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Plant Physiology and Biochemistry

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Research article

Overexpression of alfalfa Orange gene in tobacco enhances carotenoidaccumulation and tolerance to multiple abiotic stresses

Zhi Wanga,c,1, Weizhou Xub,1, Jiyue Kangc, Min Lic, Jin Huanga,c, Qingbo Kea,c, Ho Soo Kimd,Bingcheng Xua,c, Sang-Soo Kwakd,∗

a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling, 712100, Shaanxi, Chinab College of Life Science, Yulin University, Yulin, 719000, Shaanxi, Chinac Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling, 712100, Shaanxi, Chinad Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Daejeon, 34141, South Korea

A R T I C L E I N F O

Keywords:AlfalfaOrange geneAbiotic stressDroughtHeatSaltOxidative stress

A B S T R A C T

The multifunctional Orange (Or) protein plays crucial roles in carotenoid homeostasis, photosynthesis stabili-zation, and antioxidant activity in plants under various abiotic stress conditions. The Or gene has been cloned inseveral crops but not in alfalfa (Medicago sativa L.). Alfalfa is widely cultivated across the world; however, itscultivation is largely limited by various abiotic stresses, including drought. In this study, we isolated the Or genefrom alfalfa (MsOr) cv. Xinjiang Daye. The amino acid sequence of the deduced MsOr protein revealed that theprotein contained two trans-membrane domains and a DnaJ cysteine-rich zinc finger domain, and showed a highlevel of similarity with the Or protein of other plants species. The MsOr protein was localized in leaf chloroplastsof tobacco. The expression of MsOr was the highest in mature leaves and was significantly induced by abioticstresses, especially drought. To perform functional analysis of the MsOr gene, we overexpressed MsOr gene intobacco (Nicotiana benthamiana). Compared with wild-type (WT) plants, transgenic tobacco lines showed highercarotenoid accumulation and increased tolerance to various abiotic stresses, including drought, heat, salt, andmethyl viologen-mediated oxidative stress. Additionally, contents of hydrogen peroxide and malondialdehydewere lower in the transgenic lines than in WT plants, suggesting superior membrane stability and antioxidantcapacity of TOR lines under multiple abiotic stresses. These results indicate the MsOr gene as a potential targetfor the development of alfalfa cultivars with enhanced carotenoid content and tolerance to multiple environ-mental stresses.

1. Introduction

Alfalfa (Medicago sativa L.) is an excellent perennial legume and iswidely cultivated across the world. Alfalfa is an important agriculturalcrop and is also used in animal husbandry, especially in arid and semi-arid regions (Wang et al., 2015b). In the semi-arid Loess Plateau regionof China, alfalfa represents an important ecological function and is usedas the main forage crop for grain for green project which was designedto convert unsustainable farmland to grassland or forestland (Yuanet al., 2014; Wang et al., 2017). However, cultivation of alfalfa on

marginal lands under drought and salt stress results in a severe reduc-tion in its yield and quality (Li et al., 2014; Wang et al., 2017). Withcontinued global climate change, abiotic stresses such as drought, heat,and salt stress are expected to become more severe and frequent (Liet al., 2012b; IPCC, 2014). Current crop production cannot meet theincreasing demand for high quality crops (Wang et al., 2015c). There-fore, developing alfalfa varieties with high quality and greater toleranceto multiple environmental stresses is urgently needed.

Carotenoids are a class of multifunctional pigments and nutrientsand are essential for plant growth, development, and environmental

https://doi.org/10.1016/j.plaphy.2018.08.017Received 3 August 2018; Received in revised form 8 August 2018; Accepted 9 August 2018

Abbreviations: Or, Orange; TOR, transgenic tobacco plants expressing alfalfa Or gene; WT, wild-type; PC, positive control; ROS, reactive oxygen species; PSY,phytoene synthase; CHY-β, β-carotene hydroxylase; LCY-ε, lycopene ε-cyclase; MMLV, Moloney murine leukemia virus; MDA, Malondialdehyde; GFP, greenfluorescent protein; qRT-PCR, quantitative reverse transcriptase PCR; MV, methyl viologen; RWC, relative water content; PSII, photosystem II; H2O2, hydrogenperoxide; TBA, thiobarbituric acid; TCA, trichloroacetic acid; HPLC, high-performance liquid chromatography; DAPI, 4′, 6-diamidino-2-phenylindole; Fv/Fm,maximum quantum yield of PSII; OEE2-1, oxygen-evolving enhancer 2–1∗ Corresponding author.

1 The two authors contributed equally in this paper.E-mail address: sskwak@kribb.re.kr (S.-S. Kwak).

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adaptation as well as for optimal human health (Esteban et al., 2015).Carotenoids are critical components of photosynthetic and antioxidantsystems in plant cells (Ramel et al., 2013; Zarco-Tejada et al., 2013;Zhou et al., 2015; Hou et al., 2016). During photosynthesis, carotenoidsharvest light energy and protect photosynthetic organelles from excesslight energy via the xanthophyll cycle (Förster et al., 2011; Nisar et al.,2015). Carotenoids function as potent antioxidants and efficientlyscavenge intracellular reactive oxygen species (ROS) to prevent oxi-dative stress (Havaux, 2014). Carotenoids also act as substrates ofapocarotenoids, including vitamin A, and plant hormones, such asstrigolactones and abscisic acid (ABA), which play important roles ingermination, growth, branching, and stress tolerance of plants (Khoslaand Nelson, 2016). Because of their diverse roles, carotenoids representkey compounds for breeding crops with enhanced nutritional contentand environmental adaptation (Esteban et al., 2015; Kim et al., 2018).Significant efforts have been made to improve the process of car-otenogenesis in crops via the metabolic engineering of carotenoidbiosynthetic genes, such as phytoene synthase (PSY), β-carotene hy-droxylase (CHY-β), and lycopene ε-cyclase (LCY-ε) (Ye et al., 2000,Fraser et al., 2002, Han et al., 2008, Kim et al., 2012a, 2012b, 2013).

The Orange (Or) gene is crucial for carotenoid biosynthesis and ac-cumulation. Nucleotide sequence of the Or gene is highly conserved incauliflower (Brassica oleracea var. botrytis), sweetpotato (Ipomoea ba-tatas), melon (Cucumis melo var. cantalupensis), and Arabidopsis thaliana(Lu et al., 2006; Bai et al., 2014; Kang et al., 2017; Kim et al., 2018).The Or protein uniquely enhances carotenoid accumulation by stimu-lating the formation of a metabolic sink for carotenoid accumulationrather than by directly increasing carotenoid biosynthesis (Lopez et al.,2008; Li et al., 2012a). Overexpression of the cauliflower Or gene inpotato (Solanum tuberosum) under the control of a tuber-specific pro-moter induces the formation of chromoplasts with significant levels ofβ-carotene in transgenic potato tubers (Lopez et al., 2008). Previously,we showed that the overexpression of the sweetpotato Or gene sig-nificantly increases β-carotene and total carotenoid contents in sweet-potato calli and storage roots as well as in alfalfa leaves (Kim et al.,2013; Park et al., 2015; Wang et al., 2015c). Moreover, the Or proteincontains a DnaJ cysteine-rich zinc-binding domain and exhibits notableholdase chaperone activity. Under heat and oxidative stress, the Orprotein directly interacts with PSY, a key limiting enzyme in carotenoidbiosynthesis, and prevents its degradation (Park et al., 2016). Notably,expression of the Or gene is induced by various environmental stressesand confers plants with enhanced tolerance against drought, heat, salt,and oxidative stress (Kim et al. 2013, 2018; Wang et al., 2015c). Takentogether, these observations suggest the use of the Or gene as an effi-cient molecular tool for breeding crop plants with outstanding nutri-tional quality and adaptability to multiple abiotic stresses. However,the Or gene of alfalfa is not yet cloned, despite the importance of alfalfaas a forage crop on marginal lands.

In this study, we isolated the Or gene from alfalfa (MsOr) cv.Xinjiang Daye, and investigated its expression profile under variousenvironmental stress conditions. To clarify the role ofMsOr in toleranceto abiotic stress, we generated transgenic tobacco (Nicotiana ben-thamiana) lines overexpressing MsOr under the control of the cauli-flower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediatedtransformation. Our results showed that overexpression of the MsOrgene improved the carotenoid content as well as the environmentaladaptability of transgenic tobacco plants.

2. Materials and methods

2.1. Plant materials and growth conditions

Alfalfa cv. Xinjiang Daye and tobacco plants were used in this study.Plants were grown in plastic pots (12 cm upper inner diameter× 9 cmlower inner diameter× 11 cm height) filled with soil, with one plantper pot. Pots were maintained in a growth chamber at 25 °C

temperature, and under a 16 h light/8 h dark photoperiod, 60% relativehumidity, and 200 μmol m−2 s−1 light intensity. One-month-oldhealthy plants were used for further analysis.

2.2. Gene cloning and phylogenetic analysis

Total RNA was extracted from healthy leaves of 1-month-old alfalfaplants using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) andtreated with RNase-free DNase I to remove any contaminating genomicDNA. Total RNA (2 μg) was used for first-strand cDNA synthesis usingMoloney murine leukemia virus (MMLV) reverse transcriptase(TOPscript™ RT DryMIX), according to the manufacturer's instructions.To isolateMsOr, a pair of forward and reverse primers (MtOr-F/R; TableS1) were designed on the basis of the sequence of the Or gene ofMedicago truncatula (TA4160_3880), a close relative of alfalfa. Thecoding sequence (CDS) of MsOr was amplified from the first-strandcDNA using SolGent™ Pfu-X DNA Polymerase (Solgent, Daejeon,Korea). The purified PCR product was cloned into pGEM-T Easy vector(Promega, Madison, WI, USA) and sequenced.

Nucleotide sequence of MsOr was compared with that of the otherOr gene family members via online BLAST searches in UniProt and theNational Center for Biotechnology Information (NCBI). The publishedOr sequences were translated to amino acid sequences using BioEdit.Alignments of the predicted amino acid sequences were conductedusing BioEdit and BoxShade server. A phylogenetic tree was con-structed with 1000 iterations using the neighbor-joining method inMolecular Evolutionary Genetics Analysis (MEGA) version 6.

2.3. Vector construction

The vector used to overexpress MsOr in tobacco was constructedusing Gateway® cloning (Invitrogen), as described previously (Kimet al., 2013). Briefly, the MsOr CDS was amplified with primers con-taining attB sites (attB-MsOr-F/R; Table S1). Linear fragments flankedby attB sequences were used to construct the entry vector MsOr-pDONR207 (Invitrogen) using BP Clonase (Invitrogen). ThepDONR207-MsOr plasmid was cloned into the plant expression vector(pGWB5), containing the CaMV 35S promoter upstream of the greenfluorescent protein (GFP) gene, using LR Clonase (Invitrogen). The re-sulting overexpression construct (pGWB5-MsOr-GFP) was transformedinto Agrobacterium tumefaciens strain GV3101 using the freeze-thawmethod (Hofgen and Willmitzer, 1988), which was used for subcellularlocalization analysis and plant transformation.

2.4. Subcellular localization of MsOr

A. tumefaciens GV3101 strain carrying the pGWB5-MsOr-GFP vectorwas infiltrated into the young leaves of 4-week-old tobacco plants.Infected plants were grown at 25 °C and under a 16 h light/8 h darkphotoperiod for 3 days. Then, infiltrated sections of leaves were cut,and nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI).The stained leaf sections were visualized under a laser scanning con-focal microscope (Leica Microsystems, Heidelberg, Germany) as de-scribed previously (Kang et al., 2017).

2.5. PCR and gene expression analysis

Healthy leaves of 1-month-old alfalfa and tobacco plants were usedfor the extraction of total RNA, as described above. For semi-quanti-tative and quantitative expression analysis of the MsOr gene in alfalfaand transgenic tobacco plants, 2 μg of total RNA was used for first-strand cDNA synthesis using reverse transcriptase PCR (RT-PCR) Kit(TOPscript™ RTDry MIX). Quantitative RT-PCR (qRT-PCR) was per-formed in a CFX Real-Time PCR Detection System (Bio-Rad, USA) usingfluorescent BRYT Green® Dye (GoTaq® qPCR Master Mix, Promega,Beijing, China), according to the manufacturer's instructions. The gene

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expression was quantified using the 2−ΔΔCT comparative method (Livakand Schmittgen, 2001). Transcript levels of MsOr were normalized re-lative to those of the Actin gene.

Genomic DNA was extracted from healthy leaves of 1-month-oldalfalfa and tobacco plants, as described previously (Kim and Hamada,2005). Purified genomic DNA was subjected to PCR amplification usingPCR premix (cat. no. K-2012, Bioneer, Daejeon, Korea) and gene-spe-cific primers (Table S1). PCR products were separated by gel electro-phoresis on 1% agarose gel and visualized under UV light after stainingwith ethidium bromide.

2.6. Generation of transgenic tobacco plants

The pGWB5-MsOr-GFP vector was transformed into tobacco plantsvia Agrobacterium-mediated transformation, as described previously(Horsch et al., 1985). Transformed plants were selected on MS mediumcontaining 30mg l−1 hygromycin. Two transgenic lines expressingMsOr (TOR1 and TOR2) were confirmed via genomic PCR and qRT-PCRanalysis using gene-specific primers (Table S1). Four-week-old plants ofwild-type (WT) tobacco and TOR1 and TOR2 transgenic lines in thesecond generation (T2) were used for the analysis of carotenoid contentand abiotic stress tolerance.

2.7. Stress treatments

To determine the expression of MsOr in response to different abioticstresses, leaves of 4-week-old alfalfa plants were treated with 5 μMmethyl viologen (MV), 200mM NaCl, 15% PEG6000, high temperature(42 °C), and low temperature (4 °C) for 1, 3, 6, 12, 24, and 48 h.

To investigate the stress tolerance of transgenic tobacco plants ex-pressing the MsOr gene, 4-week-old WT and transgenic tobacco (TOR1and TOR2) plants were treated with drought, salt, and oxidative stressesafter 1 week of irrigation with similar quantities of water. To inducedrought stress, water was withheld from plants for 7 days. For hightemperature stress, plants grown at 25 °C were transferred to a growthchamber maintained at 42 °C for 24 h, and then returned to normalconditions (25 °C, 200 μmolm−2 s−1) for recovery. To induce saltstress, plants were irrigated with 300mM NaCl once every 3 days for 2weeks. Oxidative stress was induced as previously described (Wanget al., 2017).

2.8. Relative water content (RWC)

The fourth fully expanded leaf (counting from the shoot apicalmeristem) of tobacco plants was used to determine the relative watercontent (RWC), as described previously (Wang et al., 2017). RWC wascalculated as follows:

RWC (%)= [(fresh weight - dry weight)/(turgid weight - dryweight)]× 100

2.9. Photosynthetic activity and chlorophyll content

The third fully expanded leaf (counting from the shoot apical mer-istem) was used to evaluate the photosynthetic activity. Leaves weredark-adapted for 30min, and the maximum quantum yield of photo-system II (PSII) (Fv/Fm) was estimated using a pulse amplitudemodulated chlorophyll fluorescence system (Imaging PAM, Walz,Effeltrich, Germany). The chlorophyll content of leaves was measuredusing a portable chlorophyll meter (SPAD-502, Konica Minolta SensingInc., Osaka, Japan). Relative chlorophyll content of leaves under stressconditions was compared with that of leaves under normal growthconditions.

2.10. Hydrogen peroxide (H2O2) content

Tobacco leaves located at the same position in each plant wereharvested and used for the H2O2 assay. The hydrogen peroxide (H2O2)content was measured using an ultraviolet spectrophotometer(Spectronic, Genesys™2, Milton Roy Company, Rochester, NY, USA), asdescribed previously (Loreto and Velikova, 2001).

2.11. Malondialdehyde (MDA) content

To evaluate the degree of lipid peroxidation in leaves, the mal-ondialdehyde (MDA) content of leaves was measured using the thio-barbituric acid (TBA) method (Wang et al., 2017). Briefly, 0.1 g of leaftissue was fully triturated in 10ml of 10% trichloroacetic acid (TCA),and the homogenate was centrifuged at 10 000 g for 15min. Two mil-liliters of the extract was mixed with an equal volume of TBA and in-cubated at 100 °C for 30min. The mixture was immediately cooled onice and centrifuged at 10 000 g for 20min. Absorbance was measured at450, 532, and 600 nm using an ultraviolet spectrophotometer (Spec-tronic, Genesys™2, Milton Roy Company, Rochester, NY, USA).

2.12. Ion leakage analysis

Relative membrane permeability of tobacco leaves was evaluatedthrough the loss of cytoplasmic solutes after treatment with 5 μMMV.Ion leakage was measured at 12 h intervals using an ion conductivitymeter (model DDS-307A, Rex, Shanghai, China) and compared withtotal ion leakage of autoclaved tissue samples.

2.13. Carotenoid content

Carotenoids were extracted from leaves of 1-month-old tobaccoplants and subjected to high-performance liquid chromatography(HPLC) analysis using an Agilent 1100 HPLC system (Hewlett-Packard,Palo Alto, CA, USA), as described previously (Wang et al., 2015c). Allextraction procedures were performed under subdued light to avoidpigment degradation and loss.

2.14. Statistical analysis

Each experiment included four independent biological replicates.Data were subjected to one-way analysis of variance (ANOVA) followedby Duncan's multiple range test, and data with P < 0.05 were con-sidered significant. All statistical analyses were carried out using theStatistical Package for Social Sciences (SPSS 19).

3. Results

3.1. Isolation of MsOr and protein localization

The MsOr gene was successfully isolated from the leaves of alfalfacv. Xinjiang Daye. The full-length CDS of MsOr was 939 bp in lengthand encoded a protein of 313 amino acid residues with a molecularmass of 78.4 kDa. Phylogenetic analysis of the Or protein of 15 plantspecies, including soybean (Glycine max), sweetpotato, Arabidopsis,cauliflower, rice (Oryza sativa), grape (Vitis vinifera), tomato (Solanumlycopersicum), cotton (Gossypium hirsutum), barley (Hordeum vulgare),wheat (Triticum aestivum), sorghum (Sorghum bicolor), maize (Zeamays), Japanese morning glory (Ipomoea nil), and pepper (Capsicumannuum), revealed that the MsOr protein was most closely related to theOr protein of M. truncatula (MtOr; TA4160_3880) (Fig. 1A). The de-duced MsOr protein was 98.72% similar to the Or protein of M. trun-catula, and contained two trans-membrane domains and a cysteine-richzinc finger domain with CxxCxGxGx as the repeat motif, characterizedby a DnaJ molecular chaperone (Fig. 1B).

To determine the subcellular localization of the MsOr protein, the

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pGWB5-MsOr-GFP expression vector was transiently transformed intotobacco leaves. The transfected leaves were stained with DAPI to vi-sualize nuclei. Using a fluorescent microscope, GFP fluorescence wasdetected only in the chloroplasts of leaf epidermal cells (Fig. 1C).

3.2. Expression profile of MsOr

The expression level of MsOr was significantly higher in leaves thanin stems and roots of alfalfa (P < 0.05; Fig. 2). Among the four dif-ferent development stages of leaves, MsOr gene transcripts were the

highest in mature leaves, followed by senescent, young, and immatureleaves (Fig. 2). We also evaluated the expression profile of MsOr inmature leaves under various abiotic stresses, including MV, NaCl, PEG,heat, and cold, for 48 h (Fig. 3). Under MV-induced oxidative stress,MsOr expression increased sharply, reaching a peak at 1 h following5 μMMV treatment, and then decreased. Under salt stress, MsOr ex-pression was strongly induced after 1 h of 200mM NaCl treatment,reaching a peak at 48 h. Under PEG-induced dehydration, the MsOrgene exhibited the strongest expression after 48 h of 15% PEG treat-ment. In response to cold stress (4 °C), the MsOr expression increasedafter 3 h of cold treatment and then gradually decreased. Heat (42 °C)gradually decreased the expression of MsOr over 48 h of treatment.

3.3. Generation of transgenic tobacco plants expressing the MsOr gene

Eight transgenic lines of tobacco expressing the MsOr gene underthe control of the constitutive CaMV 35S promoter were successfullygenerated via Agrobacterium-mediated transformation. The presence ofthe transgene was confirmed in all eight TOR lines using genomic PCRwith MsOr gene-specific primers (Fig. 4A). Semi-quantitative RT-PCRanalysis of the first fully expanded leaves of 1-month-old TOR and WTplants confirmed the expression of MsOr in all TOR plants (Fig. 4B).Because the expression of MsOr was higher in TOR1 and TOR2 than inthe other transgenic lines, T2 plants of TOR1 and TOR2 were used forfurther analysis.

3.4. MsOr overexpression increases the carotenoid content of tobacco leaves

To investigate the effect of MsOr gene on carotenoid accumulation,the carotenoid content of 1-month-old TOR and WT plants was quan-tified using HPLC analysis under normal growth conditions (Fig. 4C).MsOr overexpression significantly improved the total carotenoid

Fig. 1. Characterization and sequence analysis of the alfalfa Orange gene (MsOr). (A) Phylogenetic analysis of MsOr and Or genes of other plant species. Thephylogenetic tree was constructed using the neighbor-joining method in MEGA6. (B) Amino acid sequence alignment of the predicted MsOr protein and Or proteins ofother plant species using BioEdit and BoxShade server. Amino acid sequences were deduced from the nucleotide sequences of Or genes of Medicago truncatula(TA4160_3880), sweetpotato (Ipomoea batatas; accession no. HQ828087), Arabidopsis thaliana (accession no. NP_200975), and soybean (Glycine max; TA6293_3847).The Or genes of M. truncatula and G. max were deposited in the TIGR Plant Transcript Assemblies database. Conserved amino acid residues are highlighted in black.Two predicted trans-membrane domains and repeating cysteine residues in the putative DnaJ domain are indicated with boxes and stars, respectively. (C) Subcellularlocalization of MsOr::GFP fusion protein in leaf epidermal cells of tobacco. Bright, bright field microscopy images; GFP, GFP fluorescence images; Chloroplast,chloroplast auto-fluorescence images; Merged, overlay images of bright, DAPI, GFP, and chloroplast fluorescence images. Scale bars= 20 μm.

Fig. 2. MsOr expression profiles in stem, root, and leaf tissues of 1-month-oldalfalfa plants. Small letters above bars indicate statistically significant differ-ences (P < 0.05).

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content of both TOR lines by approximately 1.86-fold compared withthat of WT plants. Among the carotenoids, the content of violaxanthinwas 1.72–1.82 times higher in TOR lines than in WT plants. Both TOR1and TOR2 contained approximately 1.22-fold higher β-carotene thanthe WT plants. The average lutein content of the two TOR lines wasapproximately 58–95% higher than that of the WT plants. In addition,the antheraxanthin and zeaxanthin contents of the two TOR lines were0.82- and 6.41-fold higher than those of the WT plants, respectively.

3.5. MsOr overexpression confers tobacco plants with enhanced droughttolerance

No differences were detected between the phenotype of TOR andWT plants under normal growth condition (Fig. 5A). However, afterwithholding water supply, WT plants were more sensitive to droughtstress than the TOR plants. Leaves of WT plants exhibited wilting fromthe fourth day of withholding water supply, whereas those of TORplants showed wilting from the seventh day. Upon withholding water

Fig. 3. Expression patterns of the MsOr gene in mature leaves of 1-month-old alfalfa plants under various abiotic stresses. Asterisks indicate statistically significantdifferences (P < 0.05) compared with the mean at 0 h. ns, no significant difference.

Fig. 4. Characterization of transgenic tobacco (TOR) lines overexpressing MsOr, and analysis of carotenoid content of tobacco leaves. (A) PCR analysis of the MsOrgene from genomic DNA of transgenic plants. M, 1 kb plus size marker; PC, positive control; WT, wild-type plants; Lanes 1–8, independent transgenic lines. (B) qRT-PCR analysis of the MsOr gene in eight transgenic lines. Transcript levels of the MsOr gene were normalized relative to that of the tobacco Actin gene (internalcontrol). (C) Quantitative HPLC analysis of the total carotenoid content and individual carotenoids in the leaves of WT and TOR plants. V, violaxanthin; A,antheraxanthin; L, lutein; Z, zeaxanthin; B, β-carotene; Total, total carotenoid content. Asterisks indicate statistically significant differences between WT and TORplants (P < 0.05).

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supply for 4 days, TOR plants maintained significantly higher PSII ef-ficiency than the WT plants, and the Fv/Fm value of TOR plants wasapproximately 1.45-fold higher than that of the WT plants (Fig. 5B).The MDA and H2O2 contents of TOR plants were approximately 48%and 30% lower than those of the WT plants, respectively (Fig. 5C andD). After 7 days of withholding water supply, the RWC of TOR leaveswas approximately 1.48-fold higher than that of the WT leaves(Fig. 5E).

3.6. MsOr overexpression increases heat tolerance of transgenic tobacco

Exposure to heat stress (42 °C) for 10 h resulted in wilting oftransgenic as well as WT plants; however, WT plants wilted much morerapidly than TOR plants (Fig. 6A). Additionally, heat stress reduced theFv/Fm values of WT plants by approximately 31% and that of TORplants by only 4% (Fig. 6B). Moreover, the accumulation of H2O2 andMDA in WT plants was 1.48- and 2.34-fold higher than that in TORplants (Fig. 6C and D), respectively. Exposure of plants to heat stress for12 h reduced the RWC of WT plants by 42.9% and that of the two TORlines by 10.9% (Fig. 6E). After 2 days of recovery at 25 °C, both of theTOR lines recovered from heat stress, whereas the WT plants were al-most dead.

3.7. MsOr overexpression confers tobacco plants with enhanced salttolerance

Under salt stress, both of the TOR lines showed superior growth tothe WT plants (Fig. 7A). When treated with 300mM NaCl for 6 days,the Fv/Fm value of WT plants declined by approximately 21.1%,whereas the TOR1 and TOR2 plants exhibited only a 4.7% decline inthe Fv/Fm value (Fig. 7B). TOR plants also exhibited notably lowerH2O2 accumulation than the WT plants (Fig. 7C). Accumulation of MDAin WT plants was approximately 1.5-fold higher than that in TORplants, which was statistically significant (Fig. 7D). In addition, therelative chlorophyll content of WT plants decreased much faster than

that of the two TOR lines during salt treatment. After 10 days of300mM NaCl treatment, WT leaves exhibited severe chlorosis, whereasthe two TOR lines showed only slight yellowing in a few leaves. Con-sistent with this phenotype, the relative chlorophyll content of TORplants was approximately 1.64-fold higher than that of WT plants,which was a significant difference (Fig. 7E).

3.8. MsOr overexpression upregulates tolerance to oxidative stress

To investigate the effect of the MsOr gene on the oxidative stresstolerance of tobacco plants, leaf discs from 1-month-old WT and TORplants were exposed to 5 μMMV for 24 h (Fig. 8). Both of the TOR linesshowed notably less damage than the WT plants. Leaf discs of WT plantswere severely necrotic after 24 h of MV treatment, while those of TORlines showed less necrosis (Fig. 8A). After 12 and 24 h of MV treatment,the level of ion leakage was approximately 1.36- and 1.71-fold higher inWT plants than in TOR plants, respectively (Fig. 8B).

4. Discussion

In this study, we isolated the Or gene from alfalfa and investigatedits effects on carotenoid content, photosynthetic efficiency, and abioticstress tolerance using transgenic tobacco plants overexpressing theMsOr gene. Our results indicate the potential of the MsOr gene as a toolto improve the content of carotenoids and tolerance to multiple abioticstresses in tobacco plants.

The Or protein is highly conserved in plants (Kim et al., 2018). Incauliflower, the Or protein localizes both in plastids and nuclei (Luet al., 2006; Zhou et al., 2015). In sweetpotato, heat stress induces thetranslocation of the Or protein from nuclei to chloroplasts (Park et al.,2016). In this study, the MsOr protein overexpressed in tobacco wasdetected only in the chloroplasts of leaf epidermal cells (Fig. 1C). Si-milar to the Or proteins in other plant species, the MsOr protein waspredicted to contain two trans-membrane domains and a DnaJ cysteine-rich zinc finger domain (Fig. 1B). Most DnaJ proteins localize to

Fig. 5. Analysis of drought tolerance of wild-type (WT) and transgenic tobacco (TOR) plants overexpressing MsOr. (A) Phenotype of 1-month-old NT and TOR plantsbefore and after withholding water. (B) Maximum quantum yield of PSII (Fv/Fm), (C) Hydrogen peroxide (H2O2) content, and (D) Malondialdehyde (MDA) contentof the leaves of WT and TOR plants after 4 days of drought treatment. (E) Relative water content (RWC) of the leaves of WT and TOR plants after 7 days of droughttreatment. Asterisks indicate statistically significant differences between WT and TOR plants (P < 0.05).

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chloroplasts (Chiu et al., 2013). The DnaJ proteins belong to the familyof heat shock proteins and play an essential role in chloroplast devel-opment, photosynthesis, and abiotic stress tolerance, suggesting thatthe expression of the Or gene improves plants' adaptability to

environmental stresses (Albrecht et al., 2008; Wang et al., 2014,2015a). Expression of DnaJ genes is responsive to various abioticstresses (Kong et al., 2014). Similarly, the expression of MsOr was sig-nificantly induced by multiple abiotic stresses (Fig. 3), which is

Fig. 6. Analysis of heat tolerance of wild-type (WT) and transgenic tobacco (TOR) plants overexpressing MsOr. (A) Phenotype of the 1-month-old WT and TOR plantsbefore and after heat (42 °C) treatment. (B) Maximum quantum yield of PSII (Fv/Fm), (C) Hydrogen peroxide (H2O2) content, and (D) Malondialdehyde (MDA)content of the leaves of WT and TOR plants after 10 h of heat treatment. (E) Relative water content (RWC) of the leaves of WT and TOR plants after 12 h of heattreatment. Asterisks indicate statistically significant differences between WT and TOR plants (P < 0.05).

Fig. 7. Analysis of salt tolerance of wild-type (WT) and transgenic tobacco (TOR) plants overexpressing MsOr. (A) Phenotype of the 1-month-old WT and TOR plantsbefore and after salt (300mM NaCl) treatment. (B) Maximum quantum yield of PSII (Fv/Fm), (C) Hydrogen peroxide (H2O2) content, and (D) Malondialdehyde(MDA) content of the leaves of the WT and TOR plants after 6 days of salt treatment. (E) Relative chlorophyll content (RWC) of the leaves of WT and TOR plants after6 and 10 days of salt treatment. Asterisks indicate statistically significant differences between WT and TOR plants (P < 0.05).

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consistent with the Or gene in sweetpotato (Kim et al., 2013). Thehighly conserved protein structure and similar expression under abioticstresses suggest that the function of MsOr is similar to that of Or genesin other plant species.

Expression of Or genes improves carotenoid accumulation by sti-mulating chromoplast formation in plants (Cazzonelli and Pogson,2010; Li et al., 2012a). Since being first discovered in cauliflower, theOr gene has been increasingly receiving attention and has been widelyused in breeding crops, such as sweetpotato, sorghum, rice, potato, andmelon, with high carotenoid content (Park et al., 2015; Tzuri et al.,2015; Yuan et al., 2015; Bai et al., 2016; Cho et al., 2016). Transgenicrice and corn overexpressing the Arabidopsis Or gene accumulate in-creased levels of carotenoids by promoting chromoplast formation (Baiet al., 2016; Berman et al., 2017). The Or genes of sweetpotato andcauliflower notably increase carotenoid accumulation in potato (Lopezet al., 2008; Li et al., 2012a; Cho et al., 2016). We previously showedthat the overexpression of the sweetpotato Or gene in alfalfa sig-nificantly increases carotenoid accumulation in leaves (Wang et al.,2015c). In this study, we showed that MsOr overexpression in tobaccoincreased the accumulation of various carotenoids, including lutein, β-carotene, violaxanthin, zeaxanthin, and antheraxanthin (Fig. 4C),which is consistent with the results of previous studies. Additionally,the Or protein functions as a post-translational regulator of PSY, whichis the most important carotenoid biosynthetic enzyme (Kim et al.,2018). High chaperone activity of Or proteins in sweetpotato andArabidopsis greatly improves carotenoid biosynthesis by directly pro-tecting PSY from degradation under heat stress (Zhou et al., 2015; Parket al., 2016).

The Or gene not only promotes carotenoid accumulation, but also

increases plants' adaptation to various abiotic stresses (Wang et al.,2015c; Park et al., 2016; Kim et al., 2018). Carotenoids are importantpowerful antioxidants (Esteban et al., 2015). Carotenoids, such as β-carotene and lutein, directly scavenge ROS and modulate membranefluidity (Domonkos et al., 2013; Havaux, 2014). Because of the pow-erful antioxidant activity, carotenoids maintain ROS homeostasis andstabilize cellular membranes, thus improving plant abiotic stress tol-erance (Kim et al., 2013, 2018). Overexpression of the sweetpotato Orgene in potato and alfalfa increases carotenoid accumulation and con-fers plants with higher antioxidant activity under various environ-mental stresses (Wang et al., 2015c; Park et al., 2016; Cho et al., 2016).In this study, we showed that the MDA and H2O2 contents of TOR lineswere lower than those of the WT plants, suggesting that the membranestability and antioxidant capacity of TOR lines were higher than thoseof the WT plants under multiple abiotic stresses (Figs. 5–7). Ad-ditionally, lower ion leakage from TOR plants than from WT plantsfollowing MV treatment suggests that the MsOr overexpression in to-bacco reduces the degree of membrane lipid peroxidation and increasestolerance to oxidative stress (Fig. 8). The enhanced membrane stabilityand antioxidant activity of transgenic tobacco plants overexpressing theMsOr gene possibly resulted from the increased level of carotenoids,including lutein, β-carotene violaxanthin, zeaxanthin, and antherax-anthin (Kim et al., 2013; Wang et al., 2015c; Cho et al., 2016).

In addition to the powerful antioxidant capacity, carotenoids en-hance thermostability and protect the photosynthetic apparatus fromdamage under abiotic stress conditions (Cazzaniga et al., 2012;Domonkos et al., 2013; Havaux, 2014; Esteban et al., 2015). Xantho-phylls, including violaxanthin, zeaxanthin, and antheraxanthin, areessential for the thermal dissipation of excess excitation energy (Niyogiand Truong, 2013). Given the role of carotenoids in regulating ROShomeostasis and thermal dissipation, enhanced carotenoid accumula-tion stabilizes photosynthesis under abiotic stresses (Kim et al., 2013,2018). Transgenic plants overexpressing the Or gene exhibit greaterphotosynthetic activity and higher levels of chlorophyll and carotenoidsunder abiotic stresses (Wang et al., 2015c; Cho et al., 2016; Park et al.,2016). The Fv/Fm parameter is generally used as an indicator of plantphotosynthetic activity and adaptability under abiotic stresses (Xuet al., 2014; Kang et al., 2017). In this study, transgenic tobacco plantsoverexpressing theMsOr gene exhibited higher xanthophyll content andFv/Fm values than WT plants under drought, salt, and heat stress(Figs. 5B, 6B and 7B). When exposed to salt stress, transgenic tobaccoplants showed improved chlorophyll stability (Fig. 7E), suggesting thatMsOr maintains the photosynthetic activity of tobacco plants (Kanget al., 2017). Moreover, the holdase chaperone activity of the Or proteinpromotes plant photosynthetic efficiency by stabilizing the oxygen-evolving enhancer 2–1 (OEE2-1) protein, which plays a key role in theregulation and stabilization of PSII as well as plant tolerance to abioticstresses (Bricker et al., 2013; Ifuku, 2014). The Or protein protectsOEE2-1 (Kang et al., 2017), which possibly contributed to the higherphotosynthetic efficiency and chlorophyll stability of TOR plants underdrought, heat, and salt stresses in this study. TOR plants also exhibitedgreater RWC than the WT plants, suggesting that MsOr overexpressionincreases cell turgidity under drought and heat stress (Figs. 5E and 6E).Carotenoids are important precursors of phytohormones, includingABA, which plays a key role in transpiration and abiotic stress tolerance(Choi et al., 2012; Brunetti et al., 2015). Increased accumulation ofcarotenoids in TOR lines likely contributed to higher water holdingcapacity and enhanced adaptability to drought and heat stress.

Overall, the deduced amino acid sequence of the MsOr protein washighly similar to that of the Or proteins of other plant species, especiallythat of M. truncatula. Among the different tissues of alfalfa, MsOr wasexpressed to the highest level in mature leaves. Heterologous expres-sion of the MsOr gene in tobacco increased the accumulation of car-otenoids and tolerance to multiple abiotic stresses, including drought,heat, salt, and oxidative stress. Taken together, our results suggest thatthe MsOr gene is a valuable molecular tool for the development of crops

Fig. 8. Effect of methyl viologen (MV)-mediated oxidative stress on the leavesof wild-type (WT) and transgenic tobacco (TOR) plants overexpressing MsOr.Leaf discs were treated with 5 μMMV, followed by incubation at 25 °C under alight intensity of 150 μmol photons m−2 s−1. (A) Phenotype of WT and TORleaf discs at 24 h after MV treatment. (B) Relative membrane permeability at 0,12, and 24 h following MV treatment. Relative membrane permeability (%) wascalculated relative to the values obtained after autoclaving plant tissue samples.Data represent the mean of three biological replicates. Asterisks indicate sta-tistically significant differences between WT and TOR plants (P < 0.05).

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with superior quality and enhanced tolerance to environmental stresses.The MsOr gene is a promising target for crop breeding programs aimedat developing cultivars for sustainable agriculture in arid and semi-aridregions to cope with global climate change.

Author contributions

Z. Wang and S. Kwak: conceived and designed the experiment. Z.Wang, W. Xu, Q. Ke, J. Kang, and M. Li: performed the experiments. Z.Wang, W. Xu, Q. Ke, and H. Kim: analyzed the data. W. Xu, J. Huang, B.Xu, and S. Kwak: contributed reagents/materials/analysis tools. Z.Wang, W. Xu, and B. Xu: wrote the paper.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (31700335), the KRIBB Initiative Program,Fundamental Research Funds for the Central Universities(2452017185), and the Doctoral Scientific Research Foundation ofNorthwest A&F University, China (2452015341).

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.plaphy.2018.08.017.

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