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E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds Jun Soo Kwak a, Sung-Il Kim a, Sang Woo Park a, Jong Tae Song b, Hak Soo Seo a, c, * a
Department of Plant Science, Research Institute for Agriculture and Life Sciences, Plant Genomics and Breeding Institute, Seoul National University, Seoul, 08826, South Korea b School of Applied Biosciences, Kyungpook National University, Daegu, 41566, South Korea c Bio-MAX Institute, Seoul National University, Seoul, 08826, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2019 Accepted 17 September 2019 Available online xxx
Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity. © 2019 Published by Elsevier Inc.
Keywords: AtSIZ1 Cruciferin E3 SUMO ligase Seed Storage protein Sumoylation
1. Introduction During development, plant seeds naturally accumulate storage reserves that are mobilized during germination to provide energy and raw materials for early seedling growth. Seed storage proteins are the main source of nitrogen required for seed germination and early seedling growth. The main seed proteins in Arabidopsis thaliana (thale cress) and other crucifers are 12S globulins, also known as cruciferins. In Arabidopsis, cruciferins are encoded by
Abbreviations: AtSIZ1, Arabidopsis E3 SUMO ligase SIZ1; CRU, Cruciferin; DTT, Dithiothreitol; EDTA, Ethylenediaminetetraacetic acid; GST, Glutathione S-Trasnferase; HPLC, High-performance liquid chromatography; IPTG, Isopropyl b-D-1thiogalactopyranoside; MAG, MAIGO; MBP, Maltose binding protein; Ni2þ-NTA, Nickel-nitrilotriacetate; PAGE, Polyacrylamide gel electrophoresis; PCR, Polymerase chain reaction; PMSF, Phenylmethanesulfonyl fluoride; PSV, Protein storage vacuole; qRT-PCR, Quantitative real-time PCR; SDS, Sodium dodecyl sulfate; SUMO, Small ubiquitin-related modifier; VSR1, Vascular sorting receptor 1. * Corresponding author. Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Gwanakro 200, Daehakdong, Gwanak-gu, Seoul, 08826, South Korea. E-mail address: [email protected] (H.S. Seo).
three genes, CRUCIFERIN1 (CRU1), CRU2, and CRU3 [1]. Cruciferins are synthesized in the rough endoplasmic reticulum and then transported to protein storage vacuoles (PSVs) via a Golgidependent [2] or -independent pathway [3e5]. Cruciferins are synthesized as preproproteins and ultimately cleaved into a (30e35 kD) and b (21e25 kD) polypeptides, which are linked to each other via a single interchain disulfide bond [6]. CRU1 and CRU2 share 70% sequence identity and similar size, whereas CRU3 exhibits approximately 50% identity to CRU1 and CRU2, and harbors an extended glutamine-rich region in the center of a larger polypeptide that contributes to its divergence from the other family members. Small ubiquitin-related modifier (SUMO) is a reversible posttranslational modifier, which is covalently conjugated to a lysine residue in the substrate protein by E3 SUMO ligases, key regulators of several different biological functions in plants [7]. The Arabidopsis E3 SUMO ligase AtSIZ1 is an SP-RING finger protein harboring a DNA-binding SAP domain and zinc finger Miz domain. AtSIZ1 is involved in seed germination [8,9], flowering [10,11], epigenetic regulation [12,13], nutrient utilization [14,15], stress response [16,17], root branching [18], and secondary cell wall
https://doi.org/10.1016/j.bbrc.2019.09.064 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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formation [19]. The siz1-2 mutant displays small leaves and severe dwarfism, indicating that AtSIZ1 also plays a central role in vegetative growth and development [20e22]. Additionally, siz1-2 mutant plants exhibit defective seed development, short silique length, and lower seed amount (approximately 50% of the wild type) [14]. Interestingly, phenotypic features of the siz1-2 mutant are restored with the supply of nitrogen, particularly in the form of ammonium [14], suggesting that the vegetative and reproductive phenotypes of the siz1-2 mutant are a result of the impaired nitrate reduction pathway due to the mutation in AtSIZ1. Based on these data, we speculated that abnormal seed development in the siz1-2 mutant causes nutritional defects in seeds, which influence seed germination and seedling growth. Therefore, we investigated the role of AtSIZ1 in the regulation of the level of seed storage proteins in Arabidopsis. Here, we provide the first evidence that the amount of seed storage proteins is regulated by the sumoylation system. The levels of CRUs were decreased in the siz1-2 mutant, and CRU2 and CRU3 proteins were conjugated with SUMO by the E3 ligase activity of AtSIZ1 through direct interaction, indicating that the abundance of CRUs in seeds is regulated by AtSIZ1.
AGCGTGGTTGGAACTCTGAT-3’; CRU2, 50 -CCGGTATTTGGAGAAGGTCA-30 and 50 -GGTTGTTTCCGGCTATCAAA-3’; CRU3, 50 CAGCAGCTTCAGAACCAACA-30 and 50 -TAGCTGTTGACGCTGGTCAC3’; ACT7, 50 -ACCACTACCGCAGAAC-30 and 50 -GCTCATACGGTCAGCA3’. 2.4. Yeast two-hybrid assays Yeast two-hybrid assays were performed using the GAL4-based two-hybrid system (Clontech). Full-length AtSIZ1 cDNA was fused with the GAL4 activation domain (AD) and cloned into the pGAD424 vector (Clontech), whereas fell-length CRU1, CRU2, and CUR3 cDNAs were fused to the GAL4 DNA-binding domain (BD) and cloned into the pGBT8 vector (Clontech). The AD-AtSIZ1 construct was co-transformed with BD-CRU1, BD-CRU2, or BD-CRU3 into the yeast strain AH109 using the lithium acetate method, and yeast cells were grown on minimal medium lacking leucine and tryptophan (-Leu/-Trp). To test interactions between AtSIZ1 and CRU proteins, the transformants were plated on minimal medium lacking Leu, Trp, and histidine (-Leu/-Trp/-His) and containing 2 mM 3-amino-1, 2, 4-triazole (3-AT).
2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia-0 (WT) and T-DNA insertion knockout mutant siz1-2 were used in this study. Seeds were germinated on agar plates containing Murashige and Skoog (MS) medium supplemented with 2% sucrose and 0.8% agar (pH 5.7). To grow plants in soil, seeds were directly sown in sterile vermiculite. All plants including seedlings were grown in a growth chamber at 22 C and under a 16 h light/8 h dark cycle. 2.2. Two-dimensional gel electrophoresis Dried seeds (150 mg) were ground in liquid nitrogen, and then 1.6 ml TCAAEB buffer containing acetone, 10% TCA, and 0.07% bmercaptoethanol was added to the ground seeds. The homogenate was incubated at 20 C for 1 h and then centrifuged at 15,000 rpm for 15 min. Pellets were washed with buffer (acetone containing 0.07% b-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid [EDTA], and protease inhibitor cocktail tablets [Sigma]), solubilized in lysis buffer (9 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate, 18 mM Tris-Cl [pH 8.0], 14 mM Trizma base, protease inhibitor cocktail, 0.2% Triton X-100, and 50 mM dithiothreitol [DTT]), and then sonicated. Samples were centrifuged at 15,000 rpm at 4 C for 15 min, and supernatants were concentrated with cold acetone. The amount of protein was measured using the Bradford assay [23]. The extracted proteins were separated by two-dimensional gel electrophoresis, according to Gallardo et al. (24). 2.3. Quantification of CRU transcript levels The WT and siz1-2 mutant seeds were imbibed in distilled water for one day, and samples were collected at the indicated time points. Total RNA was extracted from each sample and quantified. First-strand cDNA was synthesized from 5 mg total RNA using the iScript™cDNA Synthesis Kit (Bio-Rad). An equal volume of cDNA was used as a template for qRT-PCR according to the manufacturer's protocol (MyiQ; Bio-Rad). The CT values of the target genes were normalized relative to the CT value of the Arabidopsis Actin7 gene (At5g09810). The primers used for quantitative PCR were as follows: CRU1, 50 -GCCGTCACACGTACTGAAGA-30 and 50 -
2.5. Construction of recombinant plasmids To produce GST (glutathione S-transferase)-CRU2-Myc and GST-CRU3-Myc constructs, full-length CRU2 and CRU3 cDNAs were amplified by PCR using a primer containing the Myc sequence. The resulting PCR products were cloned into the pET28a (Novagen) and pGEX4T-1 (Amersham Biosciences) vectors. To construct the MBP (Maltose binding protein)-AtSIZ1 fusion, full-length AtSIZ1 cDNA was amplified by PCR using gene-specific primers and cloned into the pMALc2 vector (New England Biolabs). Full-length Arabidopsis SUMO1 cDNA was amplified by PCR using genespecific primers and cloned into pET28a to produce His6AtSUMO1-GG. The SUMO E1 and E2 constructs were kindly provided by Dr H.P. Stuible [25]. All constructs were transformed into E. coli BL21/DE3 (pLysS) cells. The transformed cells were treated with Isopropyl b-D-1thiogalactopyranoside (IPTG) to induce the production of recombinant fusion proteins. All constructs were subjected to automated DNA sequencing to rule out the possibility of mutations. 2.6. Purification of recombinant proteins All recombinant proteins were expressed in E. coli strain BL21 and purified according to the manufacturer's instructions. Briefly, to purify His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, and His6AtSUMO1-GG, bacteria were lysed in a buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 1% Triton X-100, 1 mM imidazole, 5 mM DTT, 2 mM phenylmethanesulfonyl fluoride (PMSF), and proteinase inhibitor cocktail (Roche). Recombinant proteins were purified using nickel-nitrilotriacetate (Ni2þ-NTA) resins (Qiagen). To purify GST-CRU2-Myc and GST-CRU3-Myc proteins, bacteria were lysed in phosphate-buffered saline (PBS; pH 7.5) containing 1% Triton X-100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). The recombinant proteins were purified using glutathione resins (Pharmacia). To purify MBP-AtSIZ1, bacteria were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). The recombinant protein was purified using the amylose resin (New England Biolabs). Protein concentration was determined using the Bradford assay (Bio-Rad) [24].
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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2.7. Sumoylation assays In vitro sumoylation assays were performed in 30 ml reaction buffer (20 mM HEPES [pH 7.5], 5 mM MgCl2, and 2 mM ATP), each with 50 ng His6-AtSAE1b, His6-AtSAE2, and His6-AtSCE1; 8 mg His6AtSUMO1-GG; and 100 ng GST-CRU2-Myc or GST-CRU3-Myc, with or without 500 ng MBP-AtSIZ1. After incubation at 30 C for 3 h, the reaction mixtures were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE). Sumoylated GST-CRU2-Myc and GST-CRU3Myc proteins were detected by western blotting using an anti-Myc antibody (Santa Cruz Biotechnology). 2.8. Analysis of amino acid composition To determine the amino acid composition of seeds, total seed proteins were extracted using buffer containing 50 mM Tris-Cl (pH 8.0), 100 mM NaCl, 1% NP-40, and 1 mM EDTA. To precipitate the proteins, four volumes of cold acetone was added, and samples were centrifuged at 4 C for 20 min. The pellets were dried and hydrolyzed with 30 ml of 6 N HCl. After 24 h incubation at 130 C, the acid was removed by vacuum, and the hydrolyzates were derivatized using phenylisothiocyanate. The derivatized amino acids were filtered through a 0.45 mm filter, redissolved in 100 ml phosphate buffer, and analyzed by reverse phase HPLC using an Ultimate 3000 system. This experiment was repeated three times.
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Table 1 Identification of the proteins that were decreased in siz1-2 mutant seeds. Spot
3406 3407 4101 4406 5406 5407 6401 6402 6403 6505 7305 7306 7408 0103 0812 0813
Homologous protein
CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU3 (CRUCIFERIN 3) CRU1 (CRUCIFERIN 1) Unknown CRU3 (CRUCIFERIN 3) CRU3 (CRUCIFERIN 3) CRU2 (CRUCIFERIN 2) CRU2 (CRUCIFERIN 2) CRU1 (CRUCIFERIN 1) Oleosin 2 Heat shock protein 70 Heat shock protein 70B
a
NM (kDa)
52.9 52.9 19.9 52.9 52.9 58.5 52.9 48.2 58.5 44.0 50.8 50.8 52.9 21.2 71.4 71.2
pI value
7.68 7.68 7.88 7.68 7.68 6.53 7.68 5.77 6.53 6.08 6.52 6.52 7.68 9.36 5.14 5.30
Intensity WT
siz1-2
6208 1373 22,218 5274 6319 2308 6505 8787 6478 8021 12,476 5281 8407 11 0 0
5389 218 14,082 2006 3578 1065 4254 5624 4067 4602 9361 3341 3941 295 512 807
This experiment was repeated three times using WT and siz1-2 mutant seeds. Here, one result is shown. a NM: Normal mass.
an unknown protein. By contrast, the signal intensity of 3 of the 16 spots was enhanced in the siz1-2 mutant compared with the WT. Among these three proteins, one was identified as an oleosin and two were identified as heat shock proteins (Table 1).
3. Results 3.2. Increased abundance of CRU transcripts in siz1-2 seeds 3.1. Low abundance of cruciferins in siz1-2 seeds The siz1-2 mutant displays abnormal silique and seed phenotypes [14]. This suggests that the amount and composition of nutrients such as carbohydrates, lipids, and proteins may be changed in siz1-2 mutant seeds. In this study, we analyzed the amount and composition of seed storage proteins in mature seeds of WT and siz1-2 mutant plants using proteomics. Total proteins were extracted from mature WT and siz1-2 mutant seeds and analyzed by two-dimensional gel electrophoresis (Fig. 1). Next, we determined the identity of proteins corresponding to these spots by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry analysis. A total of 67 spots showed excellent reproducibility in three replicate experiments. Among these 67 spots, 16 showed differential signal intensities between the siz1-2 mutant and WT seeds (Table 1). The signal intensity of 13 of the 16 spots was decreased in siz1-2 mutant seeds compared with the WT (Table 1). Proteins corresponding to 12 of the 13 spots were identified as CRUs, including CRU1 (7 spots), CRU2 (2 spots), and CRUS (3 spots), while one spot corresponded to
Fig. 1. Proteomic analysis of seed storage proteins in WT and siz1-2 mutant. Red arrows indicate the identified protein spots showing major differences between the siz12 mutant and the WT.
A lower level of CRUs in siz1-2 seeds could result from two possible causes: (1) lower expression of CRU genes in siz1-2 seeds compared with the WT, or (2) post-translational modification of CRU proteins. To test the first possibility, we examined the transcript levels of CRU1-3 genes in siz1-2 mutant and WT seeds. Total RNA was isolated from mutant and WT seeds imbibed in water for 24 h, and transcript levels of CRU1-3 genes were analyzed by qRTPCR. The results showed that transcript levels of these genes were significantly higher in siz1-2 mutant seeds than in WT seeds (Fig. 2), while their protein levels were lower in siz1-2 seeds than in WT seeds (Table 1). However, transcript levels of these genes were similar in WT and siz1-2 seeds after imbibition. This result implies that the stability and amount of CRU1, CRU2, and CRU3 proteins are regulated at the post-translational level during seed maturation. 3.3. Sumoylation of CRU proteins by AtSIZ1 Despite the high transcript levels of CRU genes in siz1-2 mutant seeds (Fig. 2), the corresponding protein levels were reduced in mutant seeds compared with the WT (Table 1). In many cases,
Fig. 2. Expression analysis of CRU genes in the WT and siz1-2 mutant. Transcript levels of genes encoding CRU1, CRU2, and CRU3 were analyzed by qRTePCR using genespecific primers. Actin 7 gene was used to normalize qRTePCR data. Black and white bars indicate WT and siz1-2 seeds, respectively.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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either the ubiquitinated proteins are degraded by the 26S proteosome complex, or the function and stability of these proteins are altered [26,27]. However, sumoylated proteins are not degraded. Instead, these proteins adopt important cellular functions [27,28]. This led us to search the amino acid sequences of CRU proteins. The deduced amino acid sequences of CRUs displayed putative sumoylation sites (Fig. 3A). We thus examined possible interaction between AtSIZ1 and CRUs by the yeast two-hybrid assay. Results showed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3 proteins (Fig. 3B), suggesting that AtSIZ1 is involved in the sumoylation of CRU1-3 proteins via its E3 SUMO ligase activity. To confirm that AtSZ1 regulates CRU sumoylation, we generated the translational fusion constructs of the MBP gene with AtSIZ1 (MBP-AtSIZ1) and of GST with CRU genes (GST-CRU2 and GST-CRU3). The MBP-AtSIZ1 recombinant protein was co-expressed with each of the three GST-CRU fusion proteins in E. coli separately using IPTG treatment. The recombinant proteins were purified using amylose and glutathione resins, and subjected to in vitro sumoylation assays. The GST-CRU2 and GST-CRU3 fusions were sumoylated by AtSIZ1, and the reaction was dependent on the E1 and E2 activity (Fig. 3C). In addition, if AtSIZ1 was not included in the reaction, GST-CRU2 and GST-CRU3 were not sumoylated in the presence of enzymes E1 and E2. Thus, these results indicate that CRU2 and CRU3 are sumoylated by the E3 ligase activity of AtSIZ1. 3.4. Increased abundance of total proteins and amino acids in siz12 seeds Differences in the level of other seed proteins between the WT
Fig. 3. AtSIZ1 sumoylates CRU proteins. (A) Putative sumoylation sites (jKXE) identified from deduced amino acid sequences of CRU1, CRU2, and CRU3 using the SUMOplot Analysis Program. (B) Yeast two-hybrid analyses of the interaction between AtSIZ1 and CRUs. CRU1, cruciferin1; CRU2, cruciferin2; CRU3, cruciferin3. (C) In vitro sumoylation of CRU2 and CRU3 by AtSIZ1 activity. The E3 ligase activity of AtSIZ1 against CRU2 and CRU3 was determined in the presence or absence of His6-AtSAE1þ2, His6-AtUBC9, MBP-AtSIZ1, His6-AtSUMO1-GG, and GST-CRU2 or GST-CRU3. After the reaction, sumoylated CRU2 (left) and CRU3 (right) were detected by western blotting using an anti-Myc antibody.
and siz1-2 mutant seeds, and lower levels of cruciferins in siz1-2 mutant seeds compared with WT seeds, suggested the possibility that total protein content and amino acid composition may be altered in siz1-2 mutant seeds. To test this possibility, we also examined the total protein content and amino acid composition of siz1-2 mutant and WT seeds using the Bradford assay and highperformance liquid chromatography (HPLC) analysis. The results showed that the total protein and amino acid contents were slightly higher in siz1-2 mutant seeds than in WT seeds (Fig. 4A and B). This indicates that the total protein content and level of each amino acid were increased in siz1-2 mutant seeds, despite the reduction in the amount of major seed storage proteins in the mutant background. 4. Discussion Plants accumulate various proteins in seeds during the seed filling stage. Seed proteins are an important source of nitrogen and amino acids for the germinating embryo. Arabidopsis contains two major seed storage proteins: CRUs (12S globulins) and napins (2S albumins). However, CRUs are more abundant than napins. Previously, we reported that the Arabidopsis siz1-2 mutant shows a very low germination percentage compared with the WT [9]. Wan et al. show that the low level of 12S globulins in seeds causes abnormal seed germination and seedling establishment [29]. These data indicate that CRUs are important for seed germination and seedling establishment. In this study, we analyzed the total protein content of siz1-2 mutant and WT seeds. The results show decreased accumulation of CRUs in siz1-2 seeds compared with WT seeds (Table 1). Arabidopsis abi1-1 mutant seeds, with defective CRU dephosphorylation, contain high levels of CRUs and are insensitive to the inhibition of germination and growth by abscisic acid [30]. The results of this study suggest that the low germination rate of siz1-2 mutant seeds [9] is likely due to the low level of CRUs (Table 1). Despite the high transcript levels of CRU1-3 genes in siz1-2 mutant seeds (Fig. 2), the level of encoded proteins was much lower in the siz1-2 mutant compared with the WT (Table 1), strongly suggesting that the CRU protein content is regulated after translation. We speculated two possible scenarios of post-translational
Fig. 4. Total protein and amino acid content of mature seeds of the WT and siz1-2 mutant. (A) Total seed protein content of seeds determined using the Bradford assay. *** indicates a significant difference from the wild type at P < 0.001 by student's t-test. (B) Amino acid content of seeds analyzed by reverse phase HPLC of total seed proteins. Values represent mean ± standard deviation (SD) of three replicates.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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regulation of CRU protein content. First, the level of CRUs could be affected by their sorting regulators. The loss of vascular sorting receptor 1 (VSR1), a receptor that sorts storage proteins to PSVs in Arabidopsis seeds, results in abnormal accumulation of 12S globulin precursors in seeds [31]. Mutations in MAIGO1 (MAG1), MAG2, and MAG4 genes, encoding Golgi-localized tethering factors as VPS29 homologs, also result in abnormal accumulation of 12S globulin precursors in dry seeds [32,33]. Additionally, variation in the level of 12S globulins due to mutations in VSR genes results in abnormal seed germination and seedling growth [34]. Therefore, it can be inferred that CRU sorting regulators are not modified by sumoylation in siz1-2 mutant seeds, which affects the activity and stability of sorting regulators, resulting in lower levels of CRUs in siz1-2 mutant seeds compared with WT seeds. Second, the level of CRUs could be affected by post-translational modifications. CRUs are the major phosphorylated proteins in Arabidopsis seeds, and most of the phosphorylation sites found in CRUs are located on the interchain disulfide bond-containing face of the globulin trimer involved in hexamer formation [29]. The abi1-1 mutant, which is impaired in protein phosphatase 2C activity, exhibits a higher level of CRU phosphorylation compared with the WT [29]. These findings imply that the processing, assembly, and mobilization of CRU proteins are affected by phosphorylation. Additionally, our current analyses strongly indicate that the AtSIZ1-mediated sumoylation system regulates the conformation, processing, and assembly of CRU proteins via the putative sumoylation sites in CRUs (Fig. 3A) and the direct interaction between AtSIZ1 and CRUs (Fig. 3B). This is supported by the results of the in vitro sumoylation assay, confirming that AtSIZ1 exhibits E3 SUMO ligase activity against CRU2 and CRU3 proteins (Fig. 3C). This strongly suggests that AtSIZ1 controls the stability and accumulation of CRUs through its E3 SUMO ligase activity, thus regulating seed germination and seedling growth. Currently, we could not see sumoylated GST-CRU1 because its degradation during overexpression, purification and sumoylation reaction. But, there is still a possibility that CRU1 is sumoylated by E3 SUMO ligase activity of AtSIZ1. Our results indicate that the E3 SUMO ligase AtSIZ1 regulates the amount of major seed proteins via its ligase activity. In addition, our results suggest that the regulation of the level of CRUs by sumoylation influences seed germination and seedling growth. Thus, these results enhance our understanding of the regulatory mechanism of seed storage proteins in Arabidopsis. Further biochemical analysis is needed to understand how AtSIZ1 controls the accumulation of other seed proteins. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements We would like to thank Dr. H.-P. Stuible at Max Planck Institute for Plant Breeding Research for providing Arabidopsis SUMO E1 and E2 enzyme-encoding constructs. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ01327601), Rural Development Administration, Republic of Korea. References [1] P.P. Pang, R.R. Pruitt, E.M. Meyerowitz, Molecular cloning, genomic organization expression and evolution of 12S-seed storage protein genes of Arabidopsis thaliana, Plant Mol. Biol. 11 (1988) 805e820. [2] D.G. Robinson, M. Baumer, G. Hinz, I. Hohl, Vesicle transfer of storage proteins to the vacuole: the role of the golgi apparatus and multivesicular bodies,
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E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds Jun Soo Kwak a, Sung-Il Kim a, Sang Woo Park a, Jong Tae Song b, Hak Soo Seo a, c, * a
Department of Plant Science, Research Institute for Agriculture and Life Sciences, Plant Genomics and Breeding Institute, Seoul National University, Seoul, 08826, South Korea b School of Applied Biosciences, Kyungpook National University, Daegu, 41566, South Korea c Bio-MAX Institute, Seoul National University, Seoul, 08826, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2019 Accepted 17 September 2019 Available online xxx
Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity. © 2019 Published by Elsevier Inc.
Keywords: AtSIZ1 Cruciferin E3 SUMO ligase Seed Storage protein Sumoylation
1. Introduction During development, plant seeds naturally accumulate storage reserves that are mobilized during germination to provide energy and raw materials for early seedling growth. Seed storage proteins are the main source of nitrogen required for seed germination and early seedling growth. The main seed proteins in Arabidopsis thaliana (thale cress) and other crucifers are 12S globulins, also known as cruciferins. In Arabidopsis, cruciferins are encoded by
Abbreviations: AtSIZ1, Arabidopsis E3 SUMO ligase SIZ1; CRU, Cruciferin; DTT, Dithiothreitol; EDTA, Ethylenediaminetetraacetic acid; GST, Glutathione S-Trasnferase; HPLC, High-performance liquid chromatography; IPTG, Isopropyl b-D-1thiogalactopyranoside; MAG, MAIGO; MBP, Maltose binding protein; Ni2þ-NTA, Nickel-nitrilotriacetate; PAGE, Polyacrylamide gel electrophoresis; PCR, Polymerase chain reaction; PMSF, Phenylmethanesulfonyl fluoride; PSV, Protein storage vacuole; qRT-PCR, Quantitative real-time PCR; SDS, Sodium dodecyl sulfate; SUMO, Small ubiquitin-related modifier; VSR1, Vascular sorting receptor 1. * Corresponding author. Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Gwanakro 200, Daehakdong, Gwanak-gu, Seoul, 08826, South Korea. E-mail address: [email protected] (H.S. Seo).
three genes, CRUCIFERIN1 (CRU1), CRU2, and CRU3 [1]. Cruciferins are synthesized in the rough endoplasmic reticulum and then transported to protein storage vacuoles (PSVs) via a Golgidependent [2] or -independent pathway [3e5]. Cruciferins are synthesized as preproproteins and ultimately cleaved into a (30e35 kD) and b (21e25 kD) polypeptides, which are linked to each other via a single interchain disulfide bond [6]. CRU1 and CRU2 share 70% sequence identity and similar size, whereas CRU3 exhibits approximately 50% identity to CRU1 and CRU2, and harbors an extended glutamine-rich region in the center of a larger polypeptide that contributes to its divergence from the other family members. Small ubiquitin-related modifier (SUMO) is a reversible posttranslational modifier, which is covalently conjugated to a lysine residue in the substrate protein by E3 SUMO ligases, key regulators of several different biological functions in plants [7]. The Arabidopsis E3 SUMO ligase AtSIZ1 is an SP-RING finger protein harboring a DNA-binding SAP domain and zinc finger Miz domain. AtSIZ1 is involved in seed germination [8,9], flowering [10,11], epigenetic regulation [12,13], nutrient utilization [14,15], stress response [16,17], root branching [18], and secondary cell wall
https://doi.org/10.1016/j.bbrc.2019.09.064 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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formation [19]. The siz1-2 mutant displays small leaves and severe dwarfism, indicating that AtSIZ1 also plays a central role in vegetative growth and development [20e22]. Additionally, siz1-2 mutant plants exhibit defective seed development, short silique length, and lower seed amount (approximately 50% of the wild type) [14]. Interestingly, phenotypic features of the siz1-2 mutant are restored with the supply of nitrogen, particularly in the form of ammonium [14], suggesting that the vegetative and reproductive phenotypes of the siz1-2 mutant are a result of the impaired nitrate reduction pathway due to the mutation in AtSIZ1. Based on these data, we speculated that abnormal seed development in the siz1-2 mutant causes nutritional defects in seeds, which influence seed germination and seedling growth. Therefore, we investigated the role of AtSIZ1 in the regulation of the level of seed storage proteins in Arabidopsis. Here, we provide the first evidence that the amount of seed storage proteins is regulated by the sumoylation system. The levels of CRUs were decreased in the siz1-2 mutant, and CRU2 and CRU3 proteins were conjugated with SUMO by the E3 ligase activity of AtSIZ1 through direct interaction, indicating that the abundance of CRUs in seeds is regulated by AtSIZ1.
AGCGTGGTTGGAACTCTGAT-3’; CRU2, 50 -CCGGTATTTGGAGAAGGTCA-30 and 50 -GGTTGTTTCCGGCTATCAAA-3’; CRU3, 50 CAGCAGCTTCAGAACCAACA-30 and 50 -TAGCTGTTGACGCTGGTCAC3’; ACT7, 50 -ACCACTACCGCAGAAC-30 and 50 -GCTCATACGGTCAGCA3’. 2.4. Yeast two-hybrid assays Yeast two-hybrid assays were performed using the GAL4-based two-hybrid system (Clontech). Full-length AtSIZ1 cDNA was fused with the GAL4 activation domain (AD) and cloned into the pGAD424 vector (Clontech), whereas fell-length CRU1, CRU2, and CUR3 cDNAs were fused to the GAL4 DNA-binding domain (BD) and cloned into the pGBT8 vector (Clontech). The AD-AtSIZ1 construct was co-transformed with BD-CRU1, BD-CRU2, or BD-CRU3 into the yeast strain AH109 using the lithium acetate method, and yeast cells were grown on minimal medium lacking leucine and tryptophan (-Leu/-Trp). To test interactions between AtSIZ1 and CRU proteins, the transformants were plated on minimal medium lacking Leu, Trp, and histidine (-Leu/-Trp/-His) and containing 2 mM 3-amino-1, 2, 4-triazole (3-AT).
2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia-0 (WT) and T-DNA insertion knockout mutant siz1-2 were used in this study. Seeds were germinated on agar plates containing Murashige and Skoog (MS) medium supplemented with 2% sucrose and 0.8% agar (pH 5.7). To grow plants in soil, seeds were directly sown in sterile vermiculite. All plants including seedlings were grown in a growth chamber at 22 C and under a 16 h light/8 h dark cycle. 2.2. Two-dimensional gel electrophoresis Dried seeds (150 mg) were ground in liquid nitrogen, and then 1.6 ml TCAAEB buffer containing acetone, 10% TCA, and 0.07% bmercaptoethanol was added to the ground seeds. The homogenate was incubated at 20 C for 1 h and then centrifuged at 15,000 rpm for 15 min. Pellets were washed with buffer (acetone containing 0.07% b-mercaptoethanol, 2 mM ethylenediaminetetraacetic acid [EDTA], and protease inhibitor cocktail tablets [Sigma]), solubilized in lysis buffer (9 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate, 18 mM Tris-Cl [pH 8.0], 14 mM Trizma base, protease inhibitor cocktail, 0.2% Triton X-100, and 50 mM dithiothreitol [DTT]), and then sonicated. Samples were centrifuged at 15,000 rpm at 4 C for 15 min, and supernatants were concentrated with cold acetone. The amount of protein was measured using the Bradford assay [23]. The extracted proteins were separated by two-dimensional gel electrophoresis, according to Gallardo et al. (24). 2.3. Quantification of CRU transcript levels The WT and siz1-2 mutant seeds were imbibed in distilled water for one day, and samples were collected at the indicated time points. Total RNA was extracted from each sample and quantified. First-strand cDNA was synthesized from 5 mg total RNA using the iScript™cDNA Synthesis Kit (Bio-Rad). An equal volume of cDNA was used as a template for qRT-PCR according to the manufacturer's protocol (MyiQ; Bio-Rad). The CT values of the target genes were normalized relative to the CT value of the Arabidopsis Actin7 gene (At5g09810). The primers used for quantitative PCR were as follows: CRU1, 50 -GCCGTCACACGTACTGAAGA-30 and 50 -
2.5. Construction of recombinant plasmids To produce GST (glutathione S-transferase)-CRU2-Myc and GST-CRU3-Myc constructs, full-length CRU2 and CRU3 cDNAs were amplified by PCR using a primer containing the Myc sequence. The resulting PCR products were cloned into the pET28a (Novagen) and pGEX4T-1 (Amersham Biosciences) vectors. To construct the MBP (Maltose binding protein)-AtSIZ1 fusion, full-length AtSIZ1 cDNA was amplified by PCR using gene-specific primers and cloned into the pMALc2 vector (New England Biolabs). Full-length Arabidopsis SUMO1 cDNA was amplified by PCR using genespecific primers and cloned into pET28a to produce His6AtSUMO1-GG. The SUMO E1 and E2 constructs were kindly provided by Dr H.P. Stuible [25]. All constructs were transformed into E. coli BL21/DE3 (pLysS) cells. The transformed cells were treated with Isopropyl b-D-1thiogalactopyranoside (IPTG) to induce the production of recombinant fusion proteins. All constructs were subjected to automated DNA sequencing to rule out the possibility of mutations. 2.6. Purification of recombinant proteins All recombinant proteins were expressed in E. coli strain BL21 and purified according to the manufacturer's instructions. Briefly, to purify His6-AtSAE1b, His6-AtSAE2, His6-AtSCE1, and His6AtSUMO1-GG, bacteria were lysed in a buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 1% Triton X-100, 1 mM imidazole, 5 mM DTT, 2 mM phenylmethanesulfonyl fluoride (PMSF), and proteinase inhibitor cocktail (Roche). Recombinant proteins were purified using nickel-nitrilotriacetate (Ni2þ-NTA) resins (Qiagen). To purify GST-CRU2-Myc and GST-CRU3-Myc proteins, bacteria were lysed in phosphate-buffered saline (PBS; pH 7.5) containing 1% Triton X-100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). The recombinant proteins were purified using glutathione resins (Pharmacia). To purify MBP-AtSIZ1, bacteria were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). The recombinant protein was purified using the amylose resin (New England Biolabs). Protein concentration was determined using the Bradford assay (Bio-Rad) [24].
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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2.7. Sumoylation assays In vitro sumoylation assays were performed in 30 ml reaction buffer (20 mM HEPES [pH 7.5], 5 mM MgCl2, and 2 mM ATP), each with 50 ng His6-AtSAE1b, His6-AtSAE2, and His6-AtSCE1; 8 mg His6AtSUMO1-GG; and 100 ng GST-CRU2-Myc or GST-CRU3-Myc, with or without 500 ng MBP-AtSIZ1. After incubation at 30 C for 3 h, the reaction mixtures were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE). Sumoylated GST-CRU2-Myc and GST-CRU3Myc proteins were detected by western blotting using an anti-Myc antibody (Santa Cruz Biotechnology). 2.8. Analysis of amino acid composition To determine the amino acid composition of seeds, total seed proteins were extracted using buffer containing 50 mM Tris-Cl (pH 8.0), 100 mM NaCl, 1% NP-40, and 1 mM EDTA. To precipitate the proteins, four volumes of cold acetone was added, and samples were centrifuged at 4 C for 20 min. The pellets were dried and hydrolyzed with 30 ml of 6 N HCl. After 24 h incubation at 130 C, the acid was removed by vacuum, and the hydrolyzates were derivatized using phenylisothiocyanate. The derivatized amino acids were filtered through a 0.45 mm filter, redissolved in 100 ml phosphate buffer, and analyzed by reverse phase HPLC using an Ultimate 3000 system. This experiment was repeated three times.
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Table 1 Identification of the proteins that were decreased in siz1-2 mutant seeds. Spot
3406 3407 4101 4406 5406 5407 6401 6402 6403 6505 7305 7306 7408 0103 0812 0813
Homologous protein
CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU1 (CRUCIFERIN 1) CRU3 (CRUCIFERIN 3) CRU1 (CRUCIFERIN 1) Unknown CRU3 (CRUCIFERIN 3) CRU3 (CRUCIFERIN 3) CRU2 (CRUCIFERIN 2) CRU2 (CRUCIFERIN 2) CRU1 (CRUCIFERIN 1) Oleosin 2 Heat shock protein 70 Heat shock protein 70B
a
NM (kDa)
52.9 52.9 19.9 52.9 52.9 58.5 52.9 48.2 58.5 44.0 50.8 50.8 52.9 21.2 71.4 71.2
pI value
7.68 7.68 7.88 7.68 7.68 6.53 7.68 5.77 6.53 6.08 6.52 6.52 7.68 9.36 5.14 5.30
Intensity WT
siz1-2
6208 1373 22,218 5274 6319 2308 6505 8787 6478 8021 12,476 5281 8407 11 0 0
5389 218 14,082 2006 3578 1065 4254 5624 4067 4602 9361 3341 3941 295 512 807
This experiment was repeated three times using WT and siz1-2 mutant seeds. Here, one result is shown. a NM: Normal mass.
an unknown protein. By contrast, the signal intensity of 3 of the 16 spots was enhanced in the siz1-2 mutant compared with the WT. Among these three proteins, one was identified as an oleosin and two were identified as heat shock proteins (Table 1).
3. Results 3.2. Increased abundance of CRU transcripts in siz1-2 seeds 3.1. Low abundance of cruciferins in siz1-2 seeds The siz1-2 mutant displays abnormal silique and seed phenotypes [14]. This suggests that the amount and composition of nutrients such as carbohydrates, lipids, and proteins may be changed in siz1-2 mutant seeds. In this study, we analyzed the amount and composition of seed storage proteins in mature seeds of WT and siz1-2 mutant plants using proteomics. Total proteins were extracted from mature WT and siz1-2 mutant seeds and analyzed by two-dimensional gel electrophoresis (Fig. 1). Next, we determined the identity of proteins corresponding to these spots by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry analysis. A total of 67 spots showed excellent reproducibility in three replicate experiments. Among these 67 spots, 16 showed differential signal intensities between the siz1-2 mutant and WT seeds (Table 1). The signal intensity of 13 of the 16 spots was decreased in siz1-2 mutant seeds compared with the WT (Table 1). Proteins corresponding to 12 of the 13 spots were identified as CRUs, including CRU1 (7 spots), CRU2 (2 spots), and CRUS (3 spots), while one spot corresponded to
Fig. 1. Proteomic analysis of seed storage proteins in WT and siz1-2 mutant. Red arrows indicate the identified protein spots showing major differences between the siz12 mutant and the WT.
A lower level of CRUs in siz1-2 seeds could result from two possible causes: (1) lower expression of CRU genes in siz1-2 seeds compared with the WT, or (2) post-translational modification of CRU proteins. To test the first possibility, we examined the transcript levels of CRU1-3 genes in siz1-2 mutant and WT seeds. Total RNA was isolated from mutant and WT seeds imbibed in water for 24 h, and transcript levels of CRU1-3 genes were analyzed by qRTPCR. The results showed that transcript levels of these genes were significantly higher in siz1-2 mutant seeds than in WT seeds (Fig. 2), while their protein levels were lower in siz1-2 seeds than in WT seeds (Table 1). However, transcript levels of these genes were similar in WT and siz1-2 seeds after imbibition. This result implies that the stability and amount of CRU1, CRU2, and CRU3 proteins are regulated at the post-translational level during seed maturation. 3.3. Sumoylation of CRU proteins by AtSIZ1 Despite the high transcript levels of CRU genes in siz1-2 mutant seeds (Fig. 2), the corresponding protein levels were reduced in mutant seeds compared with the WT (Table 1). In many cases,
Fig. 2. Expression analysis of CRU genes in the WT and siz1-2 mutant. Transcript levels of genes encoding CRU1, CRU2, and CRU3 were analyzed by qRTePCR using genespecific primers. Actin 7 gene was used to normalize qRTePCR data. Black and white bars indicate WT and siz1-2 seeds, respectively.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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either the ubiquitinated proteins are degraded by the 26S proteosome complex, or the function and stability of these proteins are altered [26,27]. However, sumoylated proteins are not degraded. Instead, these proteins adopt important cellular functions [27,28]. This led us to search the amino acid sequences of CRU proteins. The deduced amino acid sequences of CRUs displayed putative sumoylation sites (Fig. 3A). We thus examined possible interaction between AtSIZ1 and CRUs by the yeast two-hybrid assay. Results showed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3 proteins (Fig. 3B), suggesting that AtSIZ1 is involved in the sumoylation of CRU1-3 proteins via its E3 SUMO ligase activity. To confirm that AtSZ1 regulates CRU sumoylation, we generated the translational fusion constructs of the MBP gene with AtSIZ1 (MBP-AtSIZ1) and of GST with CRU genes (GST-CRU2 and GST-CRU3). The MBP-AtSIZ1 recombinant protein was co-expressed with each of the three GST-CRU fusion proteins in E. coli separately using IPTG treatment. The recombinant proteins were purified using amylose and glutathione resins, and subjected to in vitro sumoylation assays. The GST-CRU2 and GST-CRU3 fusions were sumoylated by AtSIZ1, and the reaction was dependent on the E1 and E2 activity (Fig. 3C). In addition, if AtSIZ1 was not included in the reaction, GST-CRU2 and GST-CRU3 were not sumoylated in the presence of enzymes E1 and E2. Thus, these results indicate that CRU2 and CRU3 are sumoylated by the E3 ligase activity of AtSIZ1. 3.4. Increased abundance of total proteins and amino acids in siz12 seeds Differences in the level of other seed proteins between the WT
Fig. 3. AtSIZ1 sumoylates CRU proteins. (A) Putative sumoylation sites (jKXE) identified from deduced amino acid sequences of CRU1, CRU2, and CRU3 using the SUMOplot Analysis Program. (B) Yeast two-hybrid analyses of the interaction between AtSIZ1 and CRUs. CRU1, cruciferin1; CRU2, cruciferin2; CRU3, cruciferin3. (C) In vitro sumoylation of CRU2 and CRU3 by AtSIZ1 activity. The E3 ligase activity of AtSIZ1 against CRU2 and CRU3 was determined in the presence or absence of His6-AtSAE1þ2, His6-AtUBC9, MBP-AtSIZ1, His6-AtSUMO1-GG, and GST-CRU2 or GST-CRU3. After the reaction, sumoylated CRU2 (left) and CRU3 (right) were detected by western blotting using an anti-Myc antibody.
and siz1-2 mutant seeds, and lower levels of cruciferins in siz1-2 mutant seeds compared with WT seeds, suggested the possibility that total protein content and amino acid composition may be altered in siz1-2 mutant seeds. To test this possibility, we also examined the total protein content and amino acid composition of siz1-2 mutant and WT seeds using the Bradford assay and highperformance liquid chromatography (HPLC) analysis. The results showed that the total protein and amino acid contents were slightly higher in siz1-2 mutant seeds than in WT seeds (Fig. 4A and B). This indicates that the total protein content and level of each amino acid were increased in siz1-2 mutant seeds, despite the reduction in the amount of major seed storage proteins in the mutant background. 4. Discussion Plants accumulate various proteins in seeds during the seed filling stage. Seed proteins are an important source of nitrogen and amino acids for the germinating embryo. Arabidopsis contains two major seed storage proteins: CRUs (12S globulins) and napins (2S albumins). However, CRUs are more abundant than napins. Previously, we reported that the Arabidopsis siz1-2 mutant shows a very low germination percentage compared with the WT [9]. Wan et al. show that the low level of 12S globulins in seeds causes abnormal seed germination and seedling establishment [29]. These data indicate that CRUs are important for seed germination and seedling establishment. In this study, we analyzed the total protein content of siz1-2 mutant and WT seeds. The results show decreased accumulation of CRUs in siz1-2 seeds compared with WT seeds (Table 1). Arabidopsis abi1-1 mutant seeds, with defective CRU dephosphorylation, contain high levels of CRUs and are insensitive to the inhibition of germination and growth by abscisic acid [30]. The results of this study suggest that the low germination rate of siz1-2 mutant seeds [9] is likely due to the low level of CRUs (Table 1). Despite the high transcript levels of CRU1-3 genes in siz1-2 mutant seeds (Fig. 2), the level of encoded proteins was much lower in the siz1-2 mutant compared with the WT (Table 1), strongly suggesting that the CRU protein content is regulated after translation. We speculated two possible scenarios of post-translational
Fig. 4. Total protein and amino acid content of mature seeds of the WT and siz1-2 mutant. (A) Total seed protein content of seeds determined using the Bradford assay. *** indicates a significant difference from the wild type at P < 0.001 by student's t-test. (B) Amino acid content of seeds analyzed by reverse phase HPLC of total seed proteins. Values represent mean ± standard deviation (SD) of three replicates.
Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064
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regulation of CRU protein content. First, the level of CRUs could be affected by their sorting regulators. The loss of vascular sorting receptor 1 (VSR1), a receptor that sorts storage proteins to PSVs in Arabidopsis seeds, results in abnormal accumulation of 12S globulin precursors in seeds [31]. Mutations in MAIGO1 (MAG1), MAG2, and MAG4 genes, encoding Golgi-localized tethering factors as VPS29 homologs, also result in abnormal accumulation of 12S globulin precursors in dry seeds [32,33]. Additionally, variation in the level of 12S globulins due to mutations in VSR genes results in abnormal seed germination and seedling growth [34]. Therefore, it can be inferred that CRU sorting regulators are not modified by sumoylation in siz1-2 mutant seeds, which affects the activity and stability of sorting regulators, resulting in lower levels of CRUs in siz1-2 mutant seeds compared with WT seeds. Second, the level of CRUs could be affected by post-translational modifications. CRUs are the major phosphorylated proteins in Arabidopsis seeds, and most of the phosphorylation sites found in CRUs are located on the interchain disulfide bond-containing face of the globulin trimer involved in hexamer formation [29]. The abi1-1 mutant, which is impaired in protein phosphatase 2C activity, exhibits a higher level of CRU phosphorylation compared with the WT [29]. These findings imply that the processing, assembly, and mobilization of CRU proteins are affected by phosphorylation. Additionally, our current analyses strongly indicate that the AtSIZ1-mediated sumoylation system regulates the conformation, processing, and assembly of CRU proteins via the putative sumoylation sites in CRUs (Fig. 3A) and the direct interaction between AtSIZ1 and CRUs (Fig. 3B). This is supported by the results of the in vitro sumoylation assay, confirming that AtSIZ1 exhibits E3 SUMO ligase activity against CRU2 and CRU3 proteins (Fig. 3C). This strongly suggests that AtSIZ1 controls the stability and accumulation of CRUs through its E3 SUMO ligase activity, thus regulating seed germination and seedling growth. Currently, we could not see sumoylated GST-CRU1 because its degradation during overexpression, purification and sumoylation reaction. But, there is still a possibility that CRU1 is sumoylated by E3 SUMO ligase activity of AtSIZ1. Our results indicate that the E3 SUMO ligase AtSIZ1 regulates the amount of major seed proteins via its ligase activity. In addition, our results suggest that the regulation of the level of CRUs by sumoylation influences seed germination and seedling growth. Thus, these results enhance our understanding of the regulatory mechanism of seed storage proteins in Arabidopsis. Further biochemical analysis is needed to understand how AtSIZ1 controls the accumulation of other seed proteins. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements We would like to thank Dr. H.-P. Stuible at Max Planck Institute for Plant Breeding Research for providing Arabidopsis SUMO E1 and E2 enzyme-encoding constructs. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ01327601), Rural Development Administration, Republic of Korea. References [1] P.P. Pang, R.R. Pruitt, E.M. Meyerowitz, Molecular cloning, genomic organization expression and evolution of 12S-seed storage protein genes of Arabidopsis thaliana, Plant Mol. Biol. 11 (1988) 805e820. [2] D.G. Robinson, M. Baumer, G. Hinz, I. Hohl, Vesicle transfer of storage proteins to the vacuole: the role of the golgi apparatus and multivesicular bodies,
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Please cite this article as: J.S. Kwak et al., E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.064