Introduction
Drought, cold, high salt levels and ultraviolet (UV) radiation in the Antarctic limit plant growth. Mosses are the most widely distributed terrestrial plants in the Antarctic, and have adapted to the extreme environment with the aid of special functional genes and secondary metabolites. Flavonoids are the largest class of polyhydric phenylpropanoid secondary metabolites in plants and include flavonols, flavanones, anthocyanins and isoflavones. Many studies have shown that the accumulation of flavonoids is related to plant resistance to abiotic stress. Enhanced levels of flavonoids lead to increased salt (Oh et al. Reference Oh, Kim, Kim, Kwon and Lee2011), cold (Choi et al. Reference Choi, Kwon, Hossain, Hong, Lee and Lee2009), drought (Castellarin et al. Reference Castellarin, Matthews, Gaspero and Gambetta2007) and UV-B (Landrey et al. Reference Landrey, Chapple and Last1995) tolerance in plants. The flavonoid metabolic pathway is an indispensable branch of the phenylpropane metabolic pathway. In a three-step enzymatic reaction, phenylalanine is converted into coumaryol coenzyme A. Then, upon catalysis by chalcone synthase (CHS) and chalcone isomerase (CHI), p-coumaroyl-CoA and malonyl-CoA form naringenin, the precursor of all flavonoid compounds.
CHS (EC 2.3.1.74) is the rate-limiting enzyme in the above pathway and catalyses the biosynthesis of naringenin chalcone through the condensation of three units of malonyl-CoA with one p-coumaroyl-CoA (Fig. 1). Its expression level affects the downstream metabolism of flavonoids in plants, thereby affecting the plants’ ability to grow and resist external stresses. The downregulation of the CHS gene results in a decrease in flavonoid content during the development of yellow-seeded Brassica napus L. (Jiang et al. Reference Jiang, Shao, Li, Lu, Zhang and Wang2013). Li et al. (Reference Li, Ou-Lee, Raba, Amundson and Last1993) found that CHS and CHI mutations in Arabidopsis thaliana (L.) Heynh. led to the emergence of individuals with UV-hypersensitive phenotypes. Cold stress (4°C) activates CHS expression and induces the accumulation of anthocyanin in the leaves and stems in A. thaliana (Leyva et al. Reference Leyva, Jarillo, Salinas and Martinez-Zapater1995). The expression levels of most NtCHS genes are regulated during drought and salinity stresses, which suggests that tobacco NtCHS genes might play important roles in responding to these two stresses (Wang et al. Reference Wang, Xie, Zhang, Lin, Luo and Li2017).
Illustration of reactions catalysed by CHS and other enzymes of the CHS family.
The CHS gene has been widely cloned, and its role in resistance has been studied in multiple plant species; this gene is a single-copy gene in species such as Arabidopsis, Petroselinum crispum (Mill.) Fuss, and yellow snapdragon. Moreover, there are also multicopy CHS genes in Physcomitrella patens (Hedw.) Bruch & Schimp., morning glory, chrysanthemum and other plants, which shows that CHS genes have gone through varying degrees of duplication and differentiation and have formed a multigene family during the evolutionary process. A genome-wide investigation of CHS genes in rice (Oryza sativa L.) was performed by Han (Reference Han, Cao, Jiang and Ding2017), and the results revealed that the CHS genes exhibit both diversity and conservation in many aspects (Han et al. Reference Han, Cao, Jiang and Ding2017). Chalcone synthase genes exhibit various expression patterns in maize (Zea mays L.), indicating functional diversification (Han et al. Reference Han, Ding, Su and Jiang2016). Surprisingly, although it was the earliest land plant, Physcomitrella patens. contains seventeen CHS superfamily genes, and duplication and diversification of the CHS superfamily genes have occurred in the moss lineage (Koduri et al. Reference Koduri, Gordon, Barker, Colpitts, Ashton and Suh2010). The special environment of Antarctica has created unique species. Mosses are the dominant vegetation in the coastal regions of the Antarctic Peninsula and represent the earliest existing land plants; they have been reproducing on land for 470 million years (Roads et al. Reference Roads, Longton and Convey2014). Recent studies have also shown that Antarctic mosses were the origin of all land plants and have continued to evolve in isolation for tens of millions of years (Convey & Stevens Reference Convey and Stevens2007). Another study speculated that in the absence of sexual reproduction in extreme Antarctic conditions, any change in the nucleotide sequence of moss is most likely caused by immigration or mutation in situ and could be involved in the evolution of new species (Seppelt et al. Reference Seppelt, Green, Schwartz and Frost1992, Skotnicki et al. Reference Skotnicki, Mackenzie, Clements and Selkirk2005). Although CHS genes have been well studied in advanced plants, no studies have investigated the CHS family of genes in the Antarctic moss Pohlia nutans Lindberg. In the present study, six gene transcripts were cloned from P. nutans and a series of analyses were performed including multiple sequence alignment, structure simulation, subcellular localization, phylogenetic analysis and expression analysis.
Materials and methods
Plant material and stress treatment
The Antarctic moss P. nutans was obtained from a scientific investigation of the 24th Chinese National Antarctic Research Expedition. It was inoculated in sterile soil for culture, and placed in an illumination incubator at 16°C with 70% relative humidity and continuous light (109 μmol photons m-2 s-1). Mosses cultured under the above conditions were later used as experimental controls (Liu et al. Reference Liu, Ju and Xia2014). P. nutans samples were subjected to different treatments, such as cold (cultured at 4°C for three hours), drought (transferred to a 20% (w/v) Polyethylene Glycol (PEG) 6000 solution (Dingguo Corp., Beijing, China) for six hours), salinity (sprayed once with 200 mM NaCl; the stems and leaves were taken after six hours) and UV-B radiation (exposed under an 8 W UV-B lamp, 302 nm, reaching 80 μW cm-2 irradiance for six hours). In addition, the samples were also treated with the plant hormones abscisic acid (ABA) and methyl jasmonate (MeJA); they were sprayed with 50 μM ABA or 50 μM MeJA and incubated at 16°C for one hour. The stems and leaves of the moss plants were cut and immediately placed in a liquid nitrogen tank (-197°C) to be used as RNA extraction material.
Extraction of total RNA and rapid amplification of cDNA ends
Total RNA was obtained from the stems and leaves of P. nutans according to the modified cetyltrimethylammonium bromide method (Gambino et al. Reference Gambino, Perrone and Gribaudo2008). Both 5’- and 3’- rapid amplification of cDNA ends (RACE) were performed with a SMARTTM RACE cDNA Amplification Kit (Clontech). The above procedures were carried out according to the manufacturer’s instructions. Gene-specific primers for 5’- and 3’-RACE reactions were designed from the core cDNA sequence of chalcone synthase. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed using total RNA as the template. First, a cDNA library was constructed with 5’/3’-RACE CDS Primer A and PrimeScript™ Reverse Transcriptase (37°C for 60 min). Then, six P. nutans genes were amplified with gene-specific primers (see Supplemental Table SI at https://doi.org/10.1017/S0954102018000470), Nested Universal Primer A and TransStart™ FastPfu DNA Polymerase (TransGenBiotech, Beijing). Using the Tiangen gel purification kit (Tiangen, Beijing, China), the 5’- or 3’-RACE products were purified from the gel, ligated into a pEASY-Blunt cloning vector (TransGen Biotech, Beijing), and transferred into competent cells (Escherichia coli DH5α). The positive clones were further sequenced, and the generated sequence files were compiled. The full-length cDNA sequences of the six P. nutans genes were identified and assembled by using the SeqMan program (DNAStar 7.1, DNASTAR Inc., USA).
Gene information acquisition, multiple sequence alignment and three-dimensional structure construction
Sequencing of the transcriptome of P. nutans was completed by using Illumina ultra-high throughput sequencing technology. Data were retrieved from the NCBI Short Read Archive (SRA) (accession number SRA051595). Chal_sti_synt_N (PF02797) and Chal_sti_synt_C (PF00195) domain models built with the hidden Markov model were downloaded from the Sanger Centre (http://pfam.sanger.ac.uk/). First, using the HMMER package according to the provided instructions, homologous protein sequences were obtained with the program hmmsearch. Then the genes with the highest scores above the threshold (with the highest identity) were chosen as target genes. Finally, the six full-length gene sequences of P. nutans (Pn021, PnCHS088, Pn270, PnCHS444, PnCHS768 and Pn847) were obtained and then annotated further. The GC (guanine-cytosine) content of each nucleic acid sequence was analysed with DNASTAR software. The molecular weight and isoelectric point were calculated with the Swiss Biological Information Institute (SIB) ExPASy Compute PI/Mw online server, and functional annotation was performed based on protein sequence from NCBI basic local alignment search tool (BLAST) searches and related literature. Multiple sequence alignments were performed with the BioEdit Clustal-W program, and GeneDoc was used to show the active site. Finally, MEGA (5.0) was used to perform phylogenetic analysis. All parameters used were the default parameters for the programs.
For the construction of the three-dimensional structure, the crystal structures of Marchantia polymorpha L. stilbenecarboxylate synthase 2 (STCS2) and polyketide synthase 1 of Huperzia serrata (Thunb. ex Murray) Trevis. were used as templates, and homology modelling was performed using the SWISS-MODEL website. AutoDock software was used to predict the interactions with the substrate molecules. Finally, PyMOL software was used for visual observation and analysis. The key active amino acids were marked with a stick model.
Subcellular localization
The subcellular localization of PnCHS444 was examined with an N-terminal green fluorescent protein (GFP) tag. The gene was subcloned into a modified pBI221-GFP vector and the recombinant plasmid was transformed into Escherichia coli (Migula) Castellani and Chalmers (Zhang et al. Reference Zhang, Muyrers, Testa and Stewart2000). The gene-specific PCR primers are listed in Table SI (at https://doi.org/10.1017/S0954102018000470). Using a Biomiga Plasmid Maxprep Kit (www.biomiga.com), the recombinant plasmid was prepared. Next, the plasmid was transferred into the protoplasts of Arabidopsis. Transformed tissues were cultured in a dark room at 23°C and collected after ten hours. They were observed with a confocal laser microscope. (Zeiss LSM 700 Inverted Confocal Microscope).
Phylogenetic analysis
After obtaining the full-length sequences of the genes, the codons were used to deduce protein sequences and conduct a cophylogenetic analysis with 40 representative CHS protein sequences. These sequences were from the protein database of NCBI and included proteins from plants, fungi and bacteria. Finally, a phylogenetic tree including the six proteins and chalcone synthase family members from other plants was constructed using MEGA 6 software (MEGA, Raynham, MA, USA) with the neighbour-joining method.
Real-time PCR was used to analyse mRNA expression levels
The cDNA sequences were constructed with total RNA (500 ng) and a mixture of oligo(dT)18 (Biasini et al. Reference Biasini, Bienert, Waterhouse, Arnold, Studer and Schmidt2014) and random hexamer primers. Gene-specific primers were designed with Primer 5 (http://www.premierbiosoft.com). Real-time PCR assays were conducted using Bestar SybrGreen qPCR Master Mix (DBI Bioscience, Shanghai, China). The PCR program was 95°C for 20 s, 58°C for 20 s and 72°C for 20 s. To check the PCR specificity, the program ended with the generation of a melt curve by ramping from 65°C to 95°C in 2 s intervals. All reactions were completed in triplicate. The comparative expression levels of the selected genes were normalized to the expression of the tubulin gene. Ct (threshold cycle) values were used to quantify the mRNA expression of the six P. nutans genes.
Results
The retrieval, identification and molecular cloning of chalcone synthase family genes from the Antarctic moss Pohlia nutans
To obtain the CHS family genes of the Antarctic moss P. nutans, a homologous gene mode was used to screen the transcriptome database. Using HMMER software, homologous protein sequences were obtained with the program hmmsearch. After comparing their annotations in the NCBI database, the CHS family genes of P. nutans were filtered and manually identified. The target sequence was subjected to a BLAST search on the NCBI website and annotated. However, only Pn021 had a full-length nucleotide sequence. For the other five genes, full-length gene amplification was performed under the guidance of RACE technology and the full-length cDNAs of the CHS genes were further sequenced. The sequences have been deposited in the NCBI database, and their accession numbers are given in Table I.
Sequence information of the six P. nutans genes.
The Swiss Institute of Bioinformatics (SIB) online server was used for analysis; the nucleic acid GC content of the six genes ranged from 52.0–65.0%. In addition, the lengths of the six amino acid sequences ranged from 355–423 residues. The molecular weights of the six proteins ranged from 38.4–45.5 KDa, and the range of isoelectric points (pI) was 6.28–6.87 (Table I).
Multiple sequence alignment of the chalcone synthase superfamily
To compare the sequences with several known CHSs from other species, the CHS homologous genes of other species were found in the public NCBI EST and genomic databases (Fig. 2). The CHS homologous protein sequences of six species, including Medicago sativa L., A. thaliana, Pinus sylvestris L., Psilotum nudum (L.) P.Beauv., Marchantia paleacea L. and P. patens were compared to those of the six P. nutans genes. The identity of the six P. nutans genes at the amino acid level with the CHS genes of other species was 30.0–65.0%. The conserved amino acids in the six P. nutans genes are consistent with those conserved in the type III polyketide synthases (PKSs). PnCHS088, PnCHS444 and PnCHS768 contain two typical polypeptide sequences of the CHS family (GCFAGGTVLR and GFGPGL), the catalytic triad (Cys164, His303 and Asn336) and phenylalanine residues (Phe221 and Phe271) associated with substrate-specific binding. All of the above amino acid residues are important for the cyclization reactions in CHS/STS-type PKSs, and these amino acid residues are important for starter substrate selectivity. However, the Cys177 catalytic site of Pn021 was replaced by Phe177, and ALGPG was substituted with GLGPG. GFGPG in Pn847 was replaced with SFGPG, which may have caused differences in the starter molecules. Additionally, Pn270 and Pn847 appeared as 2’-oxoalkylresorcinol synthase (ORS) orthologues in BLAST searches.
Amino acid alignment showing the functional residues in the six P. nutans proteins. Identical amino acid residues are shaded in black; similar residues are shaded in grey. The active site residues, catalytic site, CoA binding site and other highly conserved amino acid sequences are marked with blue symbols, and the signature GFGPG loop is shown in the blue box. Reference sequences: Medicago sativa, P30074; Arabidopsis thaliana, CAI30418; Pinus sylvestris, CAA43166; Psilotum nudum, BAA87922; Marchantia paleacea, BAD42328; and Physcomitrella patens, CHS13, ABU87504.
Three-dimensional structure simulation for PnCHS444
To characterize the spatial structural properties of PnCHS444, a 3D homology model was developed based on the crystal structures of M. polymorpha. STCS2 and polyketide synthase 1 from Huperzia serrata. The three-dimensional structure of PnCHS444 was predicted with the SWISS-MODEL server.
As shown in Fig. 3, the 3D structure homology of PnCHS444 exhibits an alpha beta 3-layer (αβα) sandwich architecture and possesses the typical catalytic site containing the conserved residues of Cys188, His327 and Asn360. The 3D model results of the other five proteins also demonstrated similar structures, and so the structure of PnCHS444 was used as the representative structure. In general, the 3D structures of homologous proteins are more conserved than their amino acid sequences. There was no significant difference in the core conserved sites. In the 3D structure of CHS, the CoA binding tunnel binds p-coumaroyl-CoA, which provides access to the different catalytic site residues (marked as red in Fig. 3), catalysing the loading of the p-coumaroyl starter unit and the decarboxylative condensation reactions to extend the polyketide.
3D structure modelling of PnCHS444. α-helices are in yellow, β-sheets are in blue, and loops are in light green. The superimposed catalytic site residues for the 3D structure are in red and are annotated individually.
Subcellular localization of PnCHS444 in Arabidopsis mesophyll protoplasts
The distribution of PnCHS444 in the cells provides an important reference. A subcellular localization vector for PnCHS444 was constructed, and its production was significantly increased upon UV-B irradiation. The data revealed that GFP fluorescence was spread over the entire protoplast in the p35S::GFP/pBI221-carrying (i.e. vector control) protoplasts (Fig. 4a). However, for p35S::PnCHS444::GFP containing protoplasts, the fluorescence of the PnCHS444-GFP fusion protein was only distributed in the cell membrane and in the membranes of the endothelial organelles (Fig. 4e).
Subcellular localization of PnCHS444 in Arabidopsis protoplasts. a, b, c & d. The control p35S::GFP/pBI221 vector. e, f, g & h. The constructed p35S::PnCHS444::GFP/pBI221 vector. a. The fluorescence of the control vector was spread over the entire cell. e. The fluorescence of the fusion protein was only distributed in the cell membrane and the membranes of the endothelial organelles. b & f. Fluorescence of the chloroplast. d & h. The combined fluorescence of GFP and the chloroplast. c & g. Bright field of the protoplast. The confocal images were taken ten hours after protoplast transfection.
Phylogenetic analysis of the chalcone synthase superfamily
Phylogenetic analysis was performed with deduced amino acid sequences of the six P. nutans proteins of the CHS family, as well as for a range of catalytically verified and putative chalcone synthases from other species, including advanced plants, ferns, bryophytes, algae and fungi. Most species have multiple CHS family genes. These multiple genes are prone to mutation or ectopic exchange with other related gene copies, which is important in plant evolution.
The phylogenetic tree showed that the CHS family of advanced plants forms a sister clade to that of ferns, and the CHS family of mosses is in the transition zone of the phylogenetic tree. The CHS family of algae and fungi is located at the bottom of the phylogenetic tree. Moss enzymes clustered together and showed high sequence similarities. The six genes of P. nutans clustered in three different small branches (Fig. 5). The Pn021 branch (marked with a blue triangle) was longer than others and was separate from the rest, suggesting a larger change during the evolutionary process. However, the branch length for Pn270 was short, indicating that few nucleotide changes occurred. The branches of Pn270 and Pn847 shared the same internal nodes, indicating that Pn270 and Pn847 have closely related lineages. The PnCHS768, PnCHS444 and PnCHS088 branches shared the same node as PpCHS, a bona fide CHS from P. patens, indicating that those proteins share the same ancestor genes. The CHSs of bryophytes are clustered between those of fungi and higher plants. Even in the inner family of bryophytes, the separation of genes was not clear; the same situation was observed for the algae. However, the origin node of these branches or the root of the cluster was the same (marked with a red diamond), and it can be inferred that the higher plant chalcone genes originated from a common ancestor gene.
Phylogenetic tree of the CHS family. The numbers in the branches indicate the reliability level. The accession numbers of the enzymes included in the analysis are shown below. Physcomitrella patens (PpCHS9, A9RSI0), P. patens (CHS13, ABB84527), P. patens (CHS10, XP001781520), P. patens (CHS11, ABU87504), Plagiochasma appendiculatum Lehm. & Lindenb. (CHS, AIV42295), Arabidopsis thaliana (CHS, AED91961), Glycine max (L.) Merr. (CHS, AAO67373), Equisetum arvense L. (CHS, BAA89501), Ginkgo biloba L. (CHS, AAY52458), Picea abies (L.) H. Karst. (CHS, ABD24228), Rosa hybrid cultivar (CHS, BAC66467), Lilium speciosum Thunb. (CHS, BAE79201), Dendrobium hybrid cultivar (CHS, AAU93767), Oryza sativa Japonica Group (CHS, BAA19186), Psilotum nudum (L.) P.Beauv. (CHS, Q9SLY0), Dryopteris erythrosora (D.C.Eaton) Kuntze. (CHS, AHU87077), Ceratopteris thalictroides (L.) Brongn. (CHS, AFN02448), Nicotiana alata Link & Otto. (CHS,C5IWS6), Petunia × hybrida hort. ex E.Vilm. (CHS, P08894), Marchantia polymorpha L. (STCS, AAW30009), Pinus strobus L. (STS, CAA87013), Nicotiana sylvestris Speg. & Comes. (CHSL, CAA74847), Rheum tataricum L.f. (STS, AAP13782), Cissus rhombifolia Vahl. (STS, AAM21772), Bauhinia variegate (L.) Benth. (STS, ABF59517), Streptomyces griseus (Krainsky) Waksman and Henrici. (EGE46261), Pseudomonas Migula. (KGD87053), Aspergillus affinis Davolos, Persiani, Pietrangeli & Maggi. (AHL30726), Mycobacterium tuberculosis variant africanum Castets et al., ATCC 25420 (KBI75572), Tetraselmis F.Stein. (JAC64062), Emiliania huxleyi (Lohm.) Hay and Mohler. (EOD27900), Microcystis aeruginosa Kützing. (ELS49626), Synechococcus Nägeli. (CAE07508), Prochlorococcus marinus Chisholm. (Q7V8E2).
Expression analysis of six genes of the Antarctic moss Pohlia nutans
To investigate the responses of the six P. nutans genes to abiotic stress, their expression levels were analysed by real-time PCR. As shown in Fig. 6, the expression levels of the six P. nutans genes were markedly different. After UV treatment, the expression levels of the six P. nutans genes increased by different magnitudes. The expression levels of PnCHS088 and PnCHS444 increased by factors of 2.4 and 3.0, respectively (Fig. 6a). After simulated drought stress using PEG6000, the expression levels of PnCHS088 and Pn270 remained essentially unchanged; those of the other four genes were upregulated. The expression levels of Pn021, PnCHS444 and Pn847 increased by factors of 1.8, 1.5 and 3.3, respectively (Fig. 6b). In addition, the expression levels of the six P. nutans genes were all downregulated under low-temperature conditions (Fig. 6c). With the exception of Pn270/847, the other four P. nutans genes were all downregulated under high-salinity treatment. The expression levels of Pn021, PnCHS088, PnCHS444 and PnCHS768 were reduced to 0.50, 0.57, 0.54 and 0.62 times their previous expression levels, respectively (Fig. 6d).
Expression levels of the six P. nutans family genes after treatment of a. UV for 6 h, b. 20% PEG for 6 h, c. 4°C for 3 h, d. 200 mM NaCl for 6 h, e. 50 μM MeJA for 1 h, or f. 50 μM ABA for 1 h. The vertical bars indicate the means ± SD (n = 3). The asterisks (*) signify differences in means between the control group and the treatment group (Student’s t-test, P ≤ 0.05).
As molecular signals, phytohormones effectively control the response of stress-related proteins and affect plant resistance. The flavonoid metabolic pathway was regulated by Myb. Moreover, Myb was affected by abscisic acid and methyl jasmonate. The expression levels of PnCHS444 and Pn847 increased by factors of 3.6 and 1.7, respectively, after ABA and MeJA treatment (Fig. 6e & f).
Discussion
Flavonoids are important secondary metabolites that are synthesized by different enzymes and take part in many physiological processes in plants, including disease resistance, auxin transport and abiotic stress responses. As CHS is the rate-limiting enzyme in the flavonoid metabolic pathway, the expression levels of CHS affect the metabolism of flavonoids and the ability of plants to grow and resist external stresses. Low temperature induces CHS gene expression, resulting in anthocyanin accumulation in petunia flowers (Shvarts et al. Reference Shvarts, Borochov and Weiss1997). Overexpression of the EaCHS1 (Eupatorium adenophorum Spreng. CHS1) gene in tobacco enhances its tolerance to salinity stress (Chen et al. Reference Chen, Guo, Lin and Cheng2015). The CHS gene is actively expressed in parsley, which accumulates flavonoids in leaf epidermal cells to protect against harmful UV-B radiation (Schmelzer et al. Reference Schmelzer, Jahnen and Hahlbrock1988). The ability of Antarctic mosses to adapt to the extreme polar environment is due to distinct functional genes and different gene expression profiles that correspond to stressful conditions. In this study, six P. nutans genes were isolated, and these genes possessed a relatively conserved coding region of approximately 1.2 kb (Fig. 1 & Table I), which is approximately the same length as the coding regions of SoCHS1 (Syringa oblata Lindl, 1.17 kb), LrCHS (Lamiophlomis rotata (Bentham ex J.D. Hooker) Kudô, 1.17 kb), and PpCHS (Physcomitrella patens, 1.19 kb) and consistent with CHS genes identified in other plant lineages. PnCHS088, PnCHS444 and PnCHS768 contain two typical polypeptide sequences (GCFAGGTVLR and GFGPGL), a catalytic triad (Cys164, His303 and Asn336) and substrate-specific binding sites (Phe221 and Phe271) (Jez et al. Reference Jez, Bowman and Noel2002). However, Pn021, Pn270 and PnCHS847 are not completely conserved, showing evidence of base pair substitution or deletion. The Cys177 catalytic site of Pn021 was replaced by Phe177 (see Fig. 2), a phenomenon which is rare in the type III PKSs. Pn021 might not exhibit catalytic activity similar to typical type III PKSs, although the annotation of Pn021 upon BLAST search showed it to be one of the stilbenecarboxylate synthase (STCS) family members. Genetic mutations in P. nutans may be caused by its prolonged exposure to extreme conditions. This is consistent with Dale’s assumption that genetic variation occurring in Hennediella heimii (Hedw.) R.H. Zander is possibly due to increased exposure to UV-B irradiation (Dale et al. Reference Dale, Skotnicki, Adam and Selkirk1999). Through the construction of 3D models for the PnCHS proteins, the higher-order structures and catalytic sites of the proteins were predicted more thoroughly. For the catalytic site, the typical Cys-His-Asn catalytic triad is conserved in the CHS proteins of P. nutans, with an exception of PnCHS021 (Fig. 3). Studies have shown that four amino acids, Cys164, Phe215, His303 and Asn336 situated at the intersection of the CoA-binding tunnel and the active site cavity play an essential and distinct role during the decarboxylation of malonyl-CoA and the formation of chalcone. Cys164 plays a role as the active-site nucleophile in polyketide formation. The importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction has also been demonstrated. Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate (Jez et al. Reference Jez, Ferrer, Bowman, Dixon and Noel2000). The distribution of CHS in cells varies among species. Subcellular localization analysis showed that the distribution of PnCHS444 is similar to that of the CHS of buckwheat (Hrazdina et al. Reference Hrazdina, Zobel and Hoch1987), which is distributed in the cell membrane and in the membranes of endothelial organelles (Fig. 4). However, the CHS of grape berry is localized in the rough endoplasmic reticulum (ER) and the cytoplasm of the skin cells, with low localization in the cell wall (Tian et al. Reference Tian, Wan, Pan, Zheng and Huang2008). The CHS of Arabidopsis is predominantly distributed in the cytosol (Zhang et al. Reference Zhang, Abrahan, Colquhoun and Liu2017), and was a cytoplasmic and nuclear-localized enzyme for the local synthesis of flavonoids (Saslowsky & Winkel-Shirley Reference Saslowsky and Winkel-Shirley2001).
The importance of gene duplication in evolution is well documented (Zhang Reference Zhang2003). CHS9, CHS10, CHS11 and CHS13 from P. patens have been characterized. Gene duplications gave rise to the progenitor of PpCHS11 and the ancestor of all other CHS superfamily genes. Pn270 and Pn847 are in the same evolutionary position as PpCHS11 (Fig. 5). CHS11 is a 2’-oxoalkylresorcinol synthase (PpORS) (Kim et al. Reference Kim, Che, Wiedemann, Jepson, Rahimi and Rothwell2013). Pn270 and Pn847 may be (oxo)alkylresorcinol synthases similar to PpORS, and they would be the first enzymes belonging to the same basal clade as PpORS. PpORS and its two paralogues have thus far been the only members of the basal clade of phylogenetic trees of plant type III PKSs. According to previous studies, fungal type III polyketide synthases (Kim et al. Reference Kim, Che, Wiedemann, Jepson, Rahimi and Rothwell2013) and PpCHS11 have similar catalytic activity. Although flavonoids have been detected in microalgae (Goiris et al. Reference Goiris, Muylaert, Voorspoels, Noten, Paepe, Baart and Cooman2014), CHS-like enzymes (CHSL) extracted from the algae cannot catalyse 4-coumaroyl-CoA and malonyl-CoA to form naringin chalcone. Compared with PpORS, CHSL is only similar in terms of substrate selectivity (Baharum et al. Reference Baharum, Morita, Tomitsuka, Lee, Ng and Rahim2011), which might provide an important reference for the evolution of the CHS family genes and the initial formation of the flavonoid pathway. CHS13 is a bona fide CHS (Jiang et al. Reference Jiang, Schommer, Kim and Suh2006). PnCHS088, PnCHS444 and PnCHS768 are near P. patens CHS13 in the phylogenetic tree (Fig. 5). Although biochemical characterization is necessary, PnCHS088, PnCHS444 and PnCHS768 are probably bona fide CHS family genes. Pn021 might encode a non-CHS enzyme since it is near PpCHS9 (Koduri et al. Reference Koduri, Gordon, Barker, Colpitts, Ashton and Suh2010) in the tree, which is more similar to STCS, although this has yet to be demonstrated. The tree suggests that diversification of the type III PKSs, including the appearance of bona fide CHS genes, might have preceded the divergence of the moss species.
In nature, plants are exposed to a variety of abiotic stresses, such as drought, cold, high salt, and UV radiation. CHS is very commonly induced in many plant species under abiotic stress, resulting in changes in expression levels. The enhanced expression of CHS enhances the accumulation of flavonoids and isoflavonoids (Dao et al. Reference Dao, Linthorst and Verpoorte2011). Keeping Arabidopsis under high-intensity light conditions for 24 hours causes a fiftyfold increase in CHS activity and an accumulation of anthocyanin pigments (Feinbaum & Ausubel Reference Feinbaum and Ausubel1988). PpORS was recently shown to confer dehydration resistance (Li et al. Reference Li, Aslam, Rabbi, Vanderwel, Ashton and Suh2018). Pn847 (a putative PpORS orthologue) also showed a similar phenomenon under PEG treatment. In this study, abiotic stress promoted the upregulation of several CHS family genes in Antarctic moss. In addition, several CHS family genes were also downregulated after stress treatment (Fig. 6). The CHSA and CHSJ paralogues of Nicotiana tabacum L. have different expression patterns under abiotic stresses (Chen et al. Reference Chen, Guo, Lin and Cheng2015). Phytohormones are a class of active substances that are induced by specific environmental signals in plant cells and can regulate the physiological responses of plants. ABA and MeJA are the main determinants of enhanced plant tolerance to abiotic stress (Zhu Reference Zhu2002), and they influence the biosynthesis of flavonoids (Xu et al. Reference Xu, Dubos and Lepiniec2015). Tobacco plants accumulate more flavonoids under ABA and MeJA treatment than under control conditions (Chen et al. Reference Chen, Pan, Li, Cui, Zhang and Zhang2017). As shown in Fig. 6e & f, the mRNA production levels of six Pohlia nutans genes under ABA and MeJA treatment were different from control conditions. These results were similar to those for the family of flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase genes from P. nutans (Liu et al. Reference Liu, Ju and Xia2014).
Based on the results of this study, it is concluded that the expression profiles of these genes were influenced by diverse abiotic stresses. This study also provides an important reference regarding the adaptations of Antarctic mosses to the polar environment.
Conclusions
Pn088, Pn444 and Pn768 were identified and analysed as putative CHS genes. Pn021 was found to be similar to STCS, and the other two genes (Pn270 and Pn847) were suggested to be ORS orthologues. They are all involved in regulating abiotic stress tolerance in P. nutans. Such changes in expression are closely related to adaptation to extreme environments.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (41206176 and 41476174); the Natural Science Foundation of Shandong Province (ZR2017MD013 and ZR2014DQ012); the Basic Scientific Fund for National Public Research Institutes of China (2014T04); the Excellent Creative Team Fund of Jinan; and the Fund for Transformation of Agricultural Scientific and Technological Achievements of Shandong Province. Thanks to the School of Life Science of Shandong University for providing the advanced platform, and to the reviewers for their helpful comments that significantly improved the paper. Thanks also to Xu Li for helping with the construction of 3D models.
Supplemental material
Table SI. showing primers for RACE, plasmid construction and quantitative real-time PCR analysis will be found at http://dx.doi.org/10.1017/S0954102018000470
