The Antarctic moss 2-oxoglutarate/Fe(II)-dependent dioxygenases (Pn2-ODD2) enhanced the tolerance to drought and oxidative stress

Abstract

Background

Flavonoid biosynthesis pathway is generally thought unique to land plants and has assisted plants to adapt the terrestrial ecosystems. In this pathway, four 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs), i.e., flavone synthase I (FNSI), flavanone-3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), catalyze the hydroxylation and desaturation reactions. In bryophytes, the earliest land plant group, little is known about the biological functions of these enzymes.

Results

Here, we cloned a Pn2-ODD2 gene of flavonoid biosynthesis pathway from Antarctic moss Pohlia nutans, which was induced by exogenous NaCl, PEG and abscisic acid (ABA) treatment. Overexpression of Pn2-ODD2 increased the drought resistance in Physcomitrium patens and Arabidopsis thaliana during gametophyte growth and seed germination, respectively. Overexpressed-Pn2-ODD2 Arabidopsis also exhibited the enhanced tolerance to oxidative stress, with the downregulation of ROS generation gene and increased the total flavonoid content. Also, overexpression of Pn2-ODD2 decreased the ABA sensitivity in transgenic P. patens and Arabidopsis. Meanwhile, overexpression of Pn2-ODD2 resulted in an increase in both anthocyanins and flavonols in Arabidopsis, which was correlated with the up-regulated anthocyanin biosynthesis gene.

Conclusions

Taken together, Pn2-ODD2 conferred the resistance to drought and oxidative stress by regulating antioxidant defense system in plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06578-8.

Introduction

Flavonoids, a large class of natural polyphenolic secondary metabolites produced by plants, have identified more than 10,000 species and are thought to contribute to land plants to colonize the surrounding environments [1]. They can be divided into six groups: flavanones, flavones, isoflavones, flavanols, flavonols and anthocyanins, based on the diverse modifications at the central core skeleton (C6-C3-C6) [2, 3]. Flavonoids have been illustrated to participate in various physiological processes of plants, such as protecting plants against abiotic and biotic stress, attracting pollinators for pollination, regulating auxin transport and male fertility, and signaling during nodulation [4, 5]. In non-vascular land plants, including the liverworts (Marchantiophyta), hornworts (Anthocerotophyta) and mosses (Bryophyta), flavonoids have been also involved in the tolerance to environmental stress. Biflavonoids in Antarctic moss Ceratodon purpureus show high antioxidant and ultraviolet-screening activity [6]. The related flavone O-glycosides contributed to UV-B tolerance in Marchantia polymorpha [7]. Cell wall-bound red flavonoids, riccionidin (an auronidin) and sphagnorubin, have been reported from liverworts and mosses, respectively [8, 9]. Carella et al. (2019) had demonstrated that the accumulation of auronidin greatly promoted the resistance to Phytophthora palmivora infection in liverwort M. polymorpha [10]. In addition, evidence is emerging showing that flavonoids have potential health benefits in against cancer, diabetes, inflammation and preventing cardiovascular and neuronal diseases due to the strong antioxidant activity [11, 12]. Therefore, the flavonoid biosynthesis pathway has attracted extensive attention from biologists and chemists.

Flavonoid biosynthesis is considered to have emerged during terrestrial colonization around 550 - 470 million years ago, which originates from the general phenylpropanoid metabolism to synthesize p-coumaroyl-CoA. And p-coumaroyl-CoA and three malonyl-CoA can be further used to produce naringenin, a paramount precursor for flavonoid biosynthesis, catalyzed by chalcone synthase (CHS) and chalcone isomerase (CHI) [13, 14]. Naringenin can be converted by flavone synthase I and/or II (FNSI or FNSII) or flavanone-3-hydroxylase (F3H) to generate flavones or dihydroflavonols [15]. The resulting dihydroflavonols can serve as immediate precursors for the flavonols biosynthesis by flavonol synthase (FLS) or the biosynthesis of anthocyanins by dihydroflavonol reductase (DFR) and anthocyanin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX) [16]. Of the enzymes in flavonoid biosynthesis pathway, FNSI, F3H, FLS and ANS/LDOX belong to 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs) family. 2-ODDs are non-heme iron-containing proteins, which requires Fe²⁺ as a cofactor, and 2-oxogulatrate and O₂ as co-substrates to catalyze the oxidation of substrates [17]. Among these four enzymes, FNSI and F3H can accept flavanones as substrate, displaying a low substrate specificity, whereas FLS and ANS have substrate promiscuity. FLS can catalyze dihydroflavonols and flavanones to the corresponding products, and ANS can accept leucoanthocyanidins and dihydroflavonols as well as flavanones as the substrates [18, 19]. Furthermore, heterologous or homologous transgenic works have demonstrated that these genes play crucial roles in flavonoid biosynthesis and response to abiotic stress in plants. For example, overexpression of FNSI and FNSII from Zea mays in Arabidopsis reduced UV-B-induced damage by synthesizing the apigenin [20]. Overexpression of LcF3H from Lycium chinense enhanced the level of flavon-3-ols and promoted the tolerance to drought stress in transgenic tobacco [21]. Overexpressed-EkFLS (from Euphorbia kansui) Arabidopsis displayed the increased flavonoid accumulation and the promoted resistance to NaCl and drought stress [22]. Overexpression of RtLDOX2 from Reaumuria trigyna in Arabidopsis improved the tolerance to salt, drought and UV-B radiation by increasing anthocyanin and flavonol levels [23]. However, in early land plant bryophytes, although the gene sequences of these enzymes could be isolated, their biological functions are poorly reported.

Antarctica is thought to be one of the most inhospitable and remote places, due to its extreme climate conditions, such as low temperature, desiccation, low water availability, high salinity and strong UV-B radiation [24]. Mosses, one of the main plants of the Antarctic continent, have adapted the harsh environment due to their special protection strategies [25]. For example, Antarctic moss could repair and reduce UV-B damage by increasing the synthesis of UV absorbing compounds and antioxidant compounds (phenolics, carotenoids and bioflavonoids) to enhance the photoprotective ability [6, 26]. In Antarctic moss Leptobryum pyriforme, the transcriptomics integrated with metabolomics demonstrated that flavonoid biosynthesis pathway, Jasmonate signaling, DNA repair system and UV RESISTANCE LOCUS 8 (UVR8)-mediated signal signaling might be pivotal in the adaptation to UV-B [27]. Antarctic moss Sanionia uncinata displayed the resistance to desiccation, which is associated with the activation of protective mechanisms, including the increased antioxidant enzyme activity and the enrichment of osmotic adjustment molecules (dehydrins proteins, glycine betaine and proline) [28]. Recently, the whole-genome sequencing of Antarctic moss Pohlia nutans suggested that its genome was characterized by the numerous segmental gene duplications and the enormous expansion of gene families, which might promote neofunctionalization. Meanwhile, flavonoid biosynthesis, ROS scavenging, plant hormone signal transduction, unsaturated fatty acid biosynthesis and DNA repairing might largely contribute to the survival of moss in extreme environments [29]. Here, we focused on a Pn2-ODD2 gene from P. nutans and generated the transgenic Physcomitrium patens and Arabidopsis to characterize its functions in planta. The data showed that Pn2-ODD2, participating in flavonoid pathway, played a positive role in plant response to drought, oxidative stress and ABA treatment.

Materials and methods

Plant materials and growth conditions

The moss Pohlia nutans used in this study were collected from the Fildes Peninsula of Antarctica (S62°13.260′, W58°57.291′) in the 24th Antarctic scientific expedition of China, which were placed in vacuum-sealed plastic bags and transported to the laboratory. Then they were cultured in an illumination incubator under 12 h light /12 h dark with 70 μmol·m^- 2·s^- 1 light intensity at 16 °C, with 60-70% relative humidity. The moss material has not been deposited in a publicly available herbarium. For gene expression assay, P. nutans was treated with 200 mM NaCl, 16% PEG6000 and 50 μM ABA for 0, 6, 12, 24 and 36 h, respectively, according to previous study [30]. Physcomitrium patens was grown on BCD medium, under 16 h light/8 h dark cycle at 25 °C. For protoplast preparation, P. patens gametophytes was ground by a homogenizer, and then incubated on BCD medium containing 5 mM diammonium tartrate, covered with a cellophane. Wild-type Arabidopsis thaliana (Col-0) and transgenic lines were cultivated in a greenhouse at 22 °C, 8 h light/16 h dark photoperiod (80 μmol·m^- 2·s^- 1) and 60-70% relative humidity for about 4 weeks. Then the photoperiod was changed to 16/8 h light/dark for plant reproductive growth. The moss of P. patens used in the study was obtained and verified by Ji Luan, State Key Laboratory of Microbial Technology, Shandong University, China. The seeds of Arabidopsis Col-0 ecotype were purchased from Arashare website (https://www.arashare.cn/index/Product/index.html).

Cloning and bioinformatics analysis of Pn2-ODD2

Hmmsearch program, the DIOX_N domain (i.e., PF14226.5) and 2OG-FeII_Oxy domain (i.e., PF03171.15), was used to identify 2-oxoglutarate-dependent dioxygenase (2-ODD) from the genome of P. nutans. A total of 89 2-ODD genes were obtained. Among them, a gene originally named Poh0251780.1 (renamed as Pn2-ODD2), exhibited relatively high fold changes under UV-B and cold stress detected by differentially expressed gene (DEGs) analysis. Therefore, the Pn2-ODD2 gene was selected as a candidate gene for further analysis [29, 31]. Based on the transcriptome database of Antarctic moss P. nutans under UV-B radiation, the full-length sequence of Pn2-ODD2 was obtained and subsequently cloned by PCR with the specific primers. The conserved domain of the Pn2-ODD2 protein was analyzed by SMART online website (http://smart.embl-heidelberg.de/). DNAMAN software was used to perform the multiple sequence alignment of Pn2-ODD2 with known 2-ODDs members in other species. Subsequently, the phylogenetic tree was constructed using FastTree 36 with a Maximum likelihood (ML) method and Jones-Taylor-Thornton model and 1,000 bootstrap replicates. The following protein sequences were included: Arabidopsis LDOX (AEE84672.1), Arabidopsis F3H (NP_190692.1), Arabidopsis FLS1 (NP_001190266.1), Apocynum venetum FLS (QDY98367.1), Brassica napus F3H (AIA59794.1), Crocus sativus FLS (QBF29345.1), Dendrobium officinale ANS (AZC85762.1), Glycine max ANS (AAR26525.1), G. max F3H (NP_001236797.1), G. max FLS (NP_001237419.1), Lilium regale FLS (ASV46329.1), Malus domestica FLS (AAD26261.1), Oryza sativa ANS (CAA69252.1), P. patens probable ANS (XP_024390254.1), P. patens 2-ODD (XP_001780809.1), Prunus persica F3H (AEJ88219.1), P. persica LDOX (ABX89942.1), Rosa rugosa FLS (AIS22436.1), Solanum tuberosum FLS (ACN81826.1), Triticum aestivum ANS (BAE98274.1), Vitis vinifera LDOX (NP_001268147.1), V. vinifera F3H (CAA53579.1), Zea mays LDOX (NP_001106074.1), Ziziphus jujuba probable ANS (XP_015880653.1), Arabidopsis CHS (AAA32771.1), P. nutans FNSI (QCP71067.1), Aethusa cynapium FNSI (ABG78791.1), Apium graveolens FNSI (AAX21537.1), Daucus carota FNSI (AAX21536.1), Petroselinum crispum FNSI (AAP57393.1), Conocephalum japonicum FNSI (QEP99662.1), Marchantia emarginata FNSI (QEP99659.1), M. paleacea FNSI (QEP99658.1) and Plagiochasma appendiculatum FNSI (QEP99657.1). The online simulation of the three-dimensional structure of Pn2-ODD2 was predicted by the SWISS-MODEL website, the crystal structure of the Arabidopsis anthocyanidin synthase (2brt.pdb) as a template.

Plasmid construction and plant transformation

The coding sequence of Pn2-ODD2 was amplified and recombined into the pTFH15.3 vector harboring a rice actin promoter (obtained from Drs.Tomoaki Nishiyama and Mitsuyasu Hasebe). Then, the linearized plasmid Pn2-ODD2-pTFH15.3 by Not I was transformed into P. patens protoplast using PEG-mediated transformation method as described by Cove et al. (2009) [32], with a slight modification. After 7 days, the regenerated protoplasts were screened on BCD medium containing 25, 50 and 100 μg·mL^- 1 G418 to obtain the successful transgenic lines. Finally, the genomic PCR was used to verify stable integration of Pn2-ODD2 in P. patens.

The Pn2-ODD2 was inserted into the pRI101 plasmid carrying the 35S promoter from Cauliflower mosaic virus (CaMV) for efficient expression (stocked in our lab) to obtain transgenic Arabidopsis. Four-week-old wild-type Arabidopsis were transformed by Agrobacterium GV3101 containing the Pn2-ODD2-pRI101 vector, following the Agrobacterium-mediated floral dipping method [33]. Then, positive transgenic seedlings were selected by germinating seeds on 1/2 MS medium supplemented with kanamycin. The homozygous transgenic lines (AtOE1, AtOE2 and AtOE3) were identified for phenotypic analysis.

Plants stress tolerance analysis

For phenotype assays of transgenic P. patens, the stem tips of wild-type and transgenic P. patens gametophytes with the same size were grown on BCD medium containing 0.1, 0.2 or 0.3 M D-Mannitol and 5, 10 or 15 μM ABA for about 5-7 weeks. Then, the gametophyte phenotypes were observed and recorded. Five-week-old P. patens gametophytes were treated with 200 mM NaCl for 2 h. Then, total RNA was extracted using CTAB method for the expression analysis of stress-related genes in transgenic P. patens.

For seed germination analysis, the surface-sterilized seeds were planted on 1/2 MS medium with D-Mannitol, 3-amino-1,2,4-triazole (3-AT) and ABA, kept in the dark for 2 days at 4 °C. Then all plates were cultivated in a chamber room at 22 °C for 7-10 days. The proportion of cotyledon greening was used to express the germination rate. For root length assay, the seeds were sown on 1/2 MS medium supplemented with 0.5 or 0.75 mM H₂O₂. After vertically grown for 14 days, the root length was photographed and measured by Image J software. For the gene expression analysis under abiotic stress, two-week-old Arabidopsis seedlings were treated with 20 mM H₂O₂ and 100 μM ABA for 2 h, respectively. Then we harvested the plant samples and stored at -80 °C.

In order to observe the accumulation of anthocyanins in 5-day-old Arabidopsis, the sterilized seeds were grown on 1/2 MS medium for 5 days, with constant light at 22 °C. For the anthocyanin enrichment in 17-day-old seedlings, the seeds were sown on 1/2 MS medium in supplement with 3% (w/v) sucrose for 14 days under 16 h/8 h photoperiod cycle. Then Arabidopsis seedlings were transferred to 1/2 MS medium containing 12% (w/v) sucrose for 3 days [34]. The pigment phenotype of seedlings was recorded, and the seedlings were collected and stored for subsequent analysis.

Quantification of anthocyanins and flavonols

The extraction and quantification of anthocyanins was as follows: 0.1 g plant samples were ground into powder with liquid nitrogen, and incubated with 600 μL methanol containing 1% HCl for 24 h at 4 °C in the dark. Then 600 μL chloroform and 300 μL ddH₂O were added to remove chlorophyll, and the mixture was centrifuged for 15 min at 12,000 g. The collected supernatant was used to detect the absorbance at 340, 530 and 657 nm. The contents of total flavonoids and anthocyanins were represented as: (A340/g) and (A530-0.25*A657)/g, respectively [35].

The extraction of flavonol was performed following the previous description [22]. Homogenized plant powder was incubated with 50% methanol, then the mixture was centrifuged at 13,000 g for 15 min at 4 °C. The crude extract was hydrolyzed at 70 °C for 40 min, by adding an equal amount of 2 N HCl. Then 100% methanol was used to prevent aglycone degradation. After centrifugation (13,000 g, 15 min), the supernatants were collected for subsequent HPLC analysis (Shimadzu LC-20 A, Japan) at 365 nm, with the photodiode array (PDA) detector and a C18 column (5 μm, 4.6 × 250 mm). The column was kept at a 1 mL·min^− 1 flow rate at 30 °C. The mobile phases were solvent A (ddH₂O with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The separation program was as follows: 0 min, 5% solvent B; 30–45 min, 55-65% solvent B; 50 min, 100% solvent B; 52–60 min, 5% solvent B [18].

3,3′-diaminobenzidine (DAB) and nitrobluetetrazolium blue chloride (NBT) staining

The levels of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^-) were detected by DAB and NBT staining as described previously [36], with minor modification. In brief, the seedlings were immersed in 1 mg·mL^- 1 DAB with 10 mM Na₂HPO₄ and 0.05% Tween-20, or 1 mg·mL^- 1 NBT solution with 10 mM potassium phosphate buffer (pH 7.8) in the dark for 12 h with slight shaking. Then, a bleaching solution (ethanol: acetic acid: glycerol = 3:1:1) was used to decolorize seedlings by boiling water bath until chlorophyll was completely removed.

Total RNA extraction and quantitative real-time PCR

Total RNA was isolated from P. patens and Arabidopsis using CTAB method and TRIzol reagent, respectively. cDNA synthesis was carried out by 5 × All-In-One RT MasterMix (Abm, Canada), following the manufacturer’s protocal. The PCR reaction mixture consisted of 1 μL cDNA template, 10 μL SYBR qPCR Master Mix (Nuoweizan, Nanjing, China) and 0.5 μL primer (10 μM), using the following cycle procedure: 95 °C for 30 s, 40 cycles 95 °C for 10 s, 60 °C for 30 s. PpActin and AtTublin served as the reference gene to calculate the relative expression levels by the 2^-ΔΔCt method [37]. All primers for qRT-PCR were presented in Table S1.

Statistical analysis

All experiments were repeated at least three times for biological replicates. All data were presented as mean ± standard deviation (SD).

Results

Characterization and bioinformatics analysis of Pn2-ODD2

The full-length open reading frame (ORF) of Pn2-ODD2 consisted of 1068 bp, which encoded a protein of 355 amino acids with a predicted molecular weight of 40.7 kDa and a calculated isoelectric point of 5.46. Pn2-ODD2 contained the signature domains of the 2ODDs family: N-terminally conserved DIOX_N domain and 2-OG-FeII_Oxy domain (Fig. 1A). Multiple alignment analysis showed that Pn2-ODD2 shared homology with several known 2-ODDs from other species and possessed the same conserved domains: Fe²⁺ binding site HxDxnH (His²²⁴, Asp²²⁶ and His²⁸⁰) and the 2-oxoglutarate (2-OG) binding domain RxS (Tyr²⁰⁹, Arg²⁸⁹ and Ser²⁹¹) (Fig. 1A). The conserved active amino acids residues were represented in Fig. 1A and C. Phylogenetic analysis showed that FNSsI, F3Hs, FLSs and ANSs in flavonoids biosynthetic pathway, formed two independent branches. FLSs and ANSs were clustered in one clade, and Apiaceae FNSsI were grouped with F3Hs, away from the bryophyte/lycophyte FNSsI, which was consistent with the previous results [17, 38]. And Pn2-ODD2 had a close relationship with Physcomitrium patens 2-ODD, predicted as a probable ANS (Fig. 1B). Additionally, the relative expression patterns of Pn2-ODD2 under NaCl, PEG and ABA treatment were measured by qRT-PCR. The results indicated that the transcription level of Pn2-ODD2 was increased 4.19-fold at 6 h, decreasing thereafter to 1.37-fold at 12 h after NaCl treatment. Pn2-ODD2 exhibited a 2.24-fold increase at 12 h of PEG treatment, after which the expression levels reduced to 1.94-fold at 24 h. Also, the expression of Pn2-ODD2 were induced by ABA treatment (Fig. 1D).

Fig. 1.

Pn2-ODD2 shared homology with several known 2-ODDs from other species. A Multiple alignment of the amino acid sequences of Pn2-ODD2 with 2-ODDs from other plant species. Purple frames: Fe²⁺ binding site (HxDxnH) and 2-ODD binding site (RxS). Red triangle: the conserved amino acids residues of 2OG-FeII_Oxy domain. OsFLS: XP_015624815.1; TcFLS: EOY16220.1; GmANS: AAR26525.1; GbANS: ACC66092.1; TaANS: BAE98274.1; Pp2-ODD1: XP_001780809.1; PpANS probable: XP_024374335.1; PnFNSI: QCP71067.1. B The phylogenetic tree of Pn2-ODD2 and other known 2-ODDs constructed by Maximum likelihood (ML) method. The node symbols represent the bootstrap values. C Three-dimensional structure model of Pn2-ODD2. The reported crystal structure of anthocyanin synthase (2brt.pdb) from Arabidopsis was selected as the template for 3D structure prediction. The GMQE value was 0.68. The QMEANDisCo Global score was 0.68 ± 0.05. α-helix, β-sheet and random coils are colored red, yellow, and green, respectively. The conserved Fe²⁺ binding site (His²²⁴, Asp²²⁶ and His²⁸⁰) and the 2-oxoglutarate (2-ODD) binding domain RXS (Tyr²⁰⁹, Arg²⁸⁹ and Ser²⁹¹) were labeled by arrow. D The relative expression levels of Pn2-ODD2 under different treatment of abiotic stress measured by qRT-PCR. Asterisk (*) represents a significant difference between before and after treatment (n = 3, Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001)

Overexpression of Pn2-ODD2 promoted the resistance to osmotic stress in plants

To analyze the functions of Pn2-ODD2 in plant abiotic stress, we obtained the independent transgenic P. patens (PpOE1, PpOE2, PpOE3 and PpOE4), and confirmed the successful integration of Pn2-ODD2 into the transformed plants genome (Fig. S1). The same size stem tips of WT and transgenic P. patens were cultivated on BCD medium supplemented with 0.1, 0.2 and 0.3 M D-Mannitol. In the absence of D-Mannitol, no detectable difference in gametophytes growth was observed between WT and transgenic P. patens. However, in the presence of D-Mannitol, the growth rates of overexpressed Pn2-ODD2 P. patens were superior to that of WT plants. Under 0.1 and 0.2 M D-Mannitol treatment, the diameters of gametophytes of transgenic lines were about 1.60-fold compared with WT plants. On 0.3 M D-Mannitol medium, transgenic P. patens also displayed the larger colony size of 5.47, 5.18, 5.93 and 5.64 mm, in contrast with 3.02 mm of WT plants (Fig. 2A-B). In Arabidopsis, at 0.1 M D-Mannitol, overexpressed-Pn2-ODD2 Arabidopsis exhibited higher germination rates relative to the WT plants. Under 0.2 M D-Mannitol treatment, the germination rates of AtOE lines were 77.5%, 75.0% and 73.3%, respectively, which was about 20.0% higher than the WT plants (Fig. 2C-D).

Fig. 2.

Pn2-ODD2 conferred the resistance to osmotic stress in plants. A-B Gametophyte growth phenotype and statistical analysis of gametophyte size in WT and transgenic Pn2-ODD2 P. patens treated with D-Mannitol. C-D Phenotype of germination and statistical analysis in WT and transgenic Pn2-ODD2 Arabidopsis treated with D-Mannitol. E 3,3′-diaminobenzidine (DAB) and nitrobluetetrazolium blue chloride (NBT) staining. F-I The expression levels of ROS scavenging genes in Arabidopsis analyzed by qRT-PCR. Asterisk (*) represents a significant difference between the WT plants and AtOE lines (n = 3, Student’s t-test, *P < 0.05, **P < 0.01)

Drought stress usually induces the accumulation of reactive oxygen species (ROS) in plants. The accumulation of H₂O₂ and O₂^- were measured by DAB and NBT stainng, respectively. The result showed that AtOE lines accumulated less H₂O₂ and O₂^- level in contrast with WT plants (Fig. 2E). Furthermore, the transcript levels of ROS-scavenging related gene AtFeSOD1, AtFeSOD2, AtCu/Zn-SOD1 and AtCu/Zn-SOD2 in overexpressed Pn2-ODD2 plants were significantly up-regulated, which was an average increase of 1.66, 1.43, 1.86 and 1.64-fold compared with WT plants, respectively (Fig. 2F-I). These results suggested that Pn2-ODD2 enhanced the resistance to drought stress by increasing antioxidant capacity in plants.

Pn2-ODD2 conferred the tolerance to oxidative stress in Arabidopsis

Both salt and osmotic stress can lead to the primary oxidative stress [39]. We examined the seedling root growth in WT and AtOE lines under exogenous H₂O₂ treatment to clarify the oxidative stress response of the overexpressed-Pn2-ODD2 plants. Under non-stress condition, there was no significant difference in root growth between WT and transgenic Arabidopsis. However, at 0.5 mM H₂O₂, AtOE lines performed a 1.42-fold increase in root length, compared with WT plants. In the presence of 0.75 mM H₂O₂, the root length of AtOE lines was 1.97, 1.99 and 1.78 cm, which was approximately 50.0% longer than that of WT plants (Fig. 3A-B). Also, the lateral root numbers of overexpressed Pn2-ODD2 Arabidopsis increased by 56.3% in contrast with WT plants (Fig. 3C). Meanwhile, when treated with 20 mM H₂O₂, the transcript levels of ROS generation gene AtRbohC and AtRbohD, encoding NADPH oxidase, were decreased in AtOE lines (Fig. 3D). 3-Amino-1,2,4-triazole (3-AT), a catalase inhibitor, can induce the accumulation of endogenous H₂O₂ [40]. Under the treatment of 10 μM 3-AT for 7 days, overexpression of Pn2-ODD2 reduced the sensitivity to ROS in plants (Fig. 3E-F). In addition, overexpressed Pn2-ODD2 Arabidopsis displayed the higher levels of total flavonoids and anthocyanins compared with WT plants, which was increased about 20.0% and 30.0%, respectively (Fig. 3G-H). These results indicated that Pn2-ODD2 increased the tolerance to oxidative stress by reducing ROS levels and increasing anthocyanins accumulation.

Fig. 3.

Pn2-ODD2 increased the oxidative resistance in transgenic Arabidopsis. A Phenotypes of root growth of Arabidopsis under H₂O₂ treatment. B-C Statistical analysis of root length and lateral root number as shown in A. D The expression levels of respiratory burst oxidase homolog protein (i.e., RbohC and RbohD) in Arabidopsis under H₂O₂ treatment. E-F Phenotype of ROS sensitivity and the sensitivity quantification in WT and AtOE lines after 10 μM 3-AT treatment for 7 days. The sensitivity rate was calculated by dividing the number of seedlings showing chlorosis by the total number of seedlings. G-H The levels of total flavonoids and anthocyanins under 3-AT treatment for 7 days. Asterisk (*) represents a significant difference between the WT plants and AtOE lines (n = 3, Student’s t-test, *P < 0.05, **P < 0.01)

Pn2-ODD2 reduced the ABA sensitivity in transgenic P. patens and Arabidopsis

ABA, an important hormone in plants, is widely involved in various physiological processes such as plant growth, plant development and the adaptation to abiotic stress [41]. Here, we compared the growth of transgenic plants and WT plants under the treatment of ABA. In P. patens, overexpressed Pn2-ODD2 plants were insensitive to ABA treatment. When exposed to 5 μM and 10 μM ABA, the sizes of the transgenic gametophytes were about 1.30- and 1.40-fold compared with WT plants. At 15 μM ABA medium, the gametophytes diameters of the transgenic plants performed an increase of 37.4-62.6% in contrast with WT plants (Fig. 4A-B). In Arabidopsis, in the absence of ABA, no obvious growth difference was observed between AtOE lines and WT plants. At 0.25 μM ABA, the germination rates of AtOE lines were about 25.0% higher than that of the WT plants. Following the 0.5 μM ABA treatment, the germination rates of overexpressed-Pn2-ODD2 Arabidopsis were 28.3%, 46.7% and 38.4%, respectively, while the germination rate of WT plants was 13.4% (Fig. 4C-D).

Fig. 4.

Pn2-ODD2 reduced the ABA sensitivity in transgenic plants. A-B Gametophyte phenotype and statistical analysis of gametophyte size in WT and transgenic Pn2-ODD2 P. patens.C-D Phenotype of germination and statistical analysis in WT and transgenic Pn2-ODD2 Arabidopsis under ABA treatment. E Expression patterns of ABA-related genes in P. patens and Arabidopsis under ABA treatment. Asterisk (*) represents a significant difference between the WT plants and AtOE lines (n = 3, Student’s t-test, *P < 0.05, **P < 0.01)

To investigate the possible molecular mechanism mediating the ABA effects, the expression levels of genes related to the ABA pathway were analyzed by qRT-PCR. The results demonstrated that the expression patterns of several ABA-insensitive (ABI) genes PpABI3a, PpABI3b and PpABI5b were markedly decreased in transgenic P. patens. Meanwhile, the stress-responsive genes AtRD29A, AtRD29B and AtAREB1 were significantly up-regulated in overexpressed Pn2-ODD2 Arabidopsis compared with WT plants (Fig. 4E-F). These results showed that the reduced ABA sensitivity in transgenic plants may be associated with the increase of ABA response.

Pn2-ODD2 enhanced the anthocyanin and flavonol in transgenic Arabidopsis

In order to investigate the possible effect of Pn2-ODD2 on flavonoids metabolism, AtOE lines and WT plants were grown in a greenhouse for 5 days with constant light or induced by 12% (w/v) sucrose for 3 days to accumulate anthocyanin. The results showed that AtOE lines exhibited the higher anthocyanin levels, which was an increase of about 20.0% and 60.0% compared with WT seedlings (Fig. 5A-C). Consistent with the phenotype, the transcript patterns of genes (AtPAL, AtCHS, AtF3’H, AtDFR and AtUFGT) in anthocyanin biosynthesis were obviously up-regulated relative to WT plants (Fig. 5D-E). Furthermore, we also analyzed the flavonols compounds in 17-day-old Arabidopsis induced by 12% (w/v) sucrose. The result showed that the contents of kaempferol and quercetin in AtOE lines were significantly increased by 30.2% and 25.0% compared with WT plants, respectively (Fig. 5F-G).

Fig. 5.

Pn2-ODD2 enhanced the accumulation of anthocyanins and flavonols in Arabidopsis. A-B Visible phenotypes of anthocyanin accumulation in Arabidopsis. C The levels of anthocyanins measured by spectrophotometry analysis. D-E The transcript levels of anthocyanin biosynthesis genes in 5-day-old seedlings with constant light or in 17-day-old seedlings induced by 12% (w/v) sucrose for 3 days. F HPLC profiles of flavonols extracts (365 nm) in 17-day-old seedlings. G The contents of major flavonols (quercetin and kaempferol) in WT and transgenic Pn2-ODD2 Arabidopsis induced by sucrose. Asterisk (*) represents a significant difference between the WT plants and AtOE lines (n = 3, Student’s t-test, *P < 0.05, **P < 0.01 )

Discussion

Flavonoids are widely distributed in plants kingdom, which is involved in various physiological and biochemical processes, as well as the response to environmental stressors [42]. They are considered to have arisen during plant evolution from aquatic to terrestrial about 500 million years ago [1]. During the flavonoid biosynthesis in plants, 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs), including FNSI, F3H, FLS and ANS/LDOX, catalyze the oxidative modifications to the central C-ring to produce diverse flavonoid subgroups [43]. However, the biological functions of these enzymes in bryophytes are rarely reported and whether bryophytes synthesize anthocyanins is still controversial. A widely targeted metabolomics built on the UPLC-MS/MS platforms showed that cyanidin 3-O-(6’’-malonylglucoside), malvidin 3-O-galactoside, and peonidin O-hexoside were detected in P. nutans [29]. Similarly, six anthocyanin compounds, including cyanidin 3-O-galactoside, cyanidin 3-O-rutinoside, peonidin 3-O-glucoside chloride, peonidin O-hexoside, pelargonidin and malvidin 3-O-glucoside, were identified in the Antarctic moss Leptobryum pyriforme [27]. Further analyses confirmed the presence of anthocyanins in other Antarctic bryophytes, such as Bryum pseudotriquetrum, Ceratodon purpureus, Schistidium antarctici, and the liverwort Marchantia polymorpha [44]. However, HPLC-based analyses failed to detect anthocyanins in stressed P. patens [45]. In this study, we cloned a 2-ODD2 gene from Antarctic moss Pohlia nutans (Pn2-ODD2) and explored its roles in plant resistance to abiotic stress. Silico analysis showed that Pn2-ODD2 shared 28.3% identity with previously reported Pn2-ODD1 and contained the conserved HxDxnH motif (His²²⁴, Asp²²⁶ and His²⁸⁰) for Fe²⁺ binding and RxS motif (Tyr²⁰⁹, Arg²⁸⁹ and Ser²⁹¹) for 2-oxoglutarate (2-OG) binding, like other members of 2OG-FeII_Oxy dioxygenase family from different species. Phylogenetic analysis showed that Pn2-ODD2 was grouped with the Physcomitrium patens probable ANS, suggesting that Pn2-ODD2 might be an ANS (Fig. 1). 2-ODDs in flavonoid biosynthesis participated in plant responses to abiotic stress [46]. In cotton, when treated by different environment stress (PEG, salt, cold and heat stress), most members of Gh2ODD family were responsive to one or more stress by the transcriptome analysis [47]. In Arachis hypogaea, the expression of AhFLS was induced by different concentration of salt stress [48]. The ectopic expression of 2-ODDs genes also enhances plant tolerance to abiotic stress. For example, overexpression of PnFNSI from P. nutans conferred the resistance to drought, UV-B and oxidative stress by reducing ROS levels and increasing the ROS scavenging in Arabidopsis [49]. Overexpressed-MaANS (from Morus alba L.) tobacco displayed the increased tolerance to D-Mannitol and NaCl stress [50]. In this study, the expression of Pn2-ODD2 was up-regulated under salt, PEG and ABA treatment (Fig. 1D) and its overexpression promoted the resistance to D-Mannitol in transgenic P. patens and transgenic Arabidopsis, exhibiting the faster gametophytes growth, or the better seed germination (Fig. 2A-D). The accumulation of ROS is usually accompanied by drought stress. In the present study, when exposed to 16% PEG treatment, the levels of ROS in Arabidopsis were increased, while Pn2-ODD2-overexpressing Arabidopsis displayed less H₂O₂ and O₂^- accumulation compared with WT plants (Fig. 2E). Additionally, the transcript levels of ROS scavenging enzyme (i.e., AtFeSOD1, AtFeSOD2, AtCu/Zn-SOD1 and AtCu/Zn-SOD2) were significantly up-regulated in AtOE lines under 16% PEG treatment (Fig. 2F-I).

The overaccumulation of ROS in plant can be induced by abiotic stress, which impairs proteins, lipids and nucleic acid and even leads to cellular damage and death [51]. Plants have developed the enzymatic and non-enzymatic systems to maintain ROS homeostasis [52]. Flavonoids, one of the important non-enzymatic components, have strong antioxidant activity due to the presence of functional hydroxyl groups in their chemical structures to scavenge free radicals and chelate metal ions [53]. ROS can induce anthocyanin accumulation by up-regulating late biosynthetic and the corresponding regulatory genes. Anthocyanins regulate the ROS level and the sensitivity to ROS-generating stress [40]. In Arabidopsis, transcriptome and metabolome profiling data had revealed that flavonoid overaccumulation was essential for increased tolerance to drought and oxidative stress in plants [54]. Overexpression of RtLDOX2 from Reaumuria trigyna promoted the accumulation of flavonol and anthocyanins under abiotic stress, with a significantly increased antioxidant system [23]. In this study, overexpression of Pn2-ODD2 in Arabidopsis enhanced the tolerance to oxidative stress by the down-regulated levels of ROS production gene AtRbohC and AtRbohD (Fig. 3A-D). Under the treatment of 10 μM 3-AT (endogenous H₂O₂ treatment), overexpression of Pn2-ODD2 reduced the sensitivity to ROS in plants, and enhanced the accumulation of total flavonoids and anthocyanins (Fig. 3E-H).

Abscisic acid (ABA), a stress-responsive hormone, plays important roles in various physiological activities, including seed growth, dormancy and adaption to environmental stress [55]. In cotton, the promoter regions of Gh2ODD members mostly contained abscisic acid- and ethylene-responsive elements, which suggesting that Gh2ODD may be a target gene of ABA and ethylene [47]. The transcript pattern of GbANS gene from Ginkgo biloba was significantly promoted by ABA, salicylic acid and other stimuli [56]. Previously, overexpression of anthocyanidin reductase from Rosa rugosa increased the expressions of the ABA-biosynthesis genes encoding zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase in transgenic tobacco, suggested that the RrANR had an effect on the ABA biosynthetic pathway [57]. PnF3H, a flavanone 3-hydroxylase from the Antarctic moss P. nutans, decreased the ABA sensitivity by downregulating the expression of ABA pathway related to the seed germination and seedling growth in transgenic plants [58]. In moss, ABA can induce the differentiation of protonema cells into vegetative propagules in order to survive in detrimental environmental stress [59]. In this study, when exposed to ABA, the growth rates of transgenic gametophytes were markedly higher than those of the WT gametophytes (Fig. 4A-B). ABA-insensitive 3 (ABI3), a positive transcription factor of the ABA signaling pathway, is essential for both seed growth and dormancy; its absence reduces the seed dormancy during seed development [60]. Plants of overexpressed ABI5, encoding a basic leucine zipper transcription factor, displayed hypersensitive to ABA [61]. Here, we found that the expression levels of PpABI3a, PpABI3b and PpABI5c genes were significantly down-regulated in transgenic P. patens under ABA treatment (Fig. 4E). Furthermore, overexpression of Pn2-ODD2 in Arabidopsis also displayed decreased sensitivity to ABA during seed germination (Fig. 4C-D). ABA-responsive element-binding protein (AREB) is a basic domain/leucine zipper transcription factor, and AREB1/ABF2 was triggered in vegetative tissues by ABA treatment, dehydration or high salinity [62]. The responsive to desiccation genes RD29A and RD29B were also induced by exogenous ABA treatment, drought and high salinity, which was regulated by AREB transcription factors due to their promoter regions containing ABRE elements [63]. The transcript levels of AtAREB1, AtRD29A and AtRD29B were up-regulated in Pn2-ODD2-overexpressed Arabidopsis in comparison with WT plants (Fig. 4F), which may be correlated with the reduced ABA sensitivity in transgenic Arabidopsis.

The content of flavonoids in plants can be altered by operating the expression of genes in flavonoid biosynthesis pathway. Overexpression of CmFNS from Chrysanthemum morifolium increased the accumulation of flavones in tobacco [64]. Overexpression of RuFLS2 from blackberry (Rubus spp.) resulted in the enhanced levels of flavonoids, following the upregulated expression levels of NtF3H and NtFLS in tobacco [65]. Also, overexpressed-TcANS (from Theobroma cacao) tobacco exhibited the increased anthocyanin and proanthocyanidin accumulation, with a visible increase in pink color intensity in flower petals [66]. Here, when grown for 5 days with 24 h light, Pn2-ODD2 promoted the contents of anthocyanins, and induced the expression of genes in flavonoid biosynthesis (Fig. 5A-D). Sucrose, a main product of photosynthesis, controls various developmental and metabolic processes in plants [67]. In Arabidopsis seedlings, sucrose is the most effective activator of anthocyanin biosynthesis. Exogenous sucrose triggered the anthocyanin accumulation by elevating transcript levels of anthocyanin biosynthesis genes. Meanwhile, sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the transcription factors MYB75/PAP1 gene [68, 69]. Similarly, the stimulatory effects of sucrose on anthocyanin biosynthesis in different organs of several plant species have been reported previously, such as radish and grape berries [70, 71]. Sucrose could enhance anthocyanin production in vegetative tissue of transgenic Petunia carrying anthocyanin regulatory transcription factors [72]. In this study, when induced by 12% (w/v) sucrose for 3 days, AtOE lines displayed the enhanced accumulation of anthocyanin and up-regulated expression patterns of anthocyanin biosynthetic genes (Fig. 5A-C and 5E). Previous research had reported that overexpression of rice ANS in Nootripathu (NP, a rice mutant) resulted in an increased biosynthesis of anthocyanins, flavonol and total flavonoid, acting as a multifunctional dioxygenase in flavonoid pathway in rice [73]. In overexpressed-RtLDOX2 Arabidopsis, the versatility and mutual substitutability of RtLDOX2 in anthocyanin and flavonol biosynthesis might lead to the increased anthocyanin and flavonol levels [23]. Here, Pn2-ODD2 also enhanced the levels of kaempferol and quercetin in Arabidopsis (Fig. 5F-G). Combined with the phylogenetic analysis, Pn2-ODD2 might encode a putative ANS, which has a dual role in flavonoid pathway. More evidences are needed to further validate the conclusion.

Conclusions

In this study, we isolated a Pn2-ODD2 gene of flavonoid biosynthesis pathway from Antarctic moss Pohlia nutans. The expression of Pn2-ODD2 was triggered by various abiotic stress, including NaCl, PEG and ABA treatment. Overexpression of Pn2-ODD2 enhanced the resistance to drought and oxidative stress in Physcomitrium patens and Arabidopsis, with the larger gametophyte, the higher germination rate and the increased antioxidant ability. And Pn2-ODD2 also decreased the sensitivity to ABA in transgenic plants. Additionally, overexpression of Pn2-ODD2 led to the accumulation of anthocyanins and flavonols in Arabidopsis. Our results suggested the possible significant function of Pn2-ODD2 in P. nutans acclimation to extreme environment.

Electronic supplementary material

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Author contributions

P.Z. and S.L. conceived and designed the experiments. H.W. performed the experiments. H.W. and P.Z. conducted the data analysis. C.L. and H. L participated in the bioinformatics analysis. H.W. wrote the manuscript and P.Z. revised it. All authors read the manuscript and agreed to publish.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (ZR2022MC075), the National Natural Science Foundation of China (41976225), Scientific Fund for National Public Research Institutes of China (GY0219Q05) and Central Government Guide Local Science and Technology Development Funds (YDZX20203700002579).

Data availability

The authors confirm that all data supporting the findings of this study are included in the article and its supplementary data.

Declarations

Ethics approval and consent to participate

The experimental study used here complies with institutional, national, and international guidelines concerning plant genetic repositories.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.