Physical characteristics of the two life cycles under different Ci conditions
The effective photochemical quantum yield of photosystem II is directly related to the CO2 assimilation rate and represents the photosystem II state [22, 23]. In this work, LC had more effect on sporophytes than on gametophytes, indicated by reduced photosynthetic efficiency of YII under LC conditions. Thus, pH change under LC culture was comparatively less for sporophytes relative to gametophytes. Under HC conditions, the photosynthetic efficiency of gametophytes was higher than that of sporophytes, so the pH of HC medium rose more quickly for gametophytes, accordingly. These results indicated that the reactions of sporophytes and gametophytes to the change of Ci concentration are different; sporophytes are more sensitive to the LC relative to gametophytes, while elevated Ci enhances the photosynthetic efficiency of photosystem II in gametophytes more than in sporophytes. This result was in agreement with results of TEM. When P. yezoensis was cultured under LC, starches were firstly consumed, and then lipids. There were more lipid droplets in cells of gametophytes relative to those of sporophytes, enabling gametophytes to endure the Ci deprivation for much longer and maintain growth. Since gametophytes of Pyropia have a low carbonate compensation point, 40.7 ± 11.8 µmol/L at pH 8.2, significantly lower than the natural seawater Ci concentration (about 2.21 mM) , their growth is not limited by Ci and they use the elevated Ci to invest in lipid storage, as shown in Fig. 4. Gametophytes showed higher photosynthetic activity than sporophytes, accumulating more starches and lipid droplets than sporophytes under abundant Ci. Huan (2018) studied the response of the photosynthetic reaction in the two life stages of P. yezoensis to increasing CO2, and found that YII in gametophytes was enhanced as CO2 increased; however, there were no significant differences in YII of sporophytes . Wang (2019) also reported that net photosynthetic rates (Pn) of thalli gametophytes were about 2.9 times that of conchocelis sporophytes of P. haitanensis, and the maximal quantum yield of photosystem II (Fv/Fm) was significantly higher than that of the conchocelis at pH 8.0 . All these results suggest that gametophytes of Pyropia have a higher growth rate than sporophytes under abundant Ci conditions.
Biophysical CCM in the two different life cycles of P. yezoensis
Facing the phenomenon of rising global CO2, researchers are paying more attention to the inorganic carbonate utilization of macroalgae, including Pyropia. Work has been conducted to explore the physiological reaction to rising CO2, the pH compensation point, and the physiological reaction after adding different types of inhibitors [18, 24, 27, 28]. The pH compensation point of gametophytes and sporophytes of P. haitanensis is pH 9.9 and 9.95, respectively, and of gametophytes of P. yezoensis is pH 9.65 [18, 5]. High pH compensation point corresponds to the presence of CCMs [29, 24]. However, despite the above inferences, we still lack direct molecular evidence. In this study, we found higher abundance of most iCA genes in gametophytes than in sporophytes, which indicated that biophysical CCMs play a more important role in gametophytes relative to sporophytes. This might be one of the reasons for gametophytes growing more rapidly than sporophytes.
Since HCO3− is not freely permeable across the lipid bilayer of biological membranes, it is either transported by membrane transporter proteins or obtained in the form of CO2, which is then converted by periplasmic CA . HCO3− uptake in the two different Pyropia life stages has been elucidated by physiological reactions to different types of inhibitors, such as acetazolamide (AZ), eCA inhibitor, ethoxyzolamide (EZ), iCA inhibitor and an inhibitor of the HCO3− transporter, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS). Luo and his coworkers (2002) examined the inhibitory effect of AZ, DIDS and vanadate, an inhibitor of ATPase associated with the plasma membrane, on sporophytes of P. haitanensis. They showed inhibitory rates of 25.3% and 71.3%, respectively, after adding AZ and vanadate, and inferred that eCA is not an important part of Ci uptake in P. haitanensis conchocelis, with most Ci absorption occurring through active transport of HCO3− and CO2 . Later, Li and his coworkers (2016) suggested that the gametophytes of P. yezoensis show active HCO3− uptake by studying their reaction to enhanced CO2 in the atmosphere . Recently, Wang (2019) studied the physiological reaction to Ci utilization between gametophytes and sporophytes of P. haitanensis and found that the Pn of thallus was significantly inhibited by AZ and EZ. For conchocelis, inhibition by EZ was greater than that by AZ. Inhibition of conchocelis by DIDS was greater than that of thallus. All these results indicate that iCA and eCA play essential roles in HCO3− utilization in gametophytes, while iCA is more important than eCA in sporophytes. At the same time, the absorption of HCO3− via the DIDS-sensitive anion transport protein is less important in gametophytes than in sporophytes .
The results from our transcriptome data revalidated the above opinions. Unigenes encoding iCA were induced by LC; moreover, the transcript abundance of iCA genes was significantly higher in gametophytes relative to sporophytes. However, unigenes encoding eCA showed very low RNA amounts in sporophytes, and almost undetectable amounts in gametophytes. We identified some unigenes encoding anion exchange proteins and ABC-transporters, and these were upregulated under LC compared with NC. Moreover, the abundance of these genes encoding bicarbonate transporters was higher in sporophytes relative to gametophytes. These data are in agreement with evidence from inhibitory experiments that anion exchange proteins and ABC- transporters make a great contribution to HCO3− transportation in sporophytes of P. yezoensis.
Bicarbonate transporters are divergent within the microalgae and macroalgae. In Macrocystis pyrifera (Phaeophyta), anion exchange protein plays the main role in bicarbonate uptake, while in microalgae, such as Nannochloropsis oceanica and Chlamydomonas reinhardtii, ABC-transporters play an essential role in HCO3− transportation [31, 32, 33]. In Phaeodactylum tricornutum, the solute carrier family is the HCO3− transporter .
Biochemical CCM in P. yezoensis
Besides the C3 pathway, a C4-like pathway might exist in Pyropia. Fan et al. (2007) constructed an EST library of P. haitanensis sporophytes and found abundant PEPCK ESTs; they primarily inferred that a C4-like pathway might exist in sporophytes of P. haitanensis . Later, genes involved in the C4-like pathway were identified in P. yezoensis, except PPDK . Xie and his coworkers (2013) obtained the transcriptomes of gametophytes and sporophytes of P. haitanensis and found some key genes involved in the C4-pathway, but no unigenes encoding PPDK or MDH. They inferred that the C4-like pathway plays a role in sporophytes, according to relative expression levels of PEPCK and PEPC . In fact, there has not been enough direct evidence so far to support this opinion.
In our work, we not only found all the key genes involved in the C4-like pathway, including unigenes encoding PPDK, but we also detected the activity of these enzymes under different Ci conditions. In our work, genes encoding PEPC and NAD-MDH were all induced by LC; moreover, the abundance of PEPC transcripts in gametophytes was higher than that in sporophytes according to the transcriptome, which was verified by qRT-PCR results (Fig. 5A). Although the amounts of PEPCK unigenes and PPDK unigenes in this transcriptome were relatively low in sporophytes and gametophytes, these enzymes showed high levels of activity, especially PEPCK. All the key enzymes involved in the C4-like pathway showed high levels of activity and a similar pattern in the two life stages under HC and NC conditions, with the exception of PPDK enzyme activity.
Combining the predicted subcellular location results, we constructed an overview of the biochemical CCM of P. yezoensis (Fig. 6). We primarily suggest that the C4-like pathway might be located in the mitochondrion and belong to the PEPCK subtype, and transport concentrated CO2 to the C3 pathway in chloroplasts. Moreover, the C4-like pathway not only plays a role in sporophytes, but also plays a role in gametophytes under NC and HC conditions, unlike the previous hypothesis. This is one of the reasons that gametophytes show higher photosynthesis levels than sporophytes.