Supplementary information for Biotechnology Letters Increase in pectin deposition by overexpression of an ERF gene in cultured cells of Arabidopsis thaliana Toshitsugu Nakano1, Yuki Naito1, Koich Kakegawa2, Namie Ohtsuki1, Yayoi Tsujimoto-Inui1, Hideaki Shinshi1, Kaoru Suzuki1,* 1,*Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology

(AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

2Department of Biomass Chemistry, Forestry and Forest Products Research Institute, 1 Matsunosato,

Tsukuba, Ibaraki 305-8687, Japan. E-mail, [email protected]

*Corresponding author: Kaoru Suzuki,

E-mail, [email protected] Methods Confirmation of expression of transgnes To confirm expression of ERF or GUS genes in each transgenic cell line, reverse transcriptase-mediated polymerase chain reaction (RT-PCR) was performed. First strand cDNA was synthesized with SuperScript III (Invitrogen), oligo d(T)12–18 (Invitrogen) and the isolated total RNA. Then, PCR was performed using Ex Taq (Takara Bio. Inc., Shiga, Japan) and the gene-specific primer pairs, which were used for cloning of CDS cDNAs (Supplementary Table 1). For each cell line, -tubulin transcripts were also amplified as a constitutive expression control. Each RT-PCR product was separated by 1.3% (w/v) agarose gel electrophoresis to visualize the amplified DNAs with EtBr staining. Isolation of cell walls The cells were crushed into powder with a pestle and mortar in liquid nitrogen. Then, cell walls were prepared as described previously (Kakegawa et al. 2005). The homogenate was thawed in an ice cold 0.5 M potassium–phosphate buffer solution (pH 7.0), homogenized again with a microhomogenizer (Physcotron, Nichion, Tokyo) on ice, and centrifuged at 4°C. The precipitate was washed twice with the 0.5 M potassium–phosphate buffer (pH 7.0) and three times with water. Lipids and proteins were removed by extraction with chloroform/methanol (1:1, v/v) and phenol/acetic acid/water (2:1:1, v/v), respectively. Following the exclusion of starch by treatment with a-amylase (Sigma, St. Louis, Mo.), cell walls were freeze-dried and used in subsequent experiments. Sequential hydrolysis of cell walls The isolated cell walls were incubated with 2 M trifluoroacetic acid (TFA) at room temperature for an hour and then autoclaved at 121°C for an hour. After centrifugation, the supernatant was dried and then resolved in water. This was referred to as the TFA-soluble fraction. The residue after the hydrolysis with TFA was soaked in 72% (w/w) H2SO4 and incubated at room temperature for 3 h, and then diluted with 34 volumes of water and incubated at 100°C for 2 h. The hydrolysate was neutralized with Ba(OH)2 and centrifuged. The supernatant was referred to as 72% H2SO4-soluble fraction. Determination of sugar content and composition The amounts of total carbohydrate were determined by the phenol–sulfuric acid method with glucose as a standard (Dubois et al. 1956). In this assay, uronic acid sugars showed reduced reactivity in comparison with neutral sugars. The amounts of uronic acids were determined by the modified carbazole–sulfuric acid method of Galambos (1967) with galacturonic acid as a standard. Neutral sugars showed no reactivity in this assay. The composition of neutral and acidic glycosyl residues in the fractions obtained by sequential hydrolysis was determined as trimethylsilyl ether of methyl glycosides following methanolysis (Selvendran and Ryden 1990). The derivatives were analyzed by gas–liquid chromatography (GLC) as described by York et al. (1986). References Dubois M, Gillis KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356 Galambos JT (1967) The relation of carbazol with carbohydrates. I. Effect of borae and sulfamate on the carbazole color of sugars. Anal Chem 19:119–132 Kakegawa K, Ishii T, Matsunaga T (2007) Effects of boron deficiency in cell suspension culture of Populus alba L. Plant Cell Rep 23:573–578 Selvendran RR, Ryden P (1990) Isolation of and analysis of plant cell walls. In: Dey PM (ed) Methods in plant biochemistry. Vol 2. Carbohydrates. Academic press, London, pp. 549–597 York WS, Darvili AG, McNeil M, Albersheim P (1986) Isolation and characterization of plant cell wall and plant cell component, Methods Enzymol 118:3–40 Supplementary Table 1 Primers for PCR used in this study Name Forward Reverse GUS CDS-attB 5′- 5′- AAAAAGCAGGCTCCATGTTA AGAAAGCTGGGTTCATTGTTT CGTCCTGTAGAAACC-3′ GCCTCCCTGC-3′ At1g44830 CDS 5′- 5′- CACCATGGTGAAAACACTTC TCAGCAGAAGTTCCATAATCT AAAAGACAC-3′ GATA-3′ TUB8 5′- 5′- CGTGGATCACAGCAATACAG CCTCCTGCACTTCCACTTCGTC AGCC-3′ TTC-3′ UGP1 5′- 5′- CGTTCGATGGCCTTACTGAG GATTCTTGGTCTCGGCAACAT- A-3′ 3′ MUM4 5′- 5′- TGAGGGTTCGGGTATAGGTT AGGTCTGAGGAGATTGGCATC TC-3′ -3′ AXS2 5′- 5′-CTCCCGTCGCAGAAGATTG- GGGTGCTGAGAATGGACTC 3′ G-3′ GAE1 5′- 5′- GAAGATGAGCTGTTTCCGTC AAGAAGCGGTGAGAGCGATG- A-3′ 3′ QUA1 5′- 5′- TCTCTCCTTCTTCTTCGCTTC GATCTGAATCAACCGTGGCTT A-3′ A-3′ ACT2 5′- 5′- GATCGGTGGTTCCATTCTTG- TTCTGTGAACGATTCCTGGAC- 3′ 3′

Supplementary Fig. 1 Confirmation of overexpression of the ERF gene in A17 calli. To confirm expression of ERF or GUS genes in each transgenic cell line, reverse transcriptase-mediated polymerase chain reaction (RT-PCR) was performed. Total RNA was prepared from three independent lines of the GUS or A17 calli.

First strand cDNA was synthesized with SuperScript III (Invitrogen), oligo d(T) 12–18 (Invitrogen) and the isolated total RNA. Then, PCR was performed using Ex Taq (Takara Bio. Inc., Shiga, Japan) and the gene- specific primer pairs, which were used for cloning of a CDS of the ERF gene (At1g44830) (Supplementary Table 1). For each cell line, -tubulin transcripts (TUB8) were also amplified as a constitutive expression control. Each RT-PCR product was separated by 1.3% (w/v) agarose gel electrophoresis to visualize the amplified DNAs with ethidium bromide staining Supplementary Fig. 2 The amounts of total carbohydrates and uronic acids and the composition of glycosyl residues of TFA-soluble and -insoluble fractions of cell walls. The cell walls were isolated from the GUS and A17 calli and fractionated into TFA-soluble and -insoluble fractions. The amounts of total carbohydrates and uronic acids and the composition of glycosyl residues of each fraction were determined. The amounts of total carbohydrates (white bars) and uronic acids (black bars) were shown as sugar amounts (mg/mg CW) in A. The compositions of glycosyl residues of TFA-soluble and -insoluble fractions from the GUS (black bars) and A17 (white bars) calli were shown as the relative sugar amounts (%) in (b) and (c), respectively. Data are shown as mean ± standard deviation of values taken from three independent lines