Plant Biotechnol Rep (2010) 4:149–155 DOI 10.1007/s11816-010-0130-7 ORIGINAL ARTICLE Transcript accumulation of carotenoid biosynthesis genes in the cyanobacterium Synechocystis sp. PCC 6803 during the dark-to-light transition is mediated by photosynthetic electron transport Jee-Youn Ryu • Ji-Young Song • Youngho Chung • Young-Mok Park • Wah Soon Chow • Youn-Il Park Received: 16 December 2009 / Accepted: 27 January 2010 / Published online: 18 February 2010 Ó Korean Society for Plant Biotechnology and Springer 2010 Abstract Expression of the genes for carotenoid bio- mutations in cyanobacterial photoreceptors, such as phy- synthesis (crt) is dependent on light, but little is known tochromes (cph1, cph2 and cph3) and a cryptochrome-type about the underlying mechanism of light sensing and sig- photoreceptor (ccry), or respiratory electron transport nalling in the cyanobacterium Synechocystis sp. PCC 6803 components NDH and Cyd/CtaI. However, treatment with (hereafter, Synechocystis). In the present study, we inves- photosynthetic electron transport inhibitors significantly tigated the light-induced increase in the transcript levels of diminished the accumulation of crt gene transcripts. Synechocystis crt genes, including phytoene synthase Therefore, the light induction of the Synechocystis crt gene (crtB), phytoene desaturase (crtP), f-carotene desaturase expression is most likely mediated by photosynthetic (crtQ), and b-carotene hydroxylase (crtR), during a dark- electron transport rather than by cyanobacterial photore- to-light transition period. During the dark-to-light shift, the ceptors during the dark-to-light transition. increase in the crt transcript levels was not affected by Keywords Carotenoid biosynthesis gene Á Light induction Á Photoreceptor Á Photosynthetic electron transport Á Synechocystis & J.-Y. Ryu Á J.-Y. Song Á Y.-I. Park ( ) Abbreviations Department of Biological Sciences, College of Biological Science and Biotechnology, Chl Chlorophyll Chungnam National University, Daejeon 305-764, Korea CCCP Carbonylcyanide-3-chlorophenylhydrazone e-mail: [email protected] Cph Cyanobacterial phytochrome Ccry Cyanobacterial cryptochrome Y. Chung Proteome Analysis Team, Korea Basic Science Institute, CtaI Cyt aa3-type Cyt c oxidase Daejeon 305-333, Korea Cyd Cyt bd quinol oxidase Cyt Cytochrome Y.-M. Park DBMIB 2,5-Dibromo-3-methyl-6-isopropyl-p- Mass Spectrometer Research Center, Korea Basic Science Institute, benzoquinone Daejeon 305-333, Korea DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea Fo Instantaneous (dark) acridine yellow W. S. Chow fluorescence Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, MV Methyl viologen The Australian National University, NDH NAD(P)H dehydrogenase Canberra, ACT0200, Australia PQ Plastoquinone WT Wild-type Y.-M. Park Á Y.-I. Park Graduate School of Analytical Science and Technology, DF/Fo Relative change of acridine yellow fluorescence Chungnam National University, Daejeon 305-764, Korea induced by illumination 123 150 Plant Biotechnol Rep (2010) 4:149–155 Introduction increased cytosolic pH, which was generated by acceler- ated respiratory electron transport (Ryu et al. 2004), which Carotenoids are present in all photosynthetic organisms, in turn implies the involvement of the photosynthetic or where they not only participate in light harvesting but also respiratory electron transport process in crt gene expres- protect the photosynthetic apparatus against photo-oxida- sion, even during the dark-to-light shift period. In the tive damage (Frank and Cogdell 1996; Kirilovsky 2007). present study, various Synechocystis mutants of phyto- Photoreceptors and photosynthetic electron transport have chromes, cryptochrome, and respiratory electron transport been shown to be involved in the light induction of carot- components were used to address whether cyanobacterial enoid biosynthesis (crt) genes. Both phytochrome (von photoreceptors or respiratory electron transport act as a Lintig et al. 1997; Welsch et al. 2000) and blue light pho- trigger for the expression of crt genes upon illumination. toreceptor (Bohne and Linden 2002) are critical for crt gene Furthermore, specific inhibitors that modulate the redox expression in higher plants. Additionally, photosynthetic state of photosynthetic electron transport components in electron transport seems to be responsible for eukaryotic the thylakoid membrane were also used to search for the algal crt gene expression (Steinbrenner and Linden 2003). initial light-sensing site. Our results indicated that the Carotenoid contents in various cyanobacteria, including redox state of the PSI acceptor side of the photosynthetic Aphanocapsa (Nonnengeisser et al. 1996), Microcystis electron transport chain is closely related to expression of aeruginosa (Woitke et al. 1997), Synechocystis (Steiger the crt genes in Synechocystis during the dark-to-light et al. 1999), and Plectonema boryanum (Ivanov et al. transition period. 2000), are increased in a light intensity-dependent manner. Additionally, the expression of crt genes is also light intensity dependent, as shown in the cyanobacterium Syn- Materials and methods echocystis (Fernandez-Gonzalez et al. 1998). However, little is known about which photoreceptors are involved in Cell strains, growth conditions, and inhibitors cyanobacterial crt expression during the dark-to-light transition. Cells from wild-type (WT) Synechocystis sp. PCC 6803 The Gram-negative cyanobacterium Synechocystis is a and its mutants defective in various photoreceptors model organism that is widely used to study photosynthe- (cph1-, cph2-, cph3-, and ccry-) and respiratory electron sis, as well as adaptation to various environmental stresses flow (M55 and Cyd/CtaI) were grown in BG-11 inorganic (Douglas 1998; Burja et al. 2003). In Synechocystis, there medium buffered with 5 mM N-tris(hydroxymethyl) are several photoreceptors and photosynthetic electron methyl-2-aminoethane-sulfonic acid (TES)-NaOH (pH transport components that may be candidates for light 8.0) at a photon flux density of 30 lmol m-2 s-1 unless sensing and the signalling of crt gene expression. It has otherwise indicated (Ryu et al. 2004). For the dark-to-light been shown that three different phytochrome homologues, shift experiments, photoautotrophically grown cells in Cph1 (Slr0473), Cph2 (Sll0821), and Cph3 (Sll1124), are the exponential growth phase were incubated in the dark capable of binding to chromophores and undergoing for 16 h and shifted to 100 lmol m-2 s-1 for 30 min. reversible photoconversion in vitro (Hughes et al. 1997; When required, electron transport inhibitors (10 lM for Wilde et al. 1997). The cryptochrome-type photoreceptor is DCMU, DBMIB and CCCP, 0.1 mM for MV) were added also known to be encoded by the sll1629 gene, as its to the cell cultures 5 min before illumination. The tran- deduced amino acid sequence shows a strong sequence scription inhibitor rifampicin (Rif) was added at a final similarity to cryptochrome (Brudler et al. 2003). Addi- concentration of 300 lgml-1. Chl content was estimated tionally, the entire Synechocystis photosynthetic apparatus from the absorption spectra of intact cells (Myers et al. has the potential for light sensing and signalling for crt 1980). gene expression. Indeed, electron transfer in the photo- synthetic electron transport chain following light absorp- Mutagenesis tion leads to changes in the redox state of electron transport components such as PSII (Campbell et al. 1995; Hihara Genomic DNA used as the PCR template was isolated et al. 2003), PQ (Alfonso et al. 2000; Li and Sherman as described (Porter 1988). The cph1 (locus slr0473 in 2000), and Cyt b6f complex (Kujat and Owttrim 2000), the CyanoBase, http://www.kazusa.or.jp/cyano), cph2 thereby initiating the signal transduction cascade for the (sll0821), cph3 (sll1124), and ccry (sll1629) genes were expression of genes involved in photosynthesis and RNA amplified by PCR from the genomic DNA using the fol- metabolism. lowing primers: cph1, forward, 50-GCCAAACGCTTCA Previously, we have shown that the glucose induction of GGATAGT-30 and reverse, 50-TTCGTAACTGGCAA crt genes in Synechocystis in the dark was mediated by TCACATC-30; cph2, forward, 50-CGGAATTCATGAA 123 Plant Biotechnol Rep (2010) 4:149–155 151 CCCTAATCGATCCTT-30 and reverse, 50-CGCGGATC Minicycler using the following conditions: initial denatur- CCTAAACTTCCCCATCAACAT-30; cph3, forward, 50-C ation at 95°C for 3 min, followed by 25 (crtB, crtP, and GAGGTACCAGGGAAGTCACCGCCA-30 and reverse, crtQ), 28 (crtR)or8(rrn16Sa) cycles of 95°C for 30 s, 50-CGCGGATCCTGATGCCCCGACAAAAGC-30; and 60°C for 20 s, and 72°C for 30 s, and a final extension step ccry, forward, 50-TGCCGTCTAAATCCTCCAAA-30 and at 72°C for 7 min. These RNA concentrations were within reverse, 50-TTCCGCCAAATAAGCAGCAT-30. The the linear response range of the PCR amplification (data resulting fragments were then cloned into the pGEM-T not shown). The 16S rRNA genes were included as con- Easy vector (Promega). To inactivate cph1, a cassette trols. To detect possible DNA contamination, control conferring spectinomycin resistance (digested with SmaI) reactions were performed without reverse transcriptase, but was inserted by blunt end ligation into the unique ClaI site with Taq polymerase. The PCR products were routinely (position 2,439 of the insert) in the 4.2-kb cph1 PCR analysed on 1.4% agarose gels and viewed with a GEL- product that was cloned into the pCRII vector (Invitrogen). DOC2000 densitometer and