Journal of Plant Physiology 171 (2014) 292–300 Contents lists available at ScienceDirect Journal of Plant Physiology journa l h o me page: www.elsevier.com/locate/jplph Physiology Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata Masayuki Kakimoto a,b , Toshiki Ishikawa a , Atsuko Miyagi c , Kazuaki Saito b , Motonobu Miyazaki b , Takashi Asaeda a , Masatoshi Yamaguchi c,d , Hirofumi Uchimiya c , Maki Kawai-Yamada a,c,∗ a Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan b Saitama City Institute of Health Science and Research, 7-5-12 Suzuya, Chuo-ku, Saitama City, Saitama 338-0013, Japan c Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan a r t i c l e i n f o Article history: Received 20 June 2013 Received in revised form 21 August 2013 Accepted 4 September 2013 Available online 18 October 2013 Keywords: 2-MIB Cyanobacteria Gene expression Metabolomics Temperature-induction a b s t r a c t A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temper- atures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph–mass spectrometry (GC–MS) measurements showed higher accumulation of 2-MIB at 30 ◦ C than at 4 ◦ C or 20 ◦ C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 ◦ C and increased at 30 ◦ C, compared with at 20 ◦ C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures. © 2013 Elsevier GmbH. All rights reserved. Introduction Tastes and odors in tap water are major problems experienced by water utilities worldwide, with the number of consumer com- plaints being the highest during summer. Two main off-flavor compounds found in water are geosmin and 2-methylisoborneol (2-MIB). The sensitivity of humans to the odor of these com- pounds is very high (below 10 ng/L). Moreover, these compounds are too stable to be metabolized (Westerhoff et al., 2005). Although treatment with activated carbon removes these com- pounds (Lalezary-Craig et al., 1988; Cook et al., 2001), this approach Abbreviations: 2-MIB, 2-methylisoborneol; GPP, geranyl diphosphate; GPPMT, GPP methyltransferase; MIBS, monoterpene cyclase; SAM, S-adenosyl-l- methionine; P. galeata, Pseudanabaena galeata; CE, capillary electrophoresis; TCA, tricarboxylic acid; mtf, methyl transferase; mtc, monoterpene cyclase. ∗ Corresponding author at: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. Tel.: +81 48 858 9269; fax: +81 48 858 9269. E-mail address: mkawai@mail.saitama-u.ac.jp (M. Kawai-Yamada). is too expensive for continuous use. Furthermore, it is difficult to predict the amount of these compounds in water year to year because the intensity of the odor changes annually even though the condition of the water remains similar. Therefore, a method to predict the occurrence of geosmin and 2-MIB in tap water is needed to reduce consumer complaints and the cost of odor treatments. Geosmin and 2-MIB are volatile terpenes (sesquiterpene and monoterpene, respectively). The geosmin biosynthesis pathway has been analyzed in actinomycetes and cyanobacteria. Sesquiter- pene, farnesyl diphosphate, is converted to geosmin by the bifunctional single sesquiterpene cyclase, geosmin synthase (Cane and Watt, 2003; Gust et al., 2003; Jüttner and Watson, 2007; Agger et al., 2008; Krishnani et al., 2008). The genes encoding geosmin synthase have been identified in several organisms (Cyc2 from Streptomyces coelicolor, GeoA from S. avermitilis, and NPUNMOD from Nostoc punctuforme) (Jiang et al., 2007; Ludwig et al., 2007; Giglio et al., 2008). 2-MIB is a methylated monoterpene alcohol, with the addi- tional methyl group derived from S-adenosyl-l-methionine (SAM) (Bentley and Meganathan, 1981). Komatsu et al. (2008) identified 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.09.005