusion protein. The coding sequence of AtSCE1a was also obtained by PCR and cloned into the plasmid pSAT6-cEYFPN1 to give rise to the plasmid pSAT6-cEYFPN1-AtSCE1a expressed as the cEYFPN1-AtSCE1a fusion protein. pSAT6-nEYFPN1-AtMMS21 in
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 666–678
AtMMS21 regulates cell proliferation 677 combination with pSAT6-cEYFPN1 was coated with gold particles, as were controls. The onion epidermis was bombarded using a gene gun (PDS-100/He BiolistiK particle delivery system). Then, the bombarded samples were incubated on MS plates at 22C for 24 h. Cells with YFP fluorescence were observed using confocal laser scanning microscopy, as described previously.
Analysis of sumoylation profiles Total protein of seedlings incubated at 24C or treated by 6-BA for 1.5 h were extracted and separated by SDS–PAGE. The gel blot was probed with the SUMO1 antibody (Bioworld, http://www. bioworld.com) and detected using ECL plus (Amersham Pharmacia, http://www.gelifesciences.com).
In vitro sumoylation assay Bacterially expressed GST-AtMMS21, His-SCE1a, His-Aos1/Uba2 (SUMOE1) and His-SUMO1 were purified by Ni-NTAbeads (Qiagen, http://www.qiagen.com) or glutathione agarose (Amersham Pharmacia). The in vitro sumoylation was conducted as described by Miura et al. (2005). The immunoblots were probed with anti-SUMO1 antibody (Abcam, http://www.abcam.com).
ACKNOWLEDGEMENTS We thank Dr T. Guifoyle (University of Missouri) for providing DR5:GUS seeds, Dr T. Beeckman (Univerisity of Ghent) for CycB1:GUS seeds, F. Melchior for E1 constructs, Dr H.Q. Yang (Shanghai Jiao Tong University) for providing the BiFC vector, Dr G.J. Wu (South China Botanical Garden) for yeast two-hybrid vector, Y.H. Tang for confocal microscopy technical assistance and Dr JR Zuo for helpful discussions. This research was supported by the Program for New Century Excellent Talents in University (NCET-080644), the National Natural Science Foundation of China (30770201), the Natural Science Foundation of Guangdong (2007B020701005), and the Chinese Ministry of Science and Technology (9732009CB118504, 2007BAD59B06). We gratefully acknowledge the ABRC (Ohio State University) for the T-DNA insertion lines.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. AtMMS21 structure and T-DNA insertion diagnostic PCR. Figure S2. Semiquantitative RT-PCR analysis of expression of auxin response gene in wild-type and mms21-1 seedlings. Figure S3. Callus growth in leaf explants of wild-type and mms21-1 plants. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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