Abstract
Expression controls of the carbon acquisition system in marine diatoms in response to environmental factors are an essential issue to understand the changes in marine primary productivity. A pyrenoidal β-carbonic anhydrase, PtCA1, is one of the most important candidates to investigate the control mechanisms of the CO(2) acquisition system in the marine diatom Phaeodactylum tricornutum. A detailed functional assay was carried out on the putative core regulatory region of the ptca1 promoter using a β-glucuronidase reporter in P. tricornutum cells under changing CO(2) conditions. A set of loss-of-function assays led to the identification of three CO(2)-responsive elements, TGACGT, ACGTCA, and TGACGC, at a region -86 to -42 relative to the transcription start site. Treatment with a cyclic (c)AMP analog, dibutyryl cAMP, revealed these three elements to be under the control of cAMP; thus, we designated them, from 5' to 3', as CO(2)-cAMP-Responsive Element1 (CCRE1), CCRE2, and CCRE3. Because the sequence TGACGT is known to be a typical target of human Activating Transcription Factor6 (ATF6), we searched for genes containing a basic zipper (bZIP) region homologous to that of ATF6 in the genome of P. tricornutum. Gel-shift assays using CCRE pentamers as labeled probes showed that at least one candidate of bZIP proteins, PtbZIP11, bound specifically to CCREs. A series of gain-of-function assays with CCREs fused to a minimal promoter strongly suggested that the alternative combination of CCRE1/2 or CCRE2/3 at proper distances from the minimal promoter is required as a potential target of PtbZIP11 for an effective CO(2) response of the ptca1 gene.
Highlights
Expression controls of the carbon acquisition system in marine diatoms in response to environmental factors are an essential issue to understand the changes in marine primary productivity
Since the impaired sites of LS1 to LS3 and LS5 to LS7 corresponded, respectively, to regions around CRE1 and p300be, it was likely that these two elements are required for the CO2 response, as was assumed previously (Harada et al, 2005), while other sequences including the Skn-1 and the CRE2 elements were not required for the CO2 response of promoter region of ptca1 (Pptca1)
D, qPCR of the ptca1 and ptbZIP11 transcripts was carried out on total RNAs extracted from cells grown in 5% CO2, air, and cells acclimated from 5% CO2 to air for 3, 6, 12, 24, and 48 h
Summary
Expression controls of the carbon acquisition system in marine diatoms in response to environmental factors are an essential issue to understand the changes in marine primary productivity. The activity of CCMs is suppressed under high-CO2 conditions and induced (or derepressed) under normal air (and/or further lower CO2) conditions in a few hours (Kaplan et al, 1980; Miller et al, 1990; Colman and Rotatore, 1995; Matsuda and Colman, 1995; Johnston and Raven, 1996; Matsuda et al, 2001; Kucho et al, 2003; Vance and Spalding, 2005). This indicates that algal cells can perceive changes in ambient CO2 concentration as a signal and acclimate to new CO2 conditions in a short period.
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