Ying Zho, Xin Li, Zexin Zhng, Wenjing Pn, Sinn Li, Yun Xing,Wnying Xin,Zhnguo Zhng,Zhenbng Hu,Chunyn Liu, Xioxi Wu,Zhoming Qi, Dwei Xin,*, Qingshn Chen,*

aCollege of Agriculture,Northeast Agricultural University, Harbin 150030,Heilongjiang,China

bKey Lab of Maize Genetics and Breeding,Heilongjiang Academy of Agricultural Sciences,Harbin 150030,Heilongjiang, China

cCollege of Agriculture,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang,China

Keywords:Redox homeostasis Respiration characteristics GmGPDH12 Salt stress Osmotic stress

ABSTRACT In plants, glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) coupled to the reduction/oxidation of the nicotinamide adenine dinucleotide (NADH) pool, and plays a central role in glycerolipid metabolism and stress response. Previous studies have focused mainly on the NAD+-dependent GPDH isoforms, neglecting the role of flavin adenine dinucleotide (FAD)-dependent GPDHs. We isolated and characterized three mitochondrialtargeted FAD-GPDHs in soybean, of which one isoform (GmGPDH12) showed a significant transcriptional response to NaCl and mannitol treatments, suggesting the existence of a major FAD-GPDH isoform acting in soybean responses to salt and osmotic stress. An enzyme kinetic assay showed that the purified GmGPDH12 protein possessed the capacity to oxidize G3P to DHAP in the presence of FAD. Overexpression and RNA interference of GmGPDH12 in soybean hairy roots resulted in elevated tolerance and sensitivity to salt and osmotic stress, respectively. G3P contents were significantly lower in GmGPDH12-overexpressing hair roots and higher in knockdown hair roots, indicating that GmGPDH12 was essential for G3P catabolism. A significant perturbation in redox status of NADH,ascorbic acid (ASA) and glutathione (GSH) pools was observed in GmGPDH12-knockdown plants under stress conditions. The impaired redox balance was manifested by higher reactive oxygen species generation and consequent cell damage or death; however,overexpressing plants showed the opposite results for these traits. GmGPDH12 overexpression contributed to maintaining constant respiration rates under salt or osmotic stress by regulating mRNA levels of key mitochondrial respiratory enzymes.This study provides new evidence for the roles of mitochondria-localized GmGPDH12 in conferring resistance to salt or osmotic stress by maintaining cellular redox homeostasis, protecting cells and respiration from oxidative injury.

1.Introduction

Glycerol-3-phosphate (G3P) is an important metabolite that participates in several biochemical and physiological processes including glycolysis,lipolysis,and glycerolipid synthesis [1,2]. In plants, G3P dehydrogenase (GPDH) catalyzes the interconversion of G3P and dihydroxyacetone phosphate(DHAP) coupled to the reduction/oxidation of a nicotinamide adenine dinucleotide (NADH) pool [3,4]. G3P can be produced directly from DHAP via NAD+-dependent GPDH(EC 1.1.1.8)and degraded to DHAP by mitochondrial flavin adenine dinucleotide(FAD)-dependent GPDH(EC 1.1.99.5),with both metabolic steps essential for glycerolipid metabolism [2]. Multiple plant isoforms of NAD+-GPDH with distinct biological functions have been characterized [5-7], and reside in the cytosol or chloroplasts. In contrast, a mitochondrial FAD-GPDH isoform that catalyzes the oxidation of G3P has been described only in the model plant Arabidopsis thaliana [4]. The A. thaliana FADGPDH is strongly expressed during seed germination, when the breakdown of storage lipid into free fatty acids and glycerol is expected to occur [4]. In a further study, FADGPDH was essential for glycerol catabolism and gluconeogenesis during germination [8].

In addition to glycerol metabolism, FAD-independent GPDH has been implicated in the modulation of cellular redox homeostasis via a mitochondrial G3P shuttle [9,10].The G3P shuttle has been extensively studied in yeast and animals but poorly in plants [11,12]. In this shuttle, cytosollocalized NAD+-dependent GPDH consumes reducing power(NADH) to produce G3P via reduction of DHAP. G3P then crosses the outer mitochondrial membrane and is reoxidized to DHAP by FAD-dependent GPDH,forming FADH2in the inner mitochondrial membrane. The resulting DHAP is returned to the cytosol to complete the shuttle,which channels electrons from NADH generated in the cytosol into the mitochondrial electron transfer chain [10]. In A. thaliana, the mitochondrial FAD-GPDH encoded by AtGPDHm can form a G3P shuttle by combining with the cytosolic NAD+-GPDH encoded by AtGPDHc1, providing the first evidence for the presence of a G3P shuttle in plants [3,4]. The operation of such a shuttle is vital for maintaining the intracellular balance of NADH/NAD+in A. thaliana, similar to that found in other eukaryotic systems [10]. Loss of AtGPDHc1 results in a fluctuation of the cytosolic NADH redox state along with increased generation of reactive oxygen species (ROS) [3]. Likewise, AtGPDHmdeficient mutants(sdp6)fail to normalize the cellular homeostasis of NADH/NAD+ratio owing to the impaired G3P shuttle system [8]. In our previous study [7], plants overexpressing a cytosolic NAD+-GPDH from maize (ZmGPDH1) showed increased ability to regain intracellular redox equilibrium under stress conditions.

Salinity and osmotic stress impair plant development and grain yield by modifying protein composition and cell membrane permeability [13]. The characteristics of mitochondrial FAD-GPDH associated with osmotic stress or salt tolerance have been described in some model organisms.The enzyme activities and transcripts of Dunaliella salina mitochondrial FAD-GPDH are highly increased by salinity treatment and repressed by oxygen deficiency and cold stress[14].The expression of AtGPDHm is stimulated by salt and dehydration stress and coupled to oxygen consumption, but the response mechanism has not been investigated [4].

Given that mitochondrial FAD-GPDH genes and the G3P shuttle have to date been identified only in yeast, animals,algae, and Arabidopsis [4,10,14,15], their roles are not well understood. Soybean is a major protein and oil crop whose development is frequently affected by abiotic stresses such as high salinity and osmotic stress [16]. In the present study,three mitochondrial-targeted FAD-GPDH genes were identified and isolated from soybean on the basis of their sequence analysis and subcellular localization, and designated as GmGPDH11, GmGPDH12 and GmGPDH13. Among them,GmGPDH12 was strongly expressed in all tissues studied and was strongly stimulated by both NaCl and mannitol treatments,implying a crucial role in response to salt and osmotic stress.The prokaryotic expression of GmGPDH12 in Escherichia coli showed that this gene encoded the active FAD-dependent GPDH enzyme.

By analysis of GmGPDH12-overexpressing and GmGPDH12-RNAi transgenic soybean hair roots,we found that GmGPDH12 played a prominent role in maintaining cellular redox and ROS homeostasis as well as mitochondrial respiration under salt or osmotic stress. We present a model for the potential involvement of mitochondrial-targeted GmGPDH12 in metabolic response to oxidative stress induced by salt or osmotic stress.

2. Materials and methods

2.1. Identification of GmGPDHs

To identify GPDH genes in the soybean genome,the conserved domains of the published A. thaliana GPDHs were used as queries in a BLASTP search against a soybean genetics and genomics database (SoyBase, https://www.soybase.org/sbt)with an E-value of <10?10. Basic information of GmGPDHs,including chromosome locations, coding region lengths,protein lengths, isoelectric point, and molecular mass was obtained from SoyBase and ExPASy(http://expasy.org/).Transit peptides and transmembrane structure were predicted with TargetP 2.0 (http://www.cbs.dtu.dk/services/TargetP/)and CELLO 2.5(http://cello.life.nctu.edu.tw/)[17].

2.2. Phylogenetic and domain analysis of GmGPDHs

The amino acid sequences of GPDH-homologous proteins in A. thaliana (AtGPDH), Glycine max (GmGPDH), Zea mays(ZmGPDH), Oryza sativa (OsGPDH), Sorghum bicolor (SbGPDH),Setaria italic (SiGPDH), Phaseolus vulgaris (PvGPDH), Medicago truncatula (MtGPDH) and Brassica rapa (BrGPDH) were downloaded from Phytozome database and used to construct a phylogenetic tree with MEGA 5.0 software [18].Alignment of the amino acid sequences was performed with ClustalW and the conserved domains were confirmed.Phytozome accession numbers of these genes are listed in Table S1.

2.3. Plasmid construction and subcellular localization

The entire coding regions of the putative mitochondrial GmGPDHs were cloned from the roots of soybean cultivar SN14 and inserted into the plant expression vector pBI121 to produce a fusion protein construct with green fluorescent protein (GFP). The gene-specific primers used for cloning GmGPDHs are presented in Table S2. For co-localization studies, a mitochondrial (Mito) marker COX4 [19]was fused to the N-terminus of a far-red fluorescent protein(mkate).The recombinant plasmids GmGPDHs::GFP and mkate::COX4 were temporarily expressed in Arabidopsis mesophyll protoplasts 16 h after PEG-calcium-mediated cotransformation [20]. The empty vector was used as positive control. The subcellular locations of the expression of GFP and mkate proteins were visualized by confocal laser-scanning microscopy (LSM710,Carl Zeiss,Germany).

2.4. Prokaryotic protein expression and promoter activity assay

The coding sequence of GmGPDH12 with XhoI and NdeI sites was constructed in the prokaryotic expression vector pET-22b(+) to generate a recombinant plasmid pET-22b-GmGPDH12,which was further transformed into E. coli strain DE3 for fusion protein expression. The positive clone was selected and cultured under optimum inducing conditions (37 °C,1 mmol L?1IPTG, 4 h) to express the His-GmGPDH12 protein.His-tagged GmGPDH12 proteins were purified by Ni-NTA affinity chromatography and detected by Western blot using anti-His antibodies. The kinetic parameters of purified GmGPDH12 protein with respect to the substrate (G3P) were determined with an Eadie-Hofstee plot[4].

A 2218-bp genomic fragment from upstream of GmGPDH12 was amplified by polymerase chain reaction (PCR) and was then linked to the GUS gene in the pBI121 basic vector for an in vivo reporter gene assay. The primers used to construct this vector are listed in Table S2. The resulting recombinant plasmid (proGmGPDH12::GUS) was transformed into the wildtype A. thaliana and T2 transgenic lines were used for histochemical GUS activity assay as previously described[21]. The GUS-stained samples were visualized under a stereomicroscope.

2.5. Overexpression and suppression of GmGPDH12 in soybean hairy roots

The coding region of GmGPDH12 was cloned into the vector pSOY1 containing a CaMV35S promoter to generate a pSOY1-GmGPDH12 overexpression vector. A 189-bp ligated fragment containing the coding sequence (positions 42 to 230), was cloned and ligated into the vector pB7GWING2 to generate a pB7GWING2-GmGPDH12-RNAi suppression vector (primers see Table S2). The two plasmids were independently introduced into Agrobacterium rhizogenes strain K599 by electroporation. The resulting constructs were transformed into soybean hypocotyls to induce hair roots as described previously[22].Control hairy roots were generated by A.rhizogenes K599 infection. The transformed hair roots were screened by PCR amplification and reverse transcription-PCR (RT-PCR).More than 10-20 independent transgenic hair root lines were investigated to identify changes in biochemical and physiological parameters.

The fresh weight of hair roots and wild-type leaves and the maximum root length was recorded after 5 days of treatment with H2O2,120 mmol L?1NaCl or 200 mmol L?1mannitol. The contents of total chlorophyll,chlorophyll a,and chlorophyll b of the top secondary fully expanded leaves were measured following a reported protocol [23]and the leaves were extracted with 80% (v/v) acetone. The mitochondrial respiration of leaves and hair roots was determined by repeated measures analysis of variance with a portable photosynthesis system (LI-6400, Li-Cor, US). Air flow into the chamber was held at 400 mL min?1with a CO2concentration of 400 μmol mol?1. Soybean plants harboring transgenic hair roots were preadapted to darkness for 15 min before the measurement of mitochondrial respiration[24].

2.6. Detection of biochemical and physiological parameters

The assay of NAD+-dependent GPDH activity was performed using a method previously described [3], with slight modifications. The total volume was reduced to 1 mL with an appropriate amount of enzyme, 0.2 mmol L?1NADH,120 mmol L?1HEPES-Tris buffer (pH 6.9) and 4 mmol L?1DHAP.The reaction was conducted at 25°C for 5 min and the absorbance of the supernatant was read at 340 nm [3]. FADGPDH activity was assayed as previously described [4]. The total volume was reduced to 1 mL with an appropriate amount of enzyme, 4 mmol L?1INT, 0.1 mmol L?1FAD,1 mmol L?1KCN, 40 mmol L?1G3P and 50 mmol L?1bicine buffer(pH 8.0).The absorbance was measured at 490 nm.G3P levels were determined using a previously published method[3].

To detect the redox status of ascorbic acid (ASA) and glutathione (GSH) pools, the levels of oxidized form (DHA,GSSG) and reduced form (ASA, GSH) were measured by spectrophotometry as previously described [25]. Intracellular concentrations of NADH and NAD+were detected using a Coenzyme NADH Colorimetric Assay Kit purchased from Beijing Solarbio Technology Co. Ltd. Levels of superoxide (O2?)and hydrogen peroxide (H2O2) were determined by histochemical staining and spectrophotometry as described [26].Membrane damage was determined as malondialdehyde(MDA) content, a product of lipid peroxidation [27]. To investigate ROS-scavenging capacity, soybean samples(0.5 g)were extracted with 15 mL of 50 mmol L?1K2HPO4-KH2PO4(pH 7.0) buffer containing 0.5 mmol L?1ASA, 1.5 mmol L?1EDTA and 1% (w/v) PVP. The resulting supernatant fluid was used for the detection of antioxidase activities, including superoxide dismutase (SOD), catalase (CAT) and peroxidase(POD)[28,29].

2.7. Phenotypic analysis of the GmGPDH12 transgenic Arabidopsis

The previously characterized T-DNA insertional mutants of AtGPDHm (AT3G10370), namely sdp6 (SALK_080169) were obtained from the Arabidopsis Biological Resource Center(Ohio State University,Columbus,OH,USA).The homozygous mutants were screened by PCR and further confirmed by RTPCR(primers listed in Table S2).For complementation assays,the entire coding region of GmGPDH12 was cloned into a pBI121 binary vector under the control of the CaMV35S promoter. The overexpression construct 35S::GmGPDH12 was then mobilized into Agrobacterium strain EHA105 to transform homozygous sdp6 mutants. To investigate growth properties including germination rate,root length,and fresh weight,WT,sdp6 and T3 transgenic plants were grown on half-strength MS medium supplemented with 0, 200/300 mmol L?1mannitol and 120/150 mmol L?1NaCl for 7 days.

2.8. Expression analysis of GmGPDHs

To record the transcriptional profiles of the mitochondrial GmGPDHs during salt or osmotic stress,soybean plants at twotrifoliolate leaf stage were transplanted to 0, 200 mmol L?1mannitol or 120 mmol L?1NaCl solutions for 0,1,3,6,and 12 h.To investigate marker gene expression induced by salt/osmotic stress, 4-week-old soybean plants harboring OHR(overexpressing hairy roots), RHR (RNAi hairy roots) or CHR(control hairy roots) were treated with H2O2, 200 mmol L?1mannitol or 120 mmol L?1NaCl for 12 h. Total RNA was extracted from roots using Trizol reagent and purified by phenol/chloroform extraction.The cDNA generated from 1 μg of total RNA sample was prepared for quantitative real-time PCR(qRT-PCR). Each reaction was performed in triplicate and the expression of selected genes was analyzed by the 2?ΔΔCtmethod. GmACTIN and GmGAPDH were used as internal reference(primers see Table S2).

3.Results

3.1. Identification of mitochondrial FAD-dependent GPDHs in soybean

Thirteen GmGPDH genes were identified by BLAST search against the soybean genome using known AtGPDHs as query,and named GmGPDH1-GmGPDH13 (Table S3). Protein sequences translated from GmGPDH genes varied from 356 to 629 amino acid residues (Table S3). Phylogenic analysis of GmGPDHs with homologous proteins in O. sativa, Z. mays, A.thaliana,S.bicolor,S.italica,P.vulgaris,M.truncatula,and B.rapa indicated that plant GPDHs could be assigned to three major clusters (Fig. 1A). Cluster I corresponded to plastidic (P) type,including GmGPDH6, 7, 8, and 10, two A. thaliana P-GPDHs(AtGLY1, AtGPDHp1), and a small number of orthologous genes. Cluster I was further divided into two groups (a, b), in which GmGPDH8 and an active P-GPDH isoform (AtGLY1)were clustered as class a [6]; GmGPDH6, 7, 10 and an inactive P-GPDH isoform (AtGPDHp) were assigned to class b [5].Cluster II corresponded to a cytosolic (Cy) type, containing GmGPDH1, 2, 3, 4, 5, and 9, together with two A. thaliana Cy-GPDHs (AtGPDHc1, AtGPDHc2). Cluster III corresponded to a mitochondrial(Mito)type,comprising GmGPDH11,12,and 13,one A. thaliana mitochondrial FAD-GPDH (AtGPDHm) and orthologous genes from other species. Transit-peptide and isoform localization predictions were in agreement with the phylogenetic clades for GmGPDHs. GmGPDH6, 7, 8, and 10 were predicted as plastidic,those for GmGPDH11,12 and 13 as mitochondrial,and the others as cytosolic(Table S3).

Additionally,sequence analyses showed all GmGPDH proteins contained typical and necessary domains similar to other reported GPDH [3,15]. Specifically, GmGPDH1-10 genes encoded NAD+-GPDHs,harboring a C-terminal GPD domain(PF07479)and an N-terminal NAD+-binding domain (PF01210). GmGPDH11, 12,and 13 genes encoded FAD-GPDHs, consisting of a C-terminal alpha-glycerophosphate oxidase domain (PF02781) and an N-terminal FAD-dependent oxidoreductase domain (PF01266) (Fig.S1). Two highly conserved binding sites, G3P-binding motif(DVLSAWSGIRPLA; GLITITGGKWTTYRSMAE) and FAD-binding motif (DVLVIGGGATGCGVALDAVTRGLRVGLVER) were readily found in the protein sequence of soybean FAD-GPDHs (Fig. 1B).Based on the phylogenetic, protein structural, and subcellular localization analyses, GmGPDHs were divided into three types:six cytosolic NAD+-GPDHs (GmGPDH1, 2, 3, 4, 5 and 9), four plastidic NAD+-GPDHs (GmGPDH6, 7, 8 and 10), and three mitochondrial FAD-GPDH(GmGPDH11,12 and 13).

To further confirm the subcellular localization of candidate mitochondrial GmGPDHs, their entire coding regions were amplified, sequenced, and submitted to GenBank database under the following accession numbers: GmGPDH11(MN786868), GmGPDH12 (MN786869), and GmGPDH13(MN786870). The co-expression of GFP-tagged GmGPDHs together with the Mito-marker was measured in A. thaliana leaf mesophyll protoplasts. Single GFP alone was distributed evenly throughout the cytoplasm and excluded from the vacuoles and chloroplast; however, GmGPDH11-GFP,GmGPDH12-GFP, and GmGPDH13-GFP fusion protein each displayed the same subcellular localization as Mito-marker(Fig.1C).These results suggested that GmGPDH11,GmGPDH12 and GmGPDH were targeted specifically to mitochondria.

In all, 13 GPDH genes were identified in soybean and designated as GmGPDH1-13. Among these, only three(GmGPDH11, 12, and 13) encoded mitochondrial-targeted FAD-dependent GPDHs.

3.2. GmGPDH12 encodes the main isoform of mitochondrial FAD-dependent GPDHs in response to salinity or osmotic stress

Fig.1- Identification,subcellular localization,and expression characteristics of mitochondrial GmGPDHs.(A)Evolutionary analysis of GPDHs from G.max,A. thaliana,Z.mays, O.sativa, P.vulgaris, M.truncatula,B.rapa,and S. bicolor.(B) Multiple alignments among putative mitochondrial GmGPDHs(GmGPDH11,12, and 13) with other reported mitochondrial FAD-GPDH proteins.(C)Subcellular distribution of GFP(positive control),GmGPDH11-GFP,GmGPDH12-GFP,and GmGPDH13-GFP recombinant fusions in Arabidopsis leaf mesophyll protoplasts stained with a mkate-tagged mitochondrial(Mito) marker,COX4.The merged images include the green fluorescence (first panel),the red fluorescence (second panel),the chloroplast autofluorescence(third panel)and the corresponding bright field (fourth panel).Bars,10 μm.(D)Transcriptomic data of mitochondrial GmGPDHs in various tissues are downloaded from Phytozome database.The results are shown as heat maps.The size and color of filled circles represents transcription level, with a larger and red circle indicating high-abundance transcripts.(E)Transcripts of GmGPDH11,12,and 13 in soybean roots subjected to H2O2,200 mmol L?1 mannitol or 100 mmol L?1 NaCl solutions for 0, 1, 3,6,and 12 h.The expression of these genes under non-stress conditions was used for calibration.Asterisks above bars denote Student's t-test significance(*,P<0.05; **,P<0.01).

Transcripts of GmGPDH11, 12, and 13 genes were detected in many tissues, including root, nodules, stem, flower tissues,pod,shoot apical meristem,root hairs,leaf,and seed(Fig.1D).Of these,GmGPDH12 was the most widely expressed isoform,although the level of expression varied widely among tissues(Fig. 1D). To investigate the function of mitochondrial GmGPDHs in abiotic stress response, we first measured the expression patterns of GmGPDH11, 12, and 13 under salt and osmotic stress, which are frequently encountered in our area(Heilongjiang, China). Transcripts of mitochondrial GmGPDHs was markedly increased by salt or osmotic stress, with GmGPDH12 responding more rapidly and vigorously to both treatments than the other genes (Fig. 1E), indicating a prominent role in response to salt or osmotic stress.

To further confirm the response of GmGPDH12 to salinity,the promoter activity of GmGPDH12 gene was assayed. The highest GUS activity was observed during germination, as reflected by an intensive GUS blue coloration in 2-day-old embryos (Fig. 2A). In 4- to 14-day-old proGmGPDH12::GUS transgenic seedlings, strong GUS staining was detected in stems and leaves. In 6-week-old plants, strong GUS staining was shown in roots, flowers, and petioles (Fig. 2A). The expression of proGmGPDH12::GUS was also increased by both NaCl and mannitol treatments (Fig.2B).

We examined the catalytic characteristics of GmGPDH12 by extracting and purifying the recombinant protein from the E. coli expression plasmid of His-tagged GmGPDH12 (Fig. 2C).The kinetic parameters Vmaxand Kmof purified GmGPDH12 with spect to its substrate G3P were estimated as 58.37 μmol min?1mg?1protein and 7.09 mmol L?1, respectively (Fig. 2D).The enzyme activity was significantly elevated by addition of a flavin coenzyme, FAD (Fig. 2E), indicating that GmGPDH12 encodes an active FAD-dependent GPDH enzyme.

3.3.GmGPDH12 is involved in conferring resistance to osmotic or salt stress in transgenic soybean hair roots

Fig.2-Prokaryotic expression and promoter analysis of mitochondrial GmGPDH12.(A)GUS activity assay in distinct tissues.(a)flower;(b) cauline leaf;(c)stem;(d) root;(e)2-day-old,(f) 5-day-old,(g) 9-day-old and(h)14-day-old proGmGPDH12::GUS transgenic plants.Scale bars,100 μm.(B)GUS activity assay following salt or osmotic treatment.(a)Non-transformed wild-type(WT)A.thaliana was used as negative control.7-day-old proGmGPDH12::GUS transgenic A. thaliana treated with H2O2(b),150 mmol L?1 NaCl(c)or 200 mmol L?1 mannitol(d)for 12 h before GUS staining.Scale bars,100 μm.(C)Western blot analysis using the anti-6×His His antibody as a probe.Lane 1:Purified GmGPDH12 extracts from E. coli cells harboring pET-22b-GmGPDH12.(D)Kinetic parameters of GmGPDH12 with respect to G3P.(E)Activity of FAD-GPDH from purified GmGPDH12 in the presence(+) and absence(?)of FAD and G3P,respectively.

To further clarify how mitochondrial GmGPDH12 contributes to the response to salt and osmotic stress,composite soybean plants consisting of non-transgenic leaves and transgenic hairy roots overexpressing or silencing GmGPDH12 were generated (Fig. S2A-E). The enzymatic assay showed that the activity of FAD-GPDH in OHR was 2.2-fold higher than that in CHR, whereas the FAD-GPDH activity in RHR was 54% of that in CHR (Fig. S2E). These results suggested that GmGPDH12 gene was successfully overexpressed or suppressed in transgenic soybean hair roots, and functioned with FAD-GPDH activities. No aberrant growth phenotype was observed in composite plants harboring transgenic hairy roots or control hair roots under standard conditions (Fig. S2F), showing that overexpression and suppression of GmGPDH12 had little effect on soybean development.

Compared with CHR plants, the GmGPDH12-OHR and GmGPDH12-RHR plants displayed obvious phenotype differences after salt and osmotic-stress treatments (Fig. 3A). The RHR plants showed severe growth-inhibitory morphologies,with a visible leaf yellowing symptom.In contrast,OHR plants showed increased resistance to salt and osmotic stress, as represented by larger root elongation and higher fresh weight of both root and leaf (Fig. 3B-D). RHR plants showed lower average contents of total chlorophyll, chlorophyll a, and chlorophyll b than CHR plants under salt or osmotic stress(Fig. 3E, F). However, the chlorophyll contents in OHR plants were much higher than those in other plants. These results indicated that mitochondrial GmGPDH12 positively regulated salt and osmotic-stress tolerance in soybean.

3.4. GmGPDH12 is required for maintaining cellular redox state perturbations following treatment with salinity or osmotic stress

In controls, the NAD+-GPDH and FAD-GPDH activities were higher in GmGPDH12-OHR but lower in RHR (Fig. 5A, B). A significant induction in the two enzyme activities was detected after treatment with salinity or osmotic stress, with the increase being more noticeable in OHR (Fig. 4A, B). G3P levels were significantly reduced in OHR but increased in RHR under both control and stress conditions (Fig. 4C), suggesting that GmGPDH12 played an essential role in cellular G3P catabolism.

Fig.3- Mitochondrial GmGPDH12 increases the tolerance of transgenic soybean hair roots to salinity and osmotic stress.Phenotype(A),maximum root length(B), root fresh weight(C),and leaf fresh weight(D)of soybean plants harboring GmGPDH12-OHR,GmGPDH12-RHR or CHR after 5 days of 0,200 mmol L?1 mannitol or 120 mmol L?1 NaCl treatment.Bars,2 cm.Contents of total chlorophyll,chlorophyll a,and chlorophyll b of GmGPDH12-OHR,GmGPDH12-RHR,and CHR plants subjected to 120 mmol L?1 NaCl(E)or 200 mmol L?1 mannitol(F) Over 5 days.Values are plotted as means ± SEs(n ≥ 5). Asterisks denote Student's t-test significance (*,P< 0.05;**,P <0.01)of differences from CHR plants.

In the absence of NaCl and mannitol, NADH contents and NADH/NAD+ratios were lower in OHR but higher in RHR than in CHR (Fig. 4D). In the presence of NaCl and mannitol, a severe disturbance of NADH/NAD+ratios was detected in all transgenic lines,and the differences could also be detected in either NAD+or NADH levels.Notably,OHR maintained relative lower NADH contents than CHR, thus decreasing the NADH/NAD+ratio, even though stress caused a great rise in NADH level.In reverse,RHR accumulated higher NADH contents and lower NAD+contents than CHR, leading to an increased NADH/NAD+ratio (Fig. 4D). These results showed that mitochondrial FAD-GPDH enzyme encoding by GmGPDH12 gene participated in modulating cellular NADH/NAD+homeostasis possibly through a G3P shuttle by combination with NAD+-GPDH enzyme.

We further investigated whether GmGPDH12 overexpression or suppression could affect other metabolites involved in redox exchange, such as ascorbic acid/dehydroascorbate(ASA/DHA) and reduced/oxidized glutathione (GSH/GSSG).The levels of these metabolites did not change among CHR,OHR, and RHR under normal conditions. Under salt and osmotic stress,the OHR accumulated higher contents of GSH and ASA and lower contents of GSSG and DHA than CHR,leading to higher GSH/GSSG and ASA/DHA ratios.In contrast,a marked reduction in GSH and ASA contents was observed in the GmGPDH12-RHR relative to CHR(Fig.4E,F).Together,these results showed that the function of GmGPDH12 in salt or osmotic resistance consisted at least in part of maintaining the cellular redox balance.

3.5. GmGPDH12 participates in moderating ROS level under salinity or osmotic stress

Fig.5- Mitochondrial GmGPDH12 increases antioxidant capacity under salinity or osmotic stress.Images showing representative NBT(A),DAB(B),and Evan's blue staining(C)in leaf of GmGPDH12-OHR,GmGPDH12-RHRm and CHR plants subjected to 0,200 mmol L?1 mannitol or 120 mmol L?1 NaCl for 24 h.Bars,1 cm.O2?(D),H2O2(E)and MDA(F)contents in root and leaf of OHR,RHR,and CHR plants after 3 days of 0,200 mmol L?1 mannitol or 120 mmol L?1 NaCl treatment.Activities of CAT(G),SOD(H),and POD(I)in root and leaf of OHR,RHR,and CHR plants treated as above.Values are plotted as means ± SEs(n ≥ 5).Asterisks denote Student's t-test significance(*,P <0.05;**, P<0.01) of difference from CHR plants.

Clear changes in redox status of NADH,ASA,and GSH pools in GmGPDH12 transgenic hair roots led us to measure ROS levels under salinity and osmotic conditions. O2?and H2O2were detected by nitroblue tetrazolium (NBT) (Fig. 5A) and 3,3-diaminobenzidine (DAB) staining (Fig. 5B), respectively. For both ROS, strong staining, representing increased ROS production, was shown in GmGPDH12-RHR leaves under salt or osmotic stress. In contrast, faint and sporadic NBT and DAB staining was detected in GmGPDH12-OHR leaves under identical conditions. Cell death induced by salt or osmotic stress was also monitored by Evan's blue staining. Mild intensities of Evan's blue were observed under non-stress conditions(Fig.5C).In contrast,cell death was highly induced in RHR leaves,moderately induced in CHR leaves,and slightly induced in OHR leaves under salt and osmotic stress.Levels ofand MDA in both hairy roots and leaves of OHR plants were clearly lower than the corresponding levels in CHR plants.In contrast,there was a marked increase in H2O2,and MDA contents in RHR plants relative to CHR plants,implying a major role for GmGPDH12 in ROS elimination under salinity or osmotic stress(Fig.5D-F).

Because elevated ROS production will activate the cellular antioxidative signaling machinery in a compensatory mode,antioxidant enzyme activities were also measured [30,31]. As expected, increased activities of POD, CAT, and SOD were observed in all lines during salt and osmotic stress, with the increase in GmGPDH12-OHR plants more prominent than that in CHR plants (Fig. 5G-I). In contrast, the GmGPDH12-RHR plants showed comparatively low enzymatic ROS-scavenging capacity as compared to CHR. These findings suggested that mitochondrial GmGPDH12 might exert negative effects on ROS accumulation through the regulation of cellular redox status as well as antioxidant defense, and consequently reduce the damage to cell membranes.

3.6. Expression of genes related to cellular redox and ROS homeostasis in GmGPDH12 transgenic hair roots under salinity and osmotic stress

To deeply comprehend the molecular basis for the effects of mitochondrial GmGPDH12 on redox and ROS homeostasis,we investigated the expression levels of several known genes participating in (1) ROS-scavenging system: catalase (CAT1,CAT2), copper/zinc superoxide dismutase (SOD1, SOD2) and peroxidase (PER22) [32]; (2) ASA and GSH biosynthesis:ascorbate peroxidase (sAPX), glutathione reductase (GR1),monodehydro-ascorbate reductase (MDAR1) and L-galactose dehydrogenase (GLDH) [33,34]. In the absence of NaCl or mannitol treatment, there were no significant differences in the transcripts of all genes tested among GmGPDH12-OHR,GmGPDH12-RHR, and CHR. Nevertheless, the mRNA levels of CAT1,CAT2,SOD1,SOD2,and PER22 were significantly induced by both salinity and osmotic stress, and were higher in OHR but lower in RHR than in CHR (Fig. 6). These data also supported the finding that activities of antioxidase (CAT,POD, and SOD) were elevated in soybean hair roots overexpressing GmGPDH12 (Fig. 5). The mRNA abundance of key enzymes involved in the regulation of ASA or GSH redox state(sAPX, MDAR1, GR1 and GLDH) was markedly reduced in RHR and elevated in OHR in comparison to CHR (Fig. 6). Thus, the stress-inducible responses of these genes were impaired in GmGPDH12-RHR but increased in GmGPDH12-OHR, possibly contributing to the stress-sensitive phenotypes of RHR and stress-resistant phenotypes of OHR plants.

3.7. GmGPDH12 is essential for regulating mitochondrial respiration under salinity/osmotic stress

Because mitochondria plays a pivotal role in the orchestration of oxidative stress response [35], we also checked whether respiration characteristics were changed in GmGPDH12-OHR or GmGPDH12-RHR plants. Mitochondrial respiration rates varied negligibly between OHR, RHR, and CHR plants prior to stress treatment (Fig. 7A, B). Upon NaCl or mannitol treatment, a marked decline in mitochondrial respiration appeared in both leaves and hairy roots of all the lines. The reduction in respiration rates was more marked in RHR and less so in OHR than in CHR plants (Fig. 7A, B), indicating the importance of the GmGPDH12 gene in regulation of mitochondrial respiration.

We further assessed the effects of GmGPDH12 overexpression or suppression on the transcripts of some marker genes related to mitochondrial respiratory metabolism: (1) cytochrome c oxidase (COX15a, COX15b, COX2, COX6A) [36]; (2)alternative oxidase (AOX1a, AOX-like, AOX1, AOX2) [37]; (3)malate-OAA shuttle enzymes(MADH1,MADH2,CMDH1,IDH1)[38]. Transcripts of COX15a, COX15b, COX2, and COX6A were significantly induced by NaCl or mannitol treatment and the transcriptional patterns of these genes in OHR or RHR were generally similar to those in CHR, indicating that GmGPDH12 had no perceptible impact on cytochrome c oxidase respiration pathway (Fig. 7C). In contrast, the expression of genes encoding alternative oxidase (AOX) was markedly increased in OHR and decreased in RHR relative to CHR,except AOX-like,whose expression was unchanged between different transgenic hair roots(Fig.7D).The expression of mitochondrial and cytoplasmic malate dehydrogenase (MADH1, MADH2,CMDH1), and chloroplastic/peroxisomal isocitrate dehydrogenase (IDH1) involved in the malate-OAA shuttle were markedly stimulated in all the lines after treatment with NaCl or mannitol. However, the increase in the expression of these genes was greater in GmGPDH12-OHR than in RHR (Fig. 7E).Thus, GmGPDH12 was required to maintain mitochondrial respiration during salt or osmotic stress, possibly by upregulating the mRNA expression of respiratory enzymes.

3.8. Overexpression of GmGPDH12 rescues salinity and osmotic sensitivity of AtGPDHm-deficient mutants

To further verify these findings, GmGPDH12-transformed AtGPDHm knock-out mutant lines were generated. RT-PCR revealed that GmGPDH12 transcripts were present in each transformed plant (COM-1, COM-2), but absent in WT or homozygous AtGPDHm-deficient mutants (sdp6) (Figs. S3, S4).On media supplemented with 200 mmol L?1mannitol or 100 mmol L?1NaCl, the germination of sdp6 mutants was severely delayed compared with WT, whereas GmGPDH12-COM seeds displayed a similar germination rate to WT (Fig.8A, B). Similarly, when 7-day-old WT, sdp6 and COM plants treated with 300 mmol L?1mannitol or 150 mmol L?1NaCl for 7 days, the sdp6 seedlings displayed severe stress or injury,with a large-leaf shriveling phenotype (Fig. 8C). In contrast,the GmGPDH12 COM seedlings showed a WT-like phenotype under the same conditions (Fig. 8D, E). Cell death induced by NaCl or mannitol treatment was estimated by PI staining. As shown in Fig. 8F, the extent of cell death was less in COM plants and greater in sdp6 mutants compared to WT, again showing that GmGPDH12 overexpression helped to prevent cellular damage by oxidative stress. The functional complementation results thus confirmed that mitochondrial GmGPDH12 had the ability to restore the FAD-GPDH activities in AtGPDHm-deficient mutants,and thus rescued the salt and osmotic sensitivity of sdp6 mutants.

Fig.6- Transcripts of key genes involved in cellular redox and ROS homeostasis in GmGPDH12 transgenic hairy roots.The mRNA expression level of genes involved in the antioxidant system(CAT1,CAT2,SOD1,SOD2,and PER22)and redox homeostasis(sAPX,GR1,MDAR1,and GLDH)in OHR,RHR,and CHR subjected to 0,200 mmol L?1 mannitol,or 120 mmol L?1 NaCl for 12 h.Values are plotted as means± SEs(n ≥3). Asterisks denote Student's t-test significance (*,P< 0.05;**,P <0.01) of differences from CHR.

4.Discussion

4.1. GmGPDH12 encodes the major FAD-GPDH isoform involved in the response to salinity/osmotic stress

Sequences of thirteen GPDH genes (GmGPDH1-13) were retrieved from Soybase (Table S3). Only three proteins(GmGPDH11, 12, and 13) contained two conserved domains typical of mitochondrial FAD-dependent GPDHs [4], whereas others contained NAD+-dependent GPDH-like domains (Fig.S1). Previous studies on C. reinhardtii and D. salina GPDHs revealed a phosphoserine phosphatase (PSP) domain that functions as glycerol-3-phosphatase [39,40], but we did not find a PSP domain in soybean GPDH proteins. Similar to the yeast or A. thaliana FAD-GPDHs [4,9,15], a putative FAD-binding site and sequences involved in substrate binding were identified in GmGPDH11, 12, and 13 proteins (Fig. 1B).However, unlike the animal FAD-GPDHs [10,12], the soybean mitochondrial FAD-GPDHs lacked the EF-hand calciumbinding domain. Thus, in contrast to mitochondrial NADPH dehydrogenases which are likely to be inactive in unstressed plant cells and regulated by Ca2+[41],the activities of soybean FAD-GPDHs are not dependent on Ca2+changes.

The previously identified A. thaliana FAD-GPDH was predicted to be a mitochondrial-targeted isoform because of the existence of apparent transmembrane domains and signal peptide [4], but clear experimental evidence was lacking.Photographs of GmGPDHs-expressed mesophyll protoplasts stained with a mitochondrial tracker showed that the GmGPDH11,GmGPDH12,and GmGPDH13 proteins were localized exclusively in the mitochondria (Fig. 1C). Transcripts of the three mitochondrial GmGPDHs were stimulated by both NaCl and mannitol treatments(Fig.1D,E),with GmGPDH12 the most up-regulated member,implying a major role for soybean responses to salt and osmotic stress.This expression pattern was also confirmed by GUS histochemical staining of proGmGPDH12::GUS transgenic A. thaliana following NaCl and mannitol treatments(Fig.2B).Similar results were obtained in earlier studies:a mitochondrial FAD-GPDH gene from D.salina was essential for early salt response [14]; and the expression of AtGPDHm is also modulated by ABA, salinity and dehydration stress,though the mechanism was unclear[4].

4.2. GmGPDH12 is required for modulating the cellular redox state and ROS level under salinity or osmotic stress

Fig.7- Mitochondrial GmGPDH12 regulates respiration under salinity and osmotic stress.Mitochondrial respiration rates in root and leaf of GmGPDH12-OHR,GmGPDH12-RHR and CHR plants subjected to 120 mmol L?1 NaCl(A)or 200 mmol L?1 mannitol(B)over 5 days.The mRNA expression level of genes involved in (C)mitochondrial cytochrome c oxidase(COX15a, COX15b,COX2,and COX6A),(D)alternative oxidase(AOX1a,AOX-like,AOX1,and AOX2),and(E)malate-OAA shuttle enzymes(MMDH1,MMDH2,CMDH1,and IDH1)in OHR,RHR,and CHR subjected to 0,200 mmol L?1 mannitol or 120 mmol L?1 NaCl for 12 h.Values are plotted as means± SEs(n ≥3).Asterisks denote Student's t-test significance(*,P<0.05;**,P<0.01)of difference from CHR.

The molecular mechanism of mitochondrial GmGPDH12 in mediating salinity or osmotic adaptation was further studied by ectopic expression of GmGPDH12 in soybean hair roots or AtGPDHm-deficient mutants (sdp6), and both suggested that GmGPDH12 was involved in conferring tolerance to both salt and osmotic stress(Figs.3,8).To our knowledge,this study is the first demonstration that mitochondrial FAD-GPDH is positively associated with salt/osmotic resistance in soybean.Under salinity and osmotic stress,composite plants harboring GmGPDH12-overexpressing hair roots (OHR) showed larger elongation of root and healthier leaf than did control plants.Synchronously, the fresh weight and chlorophyll content of non-transgenic leaves in OHR plants were higher than those in control plants. Thus, this and previous studies [42,43]provide strong evidence that the function of a wild-type leaf can be influenced by transgenic hair roots in composite soybean plants, and that stress tolerance of the roots would induce better growth performance of the upper parts.

GmGPDH12 could play a key role in at least two crucial metabolic pathways under salinity and osmotic stress,including G3P dissimilation and redox homeostasis (Fig. 4).G3P is an intermediary metabolite with considerable importance for gluconeogenesis,lipolysis,and glycerolipid synthesis[1,2]. In this study, G3P content was significantly lower in GmGPDH12-OHR and higher in GmGPDH12-RNAi hair roots (Fig.4C),showing that GmGPDH12 activity favors degradation of G3P in plant cells. Consistent with its role in G3P catabolism, the highest expression level of the GUS reporter gene in proGmGPDH12::GUS transgenic A. thaliana was observed in germinating seed after 2 days of imbibition (Fig. 2-A), a period considered [2,44]to be the most rapid phase of storage lipid breakdown. Plants defective in AtGPDHm showed massive impairment in the conversion of glycerol to carbon dioxide(CO2)and tended to accumulate more G3P [8]. In Yarrowia lipolytica,inactivation of FAD-GPDH enzyme led to an increase of lipid content reaching 30%of dry weight after 24 h of growth[45].

Fig.8- Overexpression of GmGPDH12 rescues salinity and osmotic sensitivity of AtGPDHm-deficient mutants(sdp6).Constitutive pBI121-35S:GmGPDH12 transformed homozygous sdp6 mutants were obtained for complementation assays.(A)Growth performance and(B)germination rate of WT,sdp6,GmGPDH12 transformed sdp6(COM-1,COM-2)seeds treated with 0,200 mmol L?1 mannitol or 100 mmol L?1 NaCl for 7 days.(C)Growth performance,(D)root length and(E)fresh weight of 7-dayold WT,sdp6,GmGPDH12-transformed sdp6(COM-1,COM-2)seedlings treated with 0,300 mmol L?1 mannitol or 150 mmol L?1 NaCl for 7 days.Bars,1 cm.(F)PI fluorescence staining in root tips after treatment with 0, 300 mmol L?1 mannitol or 150 mmol L?1 NaCl for 12 h.Bars,100 μm.Values are plotted as means ± SEs(n ≥ 3). Asterisks denote Student's t-test significance(*, P<0.05; **,P< 0.01)of difference from WT.

FAD-GPDHs are reported to function as the mitochondrial component of a G3P shuttle that transports cytosolic reducing equivalent(NADH)into the mitochondrial respiratory electron transport chain [3,11,12]. In the present study, silencing the GmGPDH12 gene also led to a severe disturbance of NADH/NAD+homeostasis in an fashion identical to that reported in AtGPDHm knock-out mutants [8]; however, GmGPDH12-OHR showed the opposite results for these traits (Fig. 4D).Overexpression of GmGPDH12 also increased cytosolic NAD+-GPDH activity apart from a direct consequence of increased FAD-GPDH activity(Fig.4A,B).These observations support the involvement of these two enzymes in a mitochondrial G3P shuttle that effectively moderated the NADH/NAD+ratio perturbations following stress treatments, as speculated in Fig. S5. Furthermore, the impairment in maintaining cellular NADH/NAD+homeostasis in GmGPDH12-RNAi hair roots was also manifested as a higher ROS level and the consequent damage to the cell membrane, which was not observed in OHR (Fig. 5). Because a perturbation in cellular redox may promote the over-reduction of molecular oxygen, thereby inducing ROS production [30,31], it is easily conceivable that GmGPDH12 contributes to regulation of oxidative metabolism by modulating the intracellular NADH/NAD+redox state. The compensatory antioxidant response is known to be triggered by excess ROS produced during stress conditions [46]. In agreement,we found that the activity and gene expression of metabolic enzymes related to ROS elimination (CAT1, CAT2,SOD1, SOD2, PER22) [32]and ASA/GSH redox cycles (sAPX,MDAR1, GR1, GLDH) [33,34]were highly induced by salt and osmotic stress; however, the increase was much more significant in GmGPDH12-OHR (Figs. 5, 6). Indeed, there is increasing evidence that the redox status or ratio of the reduced NADH and oxidized NAD+forms regulate the expression of nuclear antioxidant/redox genes [30,47]. It is thus reasonable to speculate that the increase in NAD+/NADH ratio induced by GmGPDH12 overexpression can serve as a signal that activates the expression of genes whose products act to maintain cellular redox homeostasis and promote ROS removal,and thereby improve stress tolerance(Fig.S5).

4.3. GmGPDH12 participates in regulating respiration rate perturbations following treatment with salinity or osmotic stress

Respiration rates of mitochondria obtained from water-stressed plants were reduced, a result attributed to ROS-induced mitochondria damage[48].In the present study,respiration rates were sharply degraded by both salinity and osmotic stress, with the reduction more marked in RHR and less so in OHR than in CHR plants (Fig. 7A, B), implying a critical role of GmGPDH12 in sustaining constant respiration rates during stress. The relative importance of high respiration rates is more rapid production of adenosine triphosphate that provides essential energy for tissue tolerance, sodium exclusion, and osmotic adjustment [37]. The mRNA levels of genes encoding alternative oxidase (AOX) were much higher in OHR plants but lower in RHR than CHR(Fig.7D).The finding of no difference in transcripts of cytochrome c oxidase genes (COX15a, COX15b, COX2, COX6A) between GmGPDH12-transgenic hair roots and CHR (Fig. 7C) suggested that GmGPDH12 gene might not influence the cytochrome c oxidase pathway.Combining this finding with results of previous studies[3,4,8],we speculate that the electrons donated by the G3P shuttle are transported to O2mainly through an alternative oxidase respiration pathway,which introduces a branch into the electron transfer chain at the ubiquinone pool preventing overreduction of the downstream complexes (Fig. S5). This speculation also provides a biologically interpretable explanation for the enhanced salinity and osmotic resistance of soybean plants overexpressing GmGPDH12, as elevated AOX capacity prevents excessive reduction of the ubiquinone pool[49].

GmGPDH12 also influenced other metabolic pathway involved in cytosol-to-mitochondria transfer of reducing equivalents [38], as represented by a pronounced increase in transcripts of genes involved in the mitochondrial malate/OAA shuttle (MADH1, MADH2, CMDH1, IDH1) in GmGPDH12-OHR(Fig.7E).Deficiency in AtGPDHc1 led to a sharp change in metabolites involved in the malate/OAA shuttle, but a detailed explanation was lacking [3]. Further research may shed light on the potential interaction between the malate/OAA shuttle and the G3P shuttle.

In summary, there is a wealth of evidence that redox signals can serve as a language of interorganellar communication and induce nuclear gene expression.The central role of mitochondria as a source and target of redox regulation has been reported[47,50].The consumption of cytosolic NADH by GmGPDH12-mediated mitochondrial G3P shuttle results in very low levels of NADH (Fig. 4). Such redox changes can represent a sensor of environmental variation and act as signals that coordinate nuclear gene expression with the functional state of mitochondria but also with the physiological response to counteract the oxidative injury induced by salt and osmotic stress,as hypothesized in Fig.S5.

5.Conclusions

We report the molecular cloning, expression, and physiological characterization of three mitochondria-localized FAD-dependent GPDH genes from soybean, GmGPDH11-13, of which GmGPDH12 encoded the major FAD-GPDH isoform for soybean responses to salinity and osmotic stress. Further study suggested the role of GmGPDH12 in salt/osmotic stress tolerance through maintaining the cellular redox and ROS balance as well as mitochondrial respiration.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This study was financially supported by National Natural Science Foundation of China (31701449, 31971968, 31971899,31501332),National Key Research and Development Program of China(2016YFD 0100500,2016YFD0100300,2016YFD0100201-21,JFYS2016ZY03003792-01-21), China Postdoctoral Science Foundation (2019M661243), Postdoctoral Project of Northeast Agricultural University (NEAUBH-19002), EUCLEG (727312,2017YFE0111000), Natural Science Foundation of Heilongjiang Province (QC2017013), Special Financial Aid to Post-doctor Research Fellow in Heilongjiang (LBH-TZ1714), Heilongjiang Academy of Agricultural Sciences Funds (2019YYYF019), International Postdoctoral Exchange Fellowship Program of China Postdoctoral Council(20180004),Heilongjiang Funds for Distinguished Young Scientists(JC2016004,JC2017006).

Author contributions

Ying Zhao and Xin Li conceived and designed the experiments.Ying Zhao,Xin Li,Zexin Zhang,Wenjing Pan,Sinan Li,Yun Xing,and Wanying Xin performed the experiments.Ying Zhao,Zhanguo Zhang,Zhenbang Hu,Chunyan Liu,Zhaoming Qi, and Xiaoxia Wu analyzed the data and interpreted the results.Ying Zhao and Xin Li prepared the manuscript.Dawei Xin and Qingshan Chen revised the manuscript. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved,and approved the final version to be published.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.05.008.