Introduction

Seaweeds have adapted to the extremely high salt environment in the ocean, an environment that most of land plants never encounter. High levels of sodium (Na+) in the cell cause osmotic and ionic stress and disturb potassium (K+) uptake and functions due to their similar physicochemical properties, often resulting in a K+ deficiency response (Adams and Shin 2014). Despite the high concentrations of Na+ in seawater, cytosolic concentrations of Na+ are generally maintained at low levels in marine algae, suggesting the existence of Na+ extrusion mechanisms (Kirst 1990; Karsten 2012).

It has been long known that the marine red algae Bangiales (Rhodophyta) which include Pyropia and Porphyra (Sutherland et al. 2011) accumulate K+ in the cytoplasm and either exclude or contain Na+ preferentially in the vacuoles (Eppley 1958; Wiencke et al. 1983). In order to maintain the appropriate cytosolic K+/Na+ ratios, active K+ uptake mechanisms are considered essential. Unlike land plants and green algae (Chan et al. 2012; Pedersen et al. 2012), red algae such as Pyropia yezoensis and Porphyridium purpureum have been reported to possess animal-type Na+/K+-ATPases which extrude three ions of Na+ while taking up two ions of K+ into the cell and they are predicted to provide the driving force for Na+-driven solute transporters (Barrero-Gil et al. 2005; Bhattacharya et al. 2013). There seems a tendency that freshwater algae and land plants utilise H+ gradient generated by H+-ATPases to energise secondary transporters, whereas marine algae make use of Na+ gradient albeit with some exceptions (Chan et al. 2012) and this notion is evolutionarily quite interesting.

Recently, the 43 Mb genome of P. yezoensis was sequenced, with more than 10,000 gene models predicted (Nakamura et al. 2013). In this alga, a gene encoding K+ P-type ATPase, PyKPA1, was found to be phylogenetically related to the animal H+/K+- and Na+/K+-ATPases. Heterologous expression of PyKPA1 in the Escherichia coli strain deficient in K+ uptake demonstrated that PyKPA1 had a growth promoting effect in the K+-limited condition and that addition of Na+ further enhanced the effect of PyKPA1 (Barrero-Gil et al. 2005), suggesting PyKPA1 to be a Na+/K+-ATPase. Although gene expression of PyKPA1 was not altered in response to salt stress in P. yezoensis (Uji et al. 2012a), ectopic overexpression of PyKPA1 in rice plants increased salt tolerance by restoring growth (Kishimoto et al. 2013). Another P-type ATPase, PyKPA2, which shares a 65% sequence identity with PyKPA1 and Na+/H+ antiporters, PySOS1 and PyNhaD, have also been isolated from the genome of P. yezoensis (Barrero-Gil et al. 2005; Uji et al. 2012a, b). These membrane proteins could potentially be the major machineries in ion homeostasis and Na+ extrusion in P. yezoensis; however, ATPases may be too energetically costly to be the major K+ uptake mechanism and additional K+ transporters/channels are expected to exist. Although K+ uptake in land plants is commonly mediated by K+ channels and transporters such as AKT1, HAK5 and KUPs in a model land plant Arabidopsis thaliana (Adams and Shin 2014), proteins with similar sequences and/or function have not been reported in P. yezoensis. In order to resolve the molecular mechanisms underlying the ability of red seaweeds to survive in the marine environment, investigation of regulatory components involved in K+/Na+ homeostasis in P. yezoensis needs to be performed.

Pyropia yezoensis spends the winter in the form of gametophytes, the leafy structure commonly harvested as seaweed, and it turns into sporophytes, the filamentous structure during the summer. In autumn, sporophytes form conchosporangia from which conchospores are emitted to produce a new generation of gametophytes. There are several reports describing that different sets of genes are expressed in the extremely diverse structures observed throughout the life cycle of P. yezoensis: for instance, only 22.5% of ESTs and 1 out of 14 microRNAs are common among gametophytes and sporophytes (Asamizu et al. 2003; Shen et al. 2011; He et al. 2012). Indeed, phase-specific gene expression includes genes encoding urea transporters (PyDUR3s), an alginate lyase (PyAly) and a bromoperoxidase (PyBPO1) (Inoue et al. 2015; Matsuda et al. 2015; Kakinuma et al. 2016). Interestingly, PyKPA1 has been reported to be predominantly expressed in sporophytes while PyKPA2 is expressed specifically in gametophytes (Uji et al. 2012a) These findings strongly suggest the existence of distinct regulatory mechanisms upon K+/Na+ homeostasis in each life stage.

To identify the genes playing roles in K+ deficiency tolerance throughout the life cycle of P. yezoensis, we here constructed full-length complementary DNA (cDNA) libraries using three different stages, gametophytes, conchosporangia and sporophytes, and these libraries were then transferred into the E. coli expression vector system to isolate the genes involved in K+ deficiency response. Candidate genes and possible mechanisms by which P. yezoensis tolerates K+ deficiency are discussed.

Materials and methods

Plant material and growth conditions

The cultivation of Pyropia yezoensis strain U51 was performed as previously reported (Li et al. 2008) with a slight modification. Briefly, free-living sporophytes, free-living conchosporangia and gametophytes attached to polyvinyl alcohol (PVA) monofilaments were suspended in ESL (enriched SEALIFE) media, continuously aerated with filter-sterilised air and grown at 15 °C in a 10 h light/14 h dark photocycle with a light intensity of 60 μmol photons m−2 s−1. The sterile ESL medium was made by dissolving commercially available SEALIFE powder (Marintech Co. Ltd., Tokyo, Japan) in distilled water with added ESS2 solution (Kitade et al. 2002) and this was exchanged weekly.

RNA extraction and cDNA library construction

An excess amount of sporophyte, gametophyte and conchosporangium samples were flash frozen in liquid N2 and ground into fine powder using a mortar and a pestle. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) and 75 μg of total RNA was used to isolate messenger RNA (mRNA) using Ambion Dynabeads mRNA purification kit (Thermo Fisher Scientific). Purified mRNA was concentrated by glycogen precipitation with 1 μL of glycogen, 0.5 volumes of NH4OAc and 2.5 volumes of 100% ethanol; 0.88 ~ 3.31 μg of mRNA was used to create full-length cDNA libraries using CloneMiner II cDNA Library Construction Kit according to the manufacture’s instruction (Thermo Fisher Scientific). In short, hybridisation of Biotin-attB2-Oligo(dT) Primer to the mRNA poly(A) tail and the first strand cDNA synthesis by SuperScript III Reverse Transcriptase were followed by the second strand cDNA synthesis by E. coli Polymerase I and ligation of attB1 Adapter to the 5′ end of the cDNA. The resultant double-stranded cDNA was size fractionated by a column to remove truncated cDNA shorter than 500 bp and cloned into a Gateway entry vector pDONR222 through BP recombination reaction. The cDNA construct was then transformed into ElectroMAX DH10B T1 Phage Resistant Cells to create the final cDNA library. Titre was determined by spreading 1:10 serial dilutions (10−2, 10−3, 10−4) of each library onto LB plates containing kanamycin. Titre was calculated as colony forming unit (cfu mL−1) = colonies on plates × dilution factor / volume plated (mL) and total CFU (cfu) = average titre (cfu mL−1) × total volume of cDNA library (mL). Single colonies were picked and plasmid DNAs (pDNAs) were prepared. Each pDNA was digested by BsrG I to determine the insert size and sequenced using M13 forward and reverse universal primers and the Sanger sequencing technique (HITACHI gene analysis system with ABI PRISM 3100-21 genetic analyser).

Selection of K+ deficiency tolerance-related genes

The pDONR222 entry libraries from gametophyte and conchosporangium samples were transferred into the pBAD-DEST49 Gateway destination vector according to the manufacturer’s instructions (Thermo Fisher Scientific). Plasmid DNA was prepared from the entry library culture grown till an OD600 to be approximately 1.0. Polyethylene glycol (PEG) precipitation was performed to purify pDNA using 0.4 volumes of 30% PEG/Mg solution. The entry library was transferred into the destination vector using Gateway LR Clonase II enzyme and transformed into ElectroMAX DH10B T1 Phage Resistant Cells. Plasmid DNA of the pBAD-DEST49 library prepared from the E. coli culture with an OD600 of approximately 1.0 was transformed into an E. coli strain defective in K+ uptake, TK2463 (Epstein et al. 1993), and selected on minimal media (Ahn et al. 2004) containing 1–3 mM KCl, 0.1% arabinose and ampicillin. For functional analysis, overnight culture of TK2463 expressing pBAD-PyAMT1, PyβCA1 or PyHSP70 grown in KML media (10 g Bacto Tryptone, 10 g KCl, 5 g Bacto Yeast Extract in 1 L MilliQ water) containing ampicillin were pelleted, washed three times with autoclaved MilliQ water, resuspended in autoclaved MilliQ water and dropped onto minimal media containing 30, 1.5 or 1.25 mM KCl, 0.1% arabinose and ampicillin as fivefold serial dilutions.

Sequence analysis

Contig numbers were retrieved from the obtained sequences using the public Pyropia database (Nakamura et al. 2013). Pyropia genes were annotated using blastx function against the non-redundant protein sequences database at the NCBI search engine (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequences were aligned using Vector NTI (Thermo Fisher Scientific).

Results

Construction and validation of full-length cDNA libraries of Pyropia yezoensis

In order to gain molecular information on each stage in the life of P. yezoensis, cDNA libraries derived from gametophyte, sporophyte and conchosporangium samples, whose diverse structures are shown in Fig. 1 (a, b for gametophytes, c for sporophytes and d, e for conchosporangia), were constructed. Total CFU was greater than 107 for all the libraries with the greatest being >108 for the sporophytes library (Table 1). Twenty-four single colonies from each library were randomly picked to validate the diversity of the cDNA libraries and pDNA was digested with restriction enzyme BsrG I to determine the insert size. The recombination efficiency was 100% for all libraries and the average size of inserts was approximately 1 kb (Table 1). Inserted cDNA from each colony was also sequenced and annotated against the public protein sequences database (Table 2). For the gametophytes library, one third of genes were either not annotated or annotated to encode proteins of unknown function and most of the other genes were predicted to encode rather ubiquitous proteins involved in the general biological processes such as protein synthesis, regulation and degradation (Table 2 and Fig. 2). By contrast, more than half of the genes were annotated as unknown in the conchosporangia and sporophytes libraries. The annotated genes were for general functions such as protein synthesis and degradation.

Fig. 1
figure 1

Images of the multiple stages in the life cycle of Pyropia yezoensis. a Gametophytes. b Vegetative cells of gametophytes. c Sporophytes. d Generation of a conchosporangium from a sporophyte. e Conchosporangia. Full-length cDNA libraries were constructed with RNA extracted from gametophytes, sporophytes and conchosporangia

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Table 1 Titre, recombination % and average insert size of cDNA libraries for three life stages of the marine red alga P. yezoensis
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Table 2 Annotation of genes from cDNA libraries for three life stages of the marine red alga P. yezoensis
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Fig. 2
figure 2

Functional categories (%) of representative genes recovered from each cDNA library. Randomly selected 24 colonies from each of gametophytes, conchosporangia and sporophytes libraries were sequenced for the inserted genes and annotated based on the sequence similarities against the public protein sequences database

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Selection of genes potentially involved in K+ deficiency tolerance

In order to isolate genes responsible for efficient K+ utilisation and K+/Na+ balance in P. yezoensis, the entire entry clone libraries from gametophytes and conchosporangia were transferred into the E. coli expression vector system and transformed into TK2463, an E. coli strain defective in K+ uptake. Under the less stringent K+ deficiency conditions (2 or 3 mM KCl), 48 colonies were recovered and 45 genes were successfully sequenced from the gametophytes library (Table 3). Under the stringent condition (1 mM KCl), 23 genes from the gametophytes library and 16 genes from the conchosporangia library were revealed (Table 4). Although a large portion of genes could not be annotated for function as in the entry libraries (34.8% for gametophytes and 62.5% for conchosporangia), a higher number of the annotated genes was associated with specific functions in biological processes such as metabolism and signalling rather than general functions. Of these, three genes were annotated as β-carbonic anhydrase (βCA; two from 2 or 3 mM KCl, one from 1 mM KCl screening). All three were predicted to represent the same gene model (PyβCA1, contig_16545_g4020). Upon sequence alignment with known βCAs from Chlamydomonas reinhardtii (CrCAH4), Ostreococcus tauri (OtβCA) and Arabidopsis (AtβCA5.1), PyβCA1 was shown to possess all three conserved zinc binding sites, two cysteine residues (C) and histidine (H) (marked in blue in Fig. 3) (Provart et al. 1993; Bracey et al. 1994; Kimber and Pai 2000); however, the rest of the sequence was fairly diverse among the species (20.1, 22.0, 23.1% identity with AtβCA5.1, CrCAH4, OtβCA, respectively). Two genes recovered from stringent K+ deficiency screening were predicted to be a single ammonium transporter (PyAMT1, contig_16335_g3953) (Kakinuma et al. 2017). Sequence alignment of PyAMT1 with well-studied AtAMT1;2 (Yuan et al. 2007) and algal OtAMT (Derelle et al. 2006) indicated that many conserved amino acids such as those which form the ammonium binding site, tryptophan (W) and serine (S) (marked in red in Fig. 4), phenylalanine (F) and aspartate (D) (marked in blue) were identical among three, whereas others, such as F and threonine (T) (marked in green), highlighted the difference between algae and land plants (Pantoja 2012). The TK2463 E. coli strains expressing PyβCA1 and PyAMT1 were further analysed in the K+ deficient conditions (1.25 and 1.5 mM KCl) and compared with the strain expressing PyHSP70 as a negative control. The strains expressing PyAMT1 and, to a lesser extent, PyβCA1 grew well in K+ deficiency while the strain expressing PyHSP70 could not survive (Fig. 5). Multiple ribosomal proteins of various sizes were also selected from the gametophyte library (Tables 3 and 4).

Table 3 List of genes selected from the K+ tolerance screening of the gametophytes library expressed in the E. coli strain defective in K+ uptake under mild K+ deficiency (2 or 3 mM KCl)
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Table 4 List of genes selected from the K+ tolerance screening of the gametophytes and conchosporangia libraries expressed in the E. coli strain defective in K+ uptake under severe K+ deficiency (1 mM KCl)
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Fig. 3
figure 3

Amino acid sequence alignment of βCAs. Pyropia yezoensis PyβCA1 (contig_16545_g4020) was aligned with Arabidopsis thaliana AtβCA5.1 (At4g33580), Chlamydomonas reinhardtii CrCAH4 (GI: 159475801) and Ostreococcus tauri OtβCA (GI: 308799709). Identical amino acids among all four βCAs are highlighted as dark grey and identical amino acids between two or three among four are highlighted as light grey. The amino acids which form the conserved zinc binding site are marked in blue

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Fig. 4
figure 4

Amino acid sequence alignment of AMTs. Pyropia yezoensis PyAMT1 (contig_16335_g3953) was aligned with Arabidopsis thaliana AtAMT1;2 (At1g64780) and Ostreococcus tauri OtAMT (GI:693496005). Identical amino acids among all three AMTs are highlighted as dark grey and identical amino acids between two of three AMTs are highlighted as light grey. The amino acids which form the ammonium binding site are marked in red and other conserved amino acids reported are marked in blue (identical among three), green (identical among algae) and orange (not identical)

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Fig. 5
figure 5

Functional analysis of PyAMT1 and PyβCA1 in K+ deficiency. The E. coli strain defective in K+ uptake expressing PyAMT1 and PyβCA1 were grown in the K+ sufficient (30 mM KCl) and K+ deficient (1.5 and 1.25 mM KCl) conditions. PyHSP70 was used as a negative control

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Discussion

Full-length cDNA libraries from various life stages, including gametophytes, conchosporangia and sporophytes, were created for a model marine alga P. yezoensis with excellent recombination percentages and titre. The average insert size of approximately 1 kb corresponds with the predicted average coding sequence length in P. yezoensis (Nakamura et al. 2013). Analysis of the whole genome sequence of P. yezoensis has revealed that the function of 35% of the genes is unknown (Nakamura et al. 2013), and our results obtained from the gametophytes library was consistent with their report. It is intriguing to postulate why the conchosporangia and sporophytes libraries contain more than 50% of genes that are functionally unknown. Many of these genes do not even have any previously characterised conserved amino acid motif or domain, underlining the uniqueness of P. yezoensis genes, especially in the conchosporangia and sporophytes stages.

In the search of contributory factors in K+ deficiency tolerance in P. yezoensis, we identified a series of genes that might be involved in such response as efficient uptake and use of K+. Gametophytes and conchosporangia cDNA libraries were expressed in the E. coli system and selected in two different stringency conditions of K+ deficiency. More colonies were found in the less stringent condition (45 transformants from gametophytes) than in the more stringent condition (23 transformants from gametophytes and 16 transformants from conchosporangia). The ratios of unannotated genes were similar to the pattern in the entry libraries. Unlike the genes annotated in the entry libraries, selected genes were annotated as proteins with specific biological functions rather than ubiquitous proteins, suggesting specific pathways at work in K+ deficiency response.

There were two types of proteins selected multiple times: β-carbonic anhydrase (PyβCA1) and ammonium transporter (PyAMT1). CA catalyses the reversible reaction between CO2 and HCO3 − + H+ and is crucial for aquatic photosynthetic organisms which suffer in the low-CO2 environment to concentrate CO2 in the vicinity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Moroney et al. 2001). The existence of CA activity in marine macroalgae has been known for a long while (Bowes 1969). A βCA has previously been cloned in P. yezoensis and its expression was reported to be the lowest in gametophytes, followed by sporophytes and conchospores (Zhang et al. 2010). Although PyβCA1 selected in this screen is different from the one identified in the previous study, its expression is predicted to be low in gametophytes since the same expression pattern was also observed for P. haitanensis βCAs (Chen et al. 2016). Considering its lower abundance in the original gametophytes library and the fact that three independent transformants were recovered in the low K+ assays, it seems to point to the significance of PyβCA1 in K+ deficiency response. Furthermore, this particular βCA might be important in this response as only one gene was repeatedly isolated though multiple βCAs were expected to exist in the genome. As all three conserved amino acids which contribute to binding of the catalytic zinc ion are present in PyβCA1 (Fig. 3), it is predicted as a functional βCA. By contrast, overall sequence identity is not high among the species tested (approximately 20%) and it is possible that regulation and function of each βCA are distinct. This notion is also supported by the fact that CA is essential for E. coli growth under aerobic conditions probably due to HCO3 − requirement for amino acid, nucleotide and fatty acid synthesis (Merlin et al. 2003), indicating that expression of PyβCA1, but not the innate CA activity of E. coli, could contribute to K+ deficiency tolerance (Fig. 5). Although a direct interaction between βCA and K+ has yet to be reported in seaweeds (Escassi et al. 2002), we speculate that increased carbon source and photosynthesis by PyβCA1 might compensate the loss of K+.

Two independent transformants from the stringent K+ deficiency screen were found to carry a single AMT gene (PyAMT1). During the review process of the current paper, another group reported isolation of PyAMT1 as a functional ammonium transporter whose gene expression is dramatically induced in response to nitrogen deficiency (Kakinuma et al. 2017). It is known that ammonium is preferentially taken up over nitrate by many algae and multiple AMT genes are present and expressed in the Porphyra species (Chan et al. 2012). Amino acid sequence alignment of PyAMT1 with Arabidopsis AtAMT1;2 and green alga O. tauri OtAMT indicated that many of the functional residues were conserved but some of the amino acids were the same among algae but this was not the case with Arabidopsis. Interestingly, substitution of the H125 residue identified from bean (Phaseolus vulgaris), which is generally replaced by proline (P) in other plant homologues (marked in orange in Fig. 4), for arginine (R) renders the transporter more active (Ortiz-Ramirez et al. 2011). Since the H125 position is R in PyAMT1, this might suggest it to be an active form. Expression of PyAMT1 dramatically improved the ability of TK2463, an E. coli strain defective in K+ uptake, to survive in K+ deficiency compared to the negative control line expressing PyHSP70 (Fig. 5). Although the negative control line showed somewhat compromised growth in the sufficient K+ condition, the degree of viability under K+ deficiency between the lines expressing PyHSP70 and PyAMT1 was fairly clear. The effect of PyAMT1 was stronger than that of PyβCA1 (Fig. 5) and this point was consistent with the fact that PyAMT1 was recovered solely from the stringent screen while PyβCA1 from the mild screen as well. Interaction between K+ and ammonium is known due to their chemical similarities such as charge and size. Replacement of a nitrogen source as nitrate to ammonium in tobacco (Nicotiana tabacum) was reported to cause growth retardation and a decrease in K+ uptake (Lu et al. 2005). In Arabidopsis, ammonium has been shown to inhibit K+ deficiency-induced expression of a high-affinity K+ transporter gene, AtHAK5 (Qi et al. 2008; Rubio et al. 2008). By contrast, in ammonium-tolerant rice species, ammonium inhibits high-affinity K+ transport but promotes low-affinity K+ uptake (Szczerba et al. 2008). Tomato (Solanum lycopersicum) LeHAK5 expression is induced by ammonium although K+ concentrations in roots are not altered, and K+ uptake and accumulation are stimulated by ammonium in sorghum (Sorghum bicolor) (Alvarez-Pizarro et al. 2011). As shown in the examples from the previous reports, whether ammonium prevents or activates K+ uptake depends on the plant species. There is no information available at present on the effect of ammonium on K+ uptake in P. yezoensis, but it is possible that increased concentrations of ammonium due to PyAMT1, directly or indirectly, help accumulate K+ under K+ starvation. It would be interesting to demonstrate the functions of PyAMT1 in planta and compare those with the functions of AMTs from land plants in terms of K+ deficiency tolerance.

Taken together, our findings provided insight into the potential pathways involved in K+ uptake and response in P. yezoensis, PyAMT1 and probably ammonium being strong candidate components, although further investigation is required to clarify the roles of the selected genes in the K+ deficiency response. The cDNA libraries created will serve as a useful tool to understand the molecular mechanisms underlying K+/Na+ homeostasis in seaweeds.