Inorganic carbon limitation induces transcripts encoding components of the CO(2)-concentrating mechanism in Synechococcus sp. PCC7942 through a redox-independent pathway

Abstract

The cyanobacterial CO2-concentrating mechanism (CCM) allows photosynthesis to proceed in CO2-limited aquatic environments, and its activity is modulated in response to inorganic carbon (Ci) availability. Real-time reverse transcriptase-PCR analysis was used to examine the transcriptional regulation of more than 30 CCM-related genes in Synechococcus sp. strain PCC7942 with an emphasis on genes encoding high-affinity Ci transporters and carboxysome-associated proteins. This approach was also used to test hypotheses about sensing of Ci limitation in cyanobacteria. The transcriptional response of Synechococcus sp. to severe Ci limitation occurs rapidly, being maximal within 30 to 60 min, and three distinct temporal responses were detected: (a). a rapid, transient induction for genes encoding carboxysome-associated proteins (ccmKLMNO, rbcLS, and icfA) and the transcriptional regulator, cmpR; (b). a slow sustained induction of psbAII; and (c). a rapid sustained induction of genes encoding the inducible Ci transporters cmpABCD, sbtA, and ndhF3-D3-chpY. The Ci-responsive transcripts investigated had half-lives of 15 min or less and were equally stable at high and low Ci. Through the use of a range of physiological conditions (light and Ci levels) and inhibitors such as 3-(3,4-dichlorophenyl)-1,1dimethylurea, glycolaldehyde, dithiothreitol, and ethoxyzolamide, we found that no strict correlation exists between expression of genes known to be induced under redox stress, such as psbAII, and the expression of the Ci-responsive CCM genes. We argue that redox stress, such as that which occurs under high-light stress, is unlikely to be a primary signal for sensing of Ci limitation in cyanobacteria. We discuss the data in relation to current theories of CO2 sensing in cyanobacteria.

Figure 1.

Phosphoenolpyruvate carboxylase (ppc) transcript abundance in Ci-limited Synechococcus cells. Exponentially growing high-Ci cells were swapped to buffer equilibrated with CO₂-free air and bubbled with CO₂-free air for 3 h. The abundance of ppc transcript relative to time zero was determined by real-time PCR using cDNA pools generated from normalized, total RNA extracts from cells collected at various time points. Symbols represent the extent of induction or repression after the shift to low Ci at each time point as a percentage of the high-Ci amount (set at 100%) ± se for three separate measurements. The results of two independent experiments are shown.

Figure 2.

Early transcriptional response of Synechococcus cells subject to a severe Ci limitation. Exponentially growing high-Ci cells were swapped to CO₂-free air equilibrated buffer and bubbled with CO₂-free air for 30 min. The relative abundance of specific transcripts encoding known or putative Ci transporters (A), carboxysome associated proteins (B), or electron transport or regulatory proteins (C), was determined by real-time PCR. Bars represent the extent of induction or repression after the shift to low Ci as a percentage of the high-Ci amount (set at 100%) ± se for three separate measurements. Similar data were obtained from a replicate experiment (data not shown). Note the break in the y axis in A and C. The scale is exponential after the break in A.

Figure 3.

Time course of CCM-related transcript expression in severely Ci-limited Synechococcus cells and induction of a high-affinity CCM. A through C, Relative abundance of a subset of CCM-related transcripts as determined by real-time PCR. Symbols represent the extent of induction or repression after the shift to low Ci at each time point as a percentage of the high-Ci amount (set at 100%) ± se for three separate measurements. D, Maximum photosynthetic rate (V_max) and K_0.5(Ci) for cells collected at each time point. Similar data were obtained from a replicate experiment (data not shown). Note the break in the y axis in part C. The scale is exponential after the break in C.

Figure 4.

Relaxation of the high-affinity CCM in Synechococcus cells. Exponentially growing high-Ci cells were swapped to CO₂-free air equilibrated buffer and bubbled with CO₂-free air for 2 h (termed a H:L transition) and then returned to aeration with 1.7% CO₂ for 48 h (termed a L:H transition). Symbols represent the relative affinity for Ci, K_0.5 (Ci) at each time point or the maximum photosynthetic rate (V_max). Values are averages of at least two independent measurements ± se.

Figure 5.

Turnover of Ci-responsive CCM-related transcripts in Synechococcus cells. Exponentially growing high-Ci cells were swapped to CO₂-free air equilibrated buffer, bubbled with CO₂-free air for 30 min, and then swapped again to either CO₂-free air or 1.7% CO₂ in the presence or absence of 200 μg mL^-1 rifampicin (RIF). The relative abundance of cmpA, cmpR, ccmM, and psbAII at various time points was determined by real-time PCR. Symbols represent the abundance of transcripts at each time point as a percentage of the initial 30-min, low-Ci-induced amount (set at 100%) ± se for three separate measurements.

Figure 6.

Effect of light on CCM-related transcript abundance in Synechococcus cells. Exponentially growing high-Ci cells illuminated with 85 μmol photons m^-2 s^-1 (low light) were transferred to bubbling with CO₂-free air (low Ci) and illuminated with either 85 or 500 μmol photons m^-2 s^-1 (high light) for 30 min. A parallel culture was illuminated with 85 μmol photons m^-2 s^-1 and bubbled with 1.7% CO₂ as a control. The relative abundance of cmpA, sbtA, chpY, and psbAII transcripts was determined by real-time PCR. Bars represent the extent of induction or repression after the shift to low Ci as a percentage of the high-Ci/low-light amount (set at 100%) ± se for three separate measurements. Note the break in the y axis.

Figure 7.

Effect of redox modifiers and inhibitors of CO₂ fixation or CO₂ transport on CCM-related transcript abundance in Synechococcus cells. As determined by real-time PCR, bars represent the extent of induction or repression after the treatment as a percentage of the untreated control amount (set at 100%) ± se for three separate measurements. A, Relative abundance of cmpA, sbtA, and psbAII in high-Ci cells treated for 30 min with 20 μm 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone, 20 μm DCMU, 5 mm DTT_red, 200 μm EZ, or 10 mm GLY. B, Relative abundance of a subset of CCM-related transcripts in cells bubbled with air through a fritted sparge (rapid air; approximately 0.4 L min^-1) and treated for 30 min with 200 μm EZ. y axis scale is exponential after the break. C, Relative abundance of a subset of CCM-related transcripts in high cells swapped to medium air sparging (approximately 0.15 L min^-1) for 30 min in the presence or absence of 10 mm GLY. Note the break in the y axis in B and C.