The carbonate concentration mechanism of Pyropia yezoensis (Rhodophyta): evidence from transcriptomics and biochemical data

Abstract

Background: Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations.

Results: Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low.

Conclusion: We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

Keywords: Carbon concentrating mechanism; Enzyme activity; Photosynthetic efficiency; Pyropia yezoensis; Transcriptome.

Fig. 1

The photos of leafy thalli (gametophytes) and filamentous thalli (sporophytes). a. leafy thalli. b. filamentous thalli

Fig. 2

pH and YII changes in gametophytes and sporophytes of P. yezoensis under different Ci conditions. SNC, SHC and SLC indicate sporophytes cultivated under normal carbon (NC), high carbon (HC) and low carbon (LC) conditions, respectively. GNC, GHC and GLC indicate gametophytes cultivated under NC, HC and LC conditions, respectively. a. pH change in gametophytes and sporophytes under different Ci conditions. b. YII change in gametophytes and sporophytes under different Ci conditions. Results are represented as mean ± standard deviation

Fig. 3

Transmission electron microscope images of sporophyte (A) and gametophyte (B) cells of P. yezoensis under three Ci conditions. Sporophytes cultivated under NC (A1), LC (A2) and HC (A3); gametophytes cultivated under NC (B1), LC (B2) and HC (B3). PL: plastoglobulus; C: chloroplast; M: mitochondrion; S: starch

Fig. 4

RPKM values of some CA unigenes in gametophytes and sporophytes of P. yezoensis under three Ci conditions. Data represent the mean ± standard deviation from three biological replicates. Means followed by same lowercase letters are not significantly different at p ≤ 0.05 by one-way ANOVA and Tukey’s test. Unigene IDs are DN38784_c0_g1, DN99529_c0_g1, DN105259_c0_g1, DN105005_c0_g1, DN50495_c0_g1, DN127900_c0_g1, and DN87784_c0_g1. SNC, SLC, SHC, GNC, GLC and GHC are as in Fig. 1

Fig. 5

Expression levels of some key genes involved in biochemical CCM based on RNA-Seq assays (A) and relative expression levels verified by qPCR (B) in gametophytes and sporophytes of P. yezoensis under three Ci conditions. Data represent the mean ± standard deviation from three biological replicates. Means followed by same lowercase letters are not significantly different at p ≤ 0.05 by one-way ANOVA and Tukey’s test. ME (DN53078_c0_g1), MDH (DN74954_c0_g1)9, PPDK (DN14534 _c0_g1), PEPCK (DN101889_c0_g3), PEPC (DN107354_c0_g1), PYC (DN105351_c0_g1), CA (DN105005_c0_g1), BCT (DN101765_c1_g1)

Fig. 6

Activities of key enzymes involved in biochemical CCM (A) and the glyoxylate cycles (B) in gametophytes and sporophytes of P. yezoensis under different Ci conditions. Data represent the mean ± standard deviation from three biological replicates. Means followed by same lowercase letters are not significantly different at p ≤ 0.05 by one-way ANOVA and Tukey’s test. SNC, SLC, SHC, GNC, GLC and GHC are as in Fig. 1

Fig. 7

Diagram of the putative biochemical CCM pathway in P. yezoensis based on predicted subcellular location, enzymic activity and the pathway indicated by the transcriptome. Solid lines represent direct action, and dotted lines represent indirect action. Red arrow indicate that the expression of the gene was upregulated under HC conditions. Enzyme activity is indicated in different colors, and the deeper the color, the higher the enzyme activity. CIT:citrate; ICA: isocitrate; SUC: succinate; GLY: glyoxylate; α- KG:α-ketoglutarate