overexpression of a r2r3 myb gene mdsimyb1 increases tolerance to...

Physiologia Plantarum 150: 76–87. 2014 © 2013 Scandinavian Plant Physiology Society, ISSN 0031-9317

Overexpression of a R2R3 MYB gene MdSIMYB1 increasestolerance to multiple stresses in transgenic tobaccoand applesRong-Kai Wang†, Zhong-Hui Cao† and Yu-Jin Hao∗

National Key Laboratory of Crop Biology, National Research Center for Apple Engineering and Technology, College of Horticulture Science andEngineering, Shandong Agricultural University, Tai-An, 271018, Shandong, China

Correspondence*Corresponding author,e-mail: [email protected]

Received 6 January 2013;revised 12 April 2013

doi:10.1111/ppl.12069

MYB transcription factors (TFs) involve in plant abiotic stress tolerance andresponse in various plant species. In this study, rapid amplification of cDNAends (RACE) was conducted to isolate the R2R3-MYB TF gene MdSIMYB1 fromapples (Malus × domestica). The gene transcripts were abundant in the leaves,flowers and fruits, compared to other organs, and were induced by abioticstresses and plant hormones. We observed the subcellular localization of anMdSIMYB1-GFP fusion protein in the nucleus. Furthermore, the MdSIMYB1gene was introduced into the tobacco genome and ectopically expressed intransgenic lines. The results indicate that MdSIMYB1 transgenic tobacco seedgermination is insensitive to abscisic acid and NaCl treatment. Additionally, itwas found that the ectopic expression of MdSIMYB1 enhanced the toleranceof plants to high salinity, drought and cold tolerance by upregulating thestress-responsive genes NtDREB1A, NtERD10B and NtERD10C. Meanwhile,the transgenic tobacco exhibited robust root growth because of the enhancedexpression of the auxin-responsive genes NtIAA4.2, NtIAA4.1 and NtIAA2.5under stress conditions, which is conducive to stress tolerance. Finally,transgenic apple lines were obtained and tested. Transgenic apple lines thatwere overexpressing MdSIMYB1 exhibited a higher tolerance to abiotic stressthan the wild-type control, but suppression of MdSIMYB1 resulted in lowertolerance. Our results indicate that MdSIMYB1 may be utilized as a targetgene for enhancing stress tolerance in important crops.

Introduction

Various abiotic stresses including salinity, drought andcold greatly influence plant growth and crop productiv-ity. A variety of genes are induced by abiotic stresses todefend against abiotic stresses in higher plants (Kasuga

Abbreviations – 6-BA, 6-benzylaminopurine; ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylate; BIFC,bimolecular fluorescence complementation; CaMV, cauliflower mosaic virus; GFP, green fluorescence protein; IAA,indoleacetic acid; MeJA, methyl jasmonate; MS, Murashige and Skoog; ORF, open reading frame; PEG, polyethyleneglycol; RACE, rapid amplification of cDNA ends; RT-PCRs, reverse transcriptase-polymerase chain reaction; SA, salicylicacid; TFs, transcription factors; WT, wild-type; YFP, yellow fluorescent protein; Y2H, yeast two-hybrid.

†These authors equally contributed to this work.

et al. 2004). Generally, these stress-induced genes aredirectly or indirectly modulated by regulators that arecomponents of signaling pathway associated with abi-otic stresses (Shinozaki and Yamaguchi-Shinozaki 1997).Transcription factors (TFs) are important player in the reg-ulation of gene expression in response to developmental

76 Physiol. Plant. 150, 2014

and environmental cues (Jung et al. 2008). Among them,some members of the MYB, ERF, bZIP and WRKY TFfamilies are implicated in plant defense to abiotic stresses(Jung et al. 2008). In Arabidopsis, the MYB family includ-ing 163 members is one of the largest TF families (Yanhuiet al. 2006).

The first myb gene, i.e. an oncogene v-MYB, isidentified from avian myeloblastosis virus (Klempnaueret al. 1982). So far, it is found that MYB genesexist in almost all eukaryotes. The MYB TFs generallycontain one to three imperfect helix-turn-helix repeatsthat are responsible for the specific recognition totarget DNA (Yanhui et al. 2006). On the basis of thearrangement of those repeats, MYB TFs are dividedinto three subfamilies, i.e. R1R2R3, R2R3 and MYBrelated (which has one MYB-like domain) (Dubos et al.2010). Different structural arrangements exist in differentkingdoms, suggesting specific functions of MYB TFs inplants. Over the past decades, it has been establishedthat many MYB TFs are involved in various plant-specific processes such as secondary metabolism, cellshape determination and cell differentiation (Lippoldet al. 2009). Among them, AtMYB75 and AtMYB90are responsible for the regulation of anthocyaninsbiosynthesis (Dubos et al. 2010), while AtMYB28 forglucosinolate accumulation in Arabidopsis (Gigolashviliet al. 2007). The differentiation of cells into the non-hairfate is controlled by AtMYB66 (Bernhardt et al. 2005,Dubos et al. 2010). In addition, the anther endothecialcells show defects in secondary wall thickening inmutant myb26 which is male sterile (Yang et al. 2007).The AtMYB46 gene functions to regulate the formationof the second cell wall (Zhong et al. 2007).

In plants, lateral root formation greatly influencesroot architecture development and is highly influencedby endogenous and environmental stimuli (Fukaki andTasaka 2009, Seo et al. 2009). Under drought andabiotic stresses, lateral root initiation is suppressed byabscisic acid (ABA) signals (Seo et al. 2009). Auxin is acrucial phytohormone for the initiation of lateral roots bypromoting the development of lateral root formation (Seoet al. 2009). Several MYB genes have been identified fortheir important roles in auxin and lateral root formation.In Arabidopsis, the R2R3-MYB TF MYB77 is involved inthe plant response to auxin (Shin et al. 2007). Mutantmyb77 generates low transcript level for auxin-induciblegenes, while the overexpression of MYB77 activatesgene expression, indicating its role in the regulation oflateral root growth and development (Shin et al. 2007,Fukaki and Tasaka 2009).

In addition to their role in lateral root formation, roothairs are significant in causing the roots to become asource of water and nutrients and to serve as storage

and/or synthesis sites for important compounds in theplant (Benfey and Scheres 2000). In a previous report,members of the R2R3 MYB family were found tobe involved in many different biological processes,including root hair differentiation (Dubos et al. 2010).The CAPRICE (CPC) gene encodes a MYB-related TFthat lacks a transactivation domain and is only knownas a negative regulator for the differentiation of non-hair epidermal cells (Wada et al. 1997). As a typicalR2R3 MYB-type TF, WER regulates epidermal cell fates(Bernhardt et al. 2005). Among bHLH proteins, GL3and EGL3 redundantly regulate the specification of rootepidermal cell fate (Bernhardt et al. 2005).

Some MYB TFs regulate plant responses to biotic andabiotic stress conditions (Dubos et al. 2010). Amongthem, AtMYB30 activates the hypersensitive cell deathprogram to suppress pathogen attack (Vailleau et al.2002, Dubos et al. 2010). The ABA signals mediatedby AtMYB96 promote salicylic acid (SA) biosynthesis,and therefore induce the pathogen resistance responsein Arabidopsis (Seo and Park 2010). AtMYB15 interactswith AtICE1 to inhibit the expression of cold-responsivegenes (or CBF regulon) in a manner that is independentfrom ABA (Agarwal et al. 2006). AtMYB8 is necessary forthe basal tolerance to freezing stress, and the Arabidopsismutant myb8 is hypersensitive to high salinity andincreases the expressions of stress-related genes (Zhuet al. 2005, Lippold et al. 2009). AtMYB2 and AtMYC2bind to the cis-elements of RD22 and upregulate itstranscription, which is induced by dehydration andABA (Abe et al. 2003, Fujita et al. 2006). TransgenicArabidopsis that contain FLP and AtMYB88 haveelevated abiotic stress resistance that is provided bythe restricting divisions that occur late in the stomatalcell lineage (Xie et al. 2010).

As perennial plants, fruit trees are exposed to variousabiotic stresses for lifetime once they are planted inorchard, which make it worse for tree growth as well asfruit yield and quality under a deteriorating ecologicalenvironment (Wang et al. 2012). Adaptation to variousabiotic stresses is crucial for fruit trees throughout theirlifespan (Wang et al. 2012). There is thus an urgentneed for scientists to clarify the molecular mechanismsof how fruit trees respond to and fight against abioticstresses. Many attempts have been made to elucidatingthe molecular mechanism of R2R3-MYB roles stressesin the model plant Arabidopsis, rice and other species,however, limited is known about the role of MYB TFsin fruit trees. In this study, an R2R3-type MYB geneMdSIMYB1 was isolated from apple plants on the basis ofits salt-induced expression. Its expression was analyzedwith semi-quantitative reverse transcriptase-polymerasechain reactions (RT-PCRs). MdSIMYB1 was genetically

Physiol. Plant. 150, 2014 77

transformed into tobacco and apple to characterize itsfunction in stress tolerance.

Materials and methods

Plant materials and treatments

The root, stem leaf, flower and fruit were collected from a5-year-old ‘Gala’ apple tree. In vitro apple tissue culturesof ‘Gala’ cultivar were subcultured at a 4-week intervalon Murashige and Skoog (MS) medium plus 0.5 mg l−1 of6-benzylaminopurine (6-BA), 0.1 mg l−1 of gibberellinsand 0.2 mg l−1 of indoleacetic acid (IAA) at 25◦C under a16-h photoperiod. For root induction, 4-week-old shootcultures were shifted to MS medium supplemented with0.1 mg l−1 of IAA. The self-rooted plantlets were grownin pots containing nursery soil for further investigation.

Four-week-old apple tissue cultures were treated with200 mM NaCl, a low temperature (4◦C) and dehydrationaccording to the method described by Yamaguchi-Shinozaki and Shinozaki (1994). For the hormonetreatments, solutions containing 100 μM IAA, 100 μMABA, 150 μM methyl jasmonate (MeJA), 150 μM SAand 150 μM 1-aminocyclopropane-1-carboxylate (ACC,the immediate ethylene precursor) were sprayed on theleaves, respectively, while distilled water was used ascontrol. Tobacco (Nicotiana benthamiana) plants weregrown in pots containing nursery soil at 25◦C under a16-h photoperiod.

Semi-quantitative RT-PCRs geneexpression analysis

Apple total RNAs were extracted with a hot boratemethod as described by Yao et al. (2007), while tobaccototal RNAs with Trizol reagent (Invitrogen, Carlsbad,CA). After treated with RNase-free DNase, the first-strandcDNAs were synthesized with a PrimeScript First StrandcDNA Synthesis Kit (Takara, Dalian, China).

Semi-quantitative RT-PCRs was carried out to examinethe transcript level of MdSIMYB1 in apples and tobacco.Apple 18S rRNA and tobacco NtActin genes were usedas loading controls, respectively. Three replicates wereperformed for each semi-quantitative RT-PCR reaction.The primer sequences used in this study were shown inTable S2.

Vector construction

The full-length cDNA and the 3′-UTR cDNA ofMdSIMYB1 gene were amplified with RT-PCR, and wereused to construct overexpression and suppression vec-tors, respectively. They were digested with BamHI/SalIand inserted into the vector pBI121 downstream a

cauliflower mosaic virus (CaMV) 35S promoter. Theprimers used were listed in Table S1.

Subcellular localization

To observe the subcellular localization, the full openreading frame (ORF) of MdSIMYB1 was amplified withprimers as shown in Table S3. The PCR productwas inserted in frame into the pBI121-GFP vector.The resultant construct p35S:MdSIMYB1-GFP and thecontrol vector p35S:GFP were genetically introducedinto onion epidermal cells with an Agrobacterium-mediated transformation. Following a pre-incubation at22◦C for 24 h, the green fluorescence protein (GFP)signal was detected with a laser confocal microscope(Zeiss LSM510 Meta, Jena, Germany).

Yeast two-hybrid assay

Yeast two-hybrid (Y2H) assay was conducted witha Gal4-based two-hybrid system according to themanufacturer’s instructions (Clontech, Palo Alto, CA).AtGL3 ORF was inserted into vector pGBKT7. Theresultant vector pGBKT7-AtGL3 was used as bait. TheORFs of MdSIMYB1 and AtGL1 were cloned into vectorpGADT7. The resultant vectors pGADT7-MdSIMYB1and pGADT7-AtGL1 were used as prey, respectively.The Y2H primers were shown in Table S4. The pGBKT7-AtGL3 construct was co-transformed with pGADT7-MdSIMYB1 and pGADT7-AtGL1, respectively, into yeaststrain AH109, while pGADT7 was co-transformed withpGBKT7-AtGL3 as the control. SD/-Trp-Leu-His-Ademedium was used to select positive colonies, and β-galactosidase staining was conducted to confirm thepositive colonies.

Bimolecular fluorescence complementation assay

Vectors pSPYNE-35S and pSPYCE-35S, as well as co-transformation vector 35S:P19, were used to constructbimolecular fluorescence complementation (BiFC) plas-mids. MdSIMYB1 ORF was inserted in frame into vectorpSPYNE-35S, while AtGL3 into vector pSPYCE-35S,which contained DNA encoding the N- or C-terminal ofyellow fluorescent protein (YFP). The primers used forplasmid construction were noted in Table S5.

Different combinations of those resultant constructswere genetically introduced into onion epidermis cellswith an Agrobacterium-mediated infection method asdescribed by Walter et al. (2004). After incubatedat 24◦C for 48 h, YFP expression in onion epidermiscells was observed with a laser confocal microscope(Zeiss LSM510 Meta, Jena, Germany) with an excitation


wavelength of 488 nm and detection at 500–530 nmwith a band-path filter for YFP.

Genetic transformation and stress tolerance assayin tobacco

Leaves of wild-type tobacco were surface-sterilized,cut into small discs of explants which were thenplaced on MS medium plus 3 mg l−1 of 6-BA and0.2 mg l−1 of NAA for 3 days. The overexpression con-struct pBI121-MdSIMYB1 was genetically introducedinto these explants with an Agrobacterium-mediatedtransformation. Subsequently, the explants were trans-ferred onto MS medium plus 3 mg l−1 of 6-BA, 0.2 mg l−1

of NAA, 100 mg l−1 of kanamycin and 500 mg l−1 car-benicillin for regeneration and selection. The regener-ated shoots were then grown on MS medium containing0.1 mg l−1 IAA, 100 mg l−1 kanamycin and 500 mg l−1

carbenicillin. The self-rooted plants were shifted to potsfilled with nursery soil.

For salt tolerance assay, 5-week-old tobacco plantswere exposed to 200 mM NaCl for 2 weeks. For droughttolerance assay, 5-week-old tobacco plants were stressedby withholding water for 15 days, and then re-wateredfor 3 days. For cold treatment, 5-week-old tobacco plantswere treated with 4◦C for 6 days, and then allowed torestore growth at 22◦C for another 3 days.

Genetic transformation and stress tolerance assayin apples

Genetic transformation of MdSIMYB1 overexpressionand suppression vectors into apple was carried out withan Agrobacterium-mediated transformation using leafdiscs of ‘Gala’ apple as explants according to Kotodaet al. (2010). To get self-rooted plantlets, transgenic invitro shoots were induced on MS medium containing0.1 mg l−1 IAA. Subsequently, the rooted plantlets weregrown in pots containing nursery soil.

Both in vitro shoots and self-rooted plantlets weretreated with stresses for tolerance assay. Two-week-old in vitro shoots were treated on MS mediumsupplemented with 200 mM NaCl and 10% polyethyleneglycol (PEG), respectively, for another 14 days. To testtheir cold tolerance, they were exposed to 4◦C for 7 days,and then returned to normal conditions at 22◦C foranother 4 days.

The self-rooted plantlets were grown in pots contain-ing nursery soil for 2 months acclimation. The uniformplantlets were chosen for tolerance assays. For salt tol-erance assay, pot-grown plantlets were treated with200 mM NaCl solution twice a week. To test theirdrought tolerance, they were treated with withdrawal

of water for 15 days, and then re-watered for another6 days. For cold tolerance assay, they were treated with4◦C for 7 days, and then got recovery at 22◦C for another10 days. All experiments were repeated for three times.

Results

Molecular cloning and identification of asalt-inducible MdSIMYB1 gene

Many MYB TFs are implicated in signaling pathwaysassociated with abiotic stress (Lippold et al. 2009, Duboset al. 2010). To isolate salt-induced MYB TF genes, RT-PCR was conducted using cDNA templates preparedfrom salt-treated ‘Gala’ apple in vitro tissue cultures.The degenerate primers were synthesized according tothe conserved MYB regions. Subsequently, the positiveclones were sequenced and their expression patternswere analyzed under abiotic stresses treatment. Finally,MdSIMYB1 was chosen for further investigation due to itsremarkably high levels, especially under salt treatmentwhen compared with others. According to the 412 bpcDNA of MdSIMYB1 obtained by sequencing, 5′ RACEand 3′ RACE were amplified to isolate the full-lengthcDNA (data not shown). Sequencing indicated the full-length cDNA being 891 bp in length. The resultantfull-length cDNA was called apple salt-induced MYB1,shortened as MdSIMYB1 (MDP0000143487; GenBankaccession number KC691248). MdSIMYB1 containeda 714-bp ORF and predicted to encode a proteincontaining 237 amino acid residues with a predictedmolecular mass of 26.93 kDa and a pI of 8.04.

When compared with all the R2R3-MYB TFsin Arabidopsis, the phylogenetic tree showed thatMdSIMYB1 formed a close cluster with AtMYB112,AtMYB10, AtMYB72 and AtMYB63, and these geneshave not been associated with abiotic stresses (Fig. 1A).The R2R3-MYB conserved domain of MdSIMYB1 waslocated near the N-terminus (Fig. 1B).

MdSIMYB1 expression is induced by abiotic stressand stress-related exogenous hormones in apples

To examine the transcription levels, semi-quantitativeRT-PCRs were conducted with primers specific to the5′-UTR region of MdSIMYB1. The result indicated thatMdSIMYB1 was expressed in all five types of tissuestested. Among these tissues, the root generated the lowestlevel of MdSIMYB1 transcripts (Fig. 2A). However,MdSIMYB1 transcripts elevated in roots upon exposedto high salinity and ABA (Fig. 2B).

To examine the expression pattern of MdSIMYB1under abiotic stresses, semi-quantitative RT-PCRs werecarried out using cDNA templates prepared from in


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Fig. 1. Analysis of the similarity and structures of the MdSIMYB1 protein. (A) MdSIMYB1 phylogenetic tree with R2R3-MYB proteins in Arabidopsis.The tree was constructed with MEGA 4.0 by the neighbor-joining (NJ) method. (B) Schematic diagram of MdSIMYB1 domain structures.

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Fig. 2. Expression analysis of MdSIMYB1 gene with semi-quantitative RT-PCRs. (A) Tissue-specific expression of MdSIMYB1 under internal control.(B) MdSIMYB1 expression was induced by NaCl and ABA in adventitious roots of self-rooted plantlets. (C) The expression of MdSIMYB1 was inducedby abiotic stresses. (D) The expression of MdSIMYB1 was induced by IAA, ABA, MeJA, SA and ACC treatment. 18S rRNA was used as an internalstandard.

vitro apple shoots treated with 200 mM NaCl, cold(4◦C) and osmotic stress (10% PEG), respectively. Theresult showed that MdSIMYB1 transcripts were markedlyinduced by all stresses tested, indicating that thesestresses are the inducers of the expression of MdSIMYB1(Fig. 2C). In addition, it was found that the expressionpatterns of MdSIMYB1 were positively induced by

an immediate ethylene precursor ACC, and severalhormones including IAA, ABA, MeJA and SA (Fig. 2D).

MdSIMYB1 protein is localized in the nucleus

TFs are generally localized to the nucleus in order toexert their regulatory action (Liu et al. 1999). To observe


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Fig. 3. Subcellular localization of MdSIMYB1. An MdSIMYB1-GFP fusion was transiently expressed in onion epidermal cells. (A, D) These photographswere taken in dark field for green fluorescence. (B, E) Bright light is used to show the morphology of the cell and (C, F) in combination. (A–C) and(D–F) for p35S:MdSIMYB1-GFP and p35S:GFP plasmid control plasmid, respectively. Bars = 50 μm.

the subcellular localizations of MdSIMYB1 protein, theORF of MdSIMYB1 was fused to the N-terminus of GFPin the pBI121 vector, and its expression was drivenby a constitutive 35S CaMV promoter. The resultantconstruct p35S:MdSIMYB1-GFP was transformed intoonion epidermal cells with Agrobacterium-mediatedinfection. The GFP fluorescence was observed only inthe nucleus of transformant cells (Fig. 3), indicating thatMdSIMYB1 is localized to the nucleus in vivo.

Seed germination is insensitive to ABA and NaCl inMdSIMYB1 transgenic tobacco

To examine the function of MdSIMYB1 in seeds and inresponse to plant stress, MdSIMYB1 was transformed intotobacco. As a result, eight transgenic tobacco lines weregot. They produced MdSIMYB1 transcripts at differentlevels. Among them, three lines, i.e. M2, M7 and M8,were chosen for functional characterization. Expressionanalysis indicated that three transgenic lines producedhigh levels of MdSIMYB1, while the WT control didnot at all (Fig. 4A), demonstrating that these transgenictobacco lines ectopically expressed MdSIMYB1 gene.

The effect of ABA and NaCl on the germination of T2homozygous seeds was examined. The result showedthat the seeds of three transgenic lines exhibited agermination ratio similar to the WT control on MSmedium (Fig. 4B). On MS medium plus exogenousABA, the seed germination ratios of both WT andthree transgenic lines significantly decreased, however,three transgenic lines exhibited higher seed germinationratios than the WT control (Fig. 4C). Under 1.0 μMABA treatment, most WT seeds failed to germinate,

while approximately 80% seeds of three transgenic linesgerminated.

It was also found that the seed germination of threetransgenic lines is insensitive to high salinity comparedwith the WT control (Fig. 4D). On MS medium plus40 mM NaCl, 70–80% seeds of three transgenic linesgerminated at day 8, while only 40% WT seedsgerminated. When NaCl was elevated to 80 mM, theseed germination of both WT and transgenic lines wascompletely inhibited at day 2. However, the seed germi-nation ratios of transgenic lines were much higher at day5 than the WT, indicating that the ectopic expressionof MdSIMYB1 in tobacco leads to insensitivity of seedgermination to ABA and NaCl treatments.

Ectopic expression of MdSIMYB1 confers enhancedtolerance to abiotic stresses in tobacco

The effect of MdSIMYB1 transgene on the toleranceto salt, drought and cold in tobacco was determined.When treated with NaCl, three transgenic lines grewwell, whereas the WT plants did poorly. At 14 days afterbeing exposed to 200 mM NaCl, the WT plants startedto wilt while the transgenic lines were nearly normal(Fig. 5B).

To examine drought tolerance, plants were imposedto water deficit for 15 days. The result showed that plantgrowth was inhibited both in WT and transgenic lines.However, three transgenic lines showed less damagethan the WT control. The plant growth of three transgeniclines almost completely recovered after water deficit wasrelieved for 3 days, while the WT plants did not (Fig. 5C).These results indicate that MdSIMYB1 overexpressionconferred drought tolerance in transgenic tobacco.


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Fig. 4. MdSIMYB1 transgenic tobacco seed germination was insensitive to ABA or NaCl. (A) Expression levels of MdSIMYB1 in independenttransgenic tobacco. NtActin was used as an internal standard. (B–D) Seed germination on MS, or MS supplemented with different concentration ofABA and NaCl with transgenic MdSIMYB1 and the WT. All tests were repeated at least three times, and approximately 50 seeds were counted foreach experiment. Data are expressed as the means ± SE.

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Fig. 5. Abiotic stress tolerance of MdSIMYB1 transgenic tobacco seedlings and stress-responsive gene expression. (A–D) Tolerance of transgenictobacco under normal conditions, 200 mM NaCl for 14 days, dehydration for 15 days with a 3-day recovery, 4◦C for 6 days with a 3-day recovery. (E)Semi-quantitative RT-PCR of abiotic stress-responsive genes NtDREB1A, NtERD10B and NtERD10C in WT and transgenic tobacco lines under normalconditions. NtActin was used as an internal standard.

Tobacco plants were exposed to 4◦C for 6 days totest the effect of MdSIMYB1 ectopic expression oncold tolerance. The results showed that plant growthwas adversely influenced by cold stress both in WTand three transgenic lines. However, WT plants weremore seriously damaged than three transgenic lines. Thetransgenic and WT plants were subsequently transferredto normal conditions for 3 days of recovery. Threetransgenic lines partially recovered to grow, whileWT plants nearly died, indicating that MdSIMYB1

ectopic expression noticeably enhanced cold tolerancein transgenic tobacco (Fig. 5D).

To understand the molecular mechanisms thatunderlie MdSIMYB1 function in abiotic stress resistance,the expression levels of three known stress-inducedgenes were examined with semi-quantitative RT-PCRsin the WT and transgenic plants. The transcript levels ofNtDREB1A, NtERD10B and NtERD10C were increasedin transgenic tobacco relative to the WT under normalconditions, indicating that MdSIMYB1 enhanced stress


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Fig. 6. Abiotic stress tolerance of MdSIMYB1 transgenic adult tobacco and root phenotypes. (A, B) Above-ground and below-ground plant partsfrom the WT and transgenic tobacco under normal conditions. (C) Root dry weight of WT and transgenic tobacco under normal conditions. Thevalues are equal to the means ± SD. (D) Semi-quantitative RT-PCR of auxin-response genes NtIAA2.5, NtIAA4.2 and NtIAA4.1 in WT and transgenictobacco under normal conditions. NtActin was used as an internal standard. (E–H) Drought tolerance of transgenic tobacco for 18 days, with 200 mMNaCl for 20 days, 4◦C for 12 days and root phenotypes recorded after these stresses.

tolerance at least partially, if not completely, byregulating stress-responsive gene expression (Fig. 5E).

Ectopic expression of MdSIMYB1 in tobaccopromotes root growth and maintains a robust rootsystem under stress conditions

In addition to the increased expression of stress-responsive genes, root growth promotion is believedto enhance abiotic stress tolerance (Shukla et al.2006). To determine whether the MdSIMYB1 transgeneinfluences root growth, adult tobacco plants were usedfor this study. The results showed that MdSIMYB1transgenic plants exhibited similar appearances to theWT controls for the above-ground shoots (Fig. 6A).However, the transgenic plants generated more robustroot systems than the controls, as indicated by theroot appearances and dry weights (Fig. 6B, C). Auxinis known to play a central role in root developmentand growth (Hao et al. 2011). The expression ofMdSIMYB1 was positively induced by IAA treatment(Fig. 2D), suggesting that it could be involved in the

auxin response. Furthermore, MdSIMYB1 was studiedfor the regulation of auxin-responsive gene expression,including NtIAA4.2, NtIAA4.1 and NtIAA2.5, whichhave been associated with root growth in tobacco(Dargeviciute et al. 1998, Shukla et al. 2006). TheMdSIMYB1 transgenic tobacco plants produced manymore transcripts of NtIAA4.2, NtIAA4.1 and NtIAA2.5genes than the WT control (Fig. 6D), indicating thatMdSIMYB1 promotes root growth by regulating theexpression of auxin-responsive genes. In addition, Y2Hand BiFC assays observed that MdSIMYB1 proteininteracted with Arabidopsis AtGL3 which participatesin hair and non-hair formation in the root epidermis (Fig.S1, Bernhardt et al. 2005).

To determine whether root growth is maintained understressful conditions in the transgenic lines, adult tobaccoplants were exposed to high salt, drought and cold stress.Just as in the transgenic seedlings, the transgenic adultplants were much more tolerant than the WT control, asindicated by the growth of the above-ground shoot (Fig.6E–G). The ectopic expression of MdSIMYB1 promotedroot growth under stress conditions (Fig. 6H). As a result,


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Fig. 7. MdSIMYB1 overexpression enhances tolerance to abiotic stresses in apple. (A) Expression levels of MdSIMYB1 in independent transgenicapple lines. (B–D) Tolerance of transgenic apple in vitro shoot cultures on subculture medium plus 200 mM NaCl for 14 days, 10% PEG for 14 daysand 4◦C for 7 days with a 4-day recovery. (E–G) Tolerance of transgenic rooting apple plantlets for 200 mM NaCl for 14 days, dehydration for 15 dayswith a 6-day recovery and 4◦C for 7 days with a 10-day recovery.

the transgenic plants generated a much more robust rootsystem than the WT control under high salt, drought andcold stress.

MdSIMYB1 overexpression enhances tolerance toabiotic stresses in transgenic apple

To examine whether MdSIMYB1 confers tolerance tosalt, drought and cold in apples, MdSIMYB1 transgenicapples were obtained. Three overexpressors T1, T2 andT12, as well as one suppressor RT2, were chosen forfurther investigation. Expression analysis showed thatthree overexpressor produced much more transcripts,while the suppressor generated fewer transcripts, thanthe WT control (Fig. 7A). Correspondingly, whentransferred to rooting medium, T1, T2 and T12 plantletsgenerated more robust root systems just like transgenictobacco did, while RT2 plantlets produced poorer one,than the WT control (Fig. S2). Therefore, MdSIMYB1promotes root growth both in tobacco and apple.

To examine the tolerance to salt stress, transgenicin vitro shoots were transferred in medium containing200 mM NaCl at 2 weeks after subculture, whichpermitted growth for another 14 days (Fig. 7B). Thetransgenic self-rooted plantlets were treated twice eachweek with 200 mM NaCl solution at 2 months after theirtransfer to pots, and then were permitted to grow foranother 14 days (Fig. 7E). The result showed that the leafcolor looked normal in MdSIMYB1 overexpression lines,but WT control plants started to be yellow, indicatingthat the overexpressors got less damage than the WTcontrol. In contrast, the MdSIMYB1 suppressor RT2 gotmore serious damage than the WT control, indicatingthat MdSIMYB1 suppression reduced the tolerance tosalt in line RT2.

To induce tolerance to drought stress, in vitro shoots2 weeks after subculture were shifted to MS mediumcontaining 10% PEG and permitted to grow for 14 days(Fig. 7C). The transgenic rooting plantlets 2 monthsafter their transfer to pots were deprived of water for15 days and re-watered for another 6 days (Fig. 7F). Theoverexpression lines grew much better than the WTcontrol or the suppression line RT2. More leaves turnedyellow, especially those near the medium or the soilsurface, and some even died in the WT control and thesuppression line RT2, while three overexpression lineleaves remained close to normal in color, indicating thatMdSIMYB1 overexpression conferred enhanced droughttolerance to transgenic apples.

To test cold tolerance, in vitro shoots 2 weeks aftertheir subculture were exposed to 4◦C for 7 days, andwere then recovered under normal conditions. Fourdays later, the WT and suppressor RT2 plants exhibitednoticeable damage phenotypes such as yellow andwilted leaves, while three overexpressor looked normal(Fig. 7D). The transgenic rooted plantlets 2 monthsafter being transferred to pots were treated with 4◦Cfor 7 days, and were then permitted growth at 22◦Cfor another 10 days. The overexpressors completelyrecovered, while the WT and suppression line RT2plants eventually died (Fig. 7G), indicating MdSIMYB1overexpression enhanced cold tolerance in transgenicapples.

Discussion

In this study, a MYB TF gene MdSIMYB1 was clonedfrom apple tissues. The predicted MdSIMYB1 proteinbelongs to the R2R3 subfamily and is localized to thenucleus in a subcellular manner, just like the other


MYBs in Arabidopsis and rice (Katiyar et al. 2012).Its expression was induced by multiple abiotic stressesand stress-related hormones, suggesting its involvementin the response to and the fight against environmentalstresses. It is consistent with the fact that the expressionsof many MYB TF genes are induced by various abioticstresses in model plants (Yanhui et al. 2006).

Increasing evidence has shown that hormone signal-ing pathways associated with ABA, SA, JA and ethyleneplay crucial roles in the crosstalk between abiotic andbiotic stress signaling (Fujita et al. 2006). Among theseimportant hormones, ABA acts to inhibit seed germina-tion and early seedling development (Guo et al. 2008).ABA production is triggered under drought stress, whichsubsequently induces stress-responsive gene expression(Abe et al. 2003). MdSIMYB1 expression was induced bydehydration, salt and cold stress, as well as stress-relatedhormones, suggesting that MdSIMYB1 may be part ofthe plant response to these abiotic stresses in relation tothe signaling pathways of those hormones.

It is possible that different ABA signaling pathways areinvolved in seed germination and stress tolerance (Daiet al. 2007). In some cases of transgene investigation,seed germination and early seedling development aresensitive to exogenous ABA, despite of enhanced stresstolerance (Hu et al. 2006, Ko et al. 2006). For example,SNAC1-overexpressing rice seedlings were significantlysensitive to ABA treatment but they had improvedtolerance to drought and salt (Hu et al. 2006). AtMYC2and AtMYB2 transgenic Arabidopsis exhibited an ABA-sensitive phenotype, although they increased toleranceto osmotic stress (Abe et al. 2003). In some other cases,an enhanced tolerance to abiotic stress accompanied adecreased sensitivity to ABA for seed germination. Forexample, OsMYB3R-2 transgenic plants had enhancedtolerance to multiple stresses and decreased sensitivity ofseed germination to ABA (Dai et al. 2007). The enhancedtolerance to stresses and decreased ABA sensitivity ofseed germination also exist for other genes such asAtHD2C, CaXTH3 and AtTPS1 (Dai et al. 2007). In thisstudy, the seed germination of MdSIMYB1 transgenictobacco became more insensitive to ABA and NaCltreatments than the control.

In addition to germination insensitivity, the ectopicexpression of MdSIMYB1 increased the expression ofNtDREB1A, NtERD10B and NtERD10C, which arestress-responsive genes in tobacco (Park et al. 2001,Shukla et al. 2006). The overexpression of genes suchas CBF/DREB1 and DREB1A confers stress tolerance totransgenic plants (Kasuga et al. 2004, Ito et al. 2006).The high transcript levels of NtDREB1A, NtERD10B andNtERD10C in transgenic tobacco plants indicate thatMdSIMYB1 conferred tolerance to abiotic stresses at

least partially, if not all, by upregulating stress-responsivegenes such as NtDREB1A, NtERD10B and NtERD10C.

Auxin plays a crucial role in the root initiationand growth of higher plants (Tripathi et al. 2009).Lateral root formation and root structure adaptationare related to biotic and abiotic stress (Peterson 1992).Many genes are at least partially involved in stresstolerance by controlling auxin transport or response topromote root formation and growth. For example, theoverexpression of vacuolar H+-pyrophosphatase geneAVP1 in Arabidopsis and tomato increased tolerance tosoil water deficits by regulating auxin transport andthereby affected auxin-dependent root growth (Parket al. 2005). A T-DNA insertion into the ArabidopsisCIPK6 gene caused a reduction in gene expression,the emergence of lateral roots and sensitivity tosalt stress (Tripathi et al. 2009). In this study, theexpression of MdSIMYB1 was positively induced by IAAtreatment, suggesting that it could be involved in theauxin response. The ectopic expression of MdSIMYB1enhanced the transcript level of auxin signaling genessuch as NtIAA4.2, NtIAA4.1 and NtIAA2.5, indicatingthat MdSIMYB1 most likely promotes root growth byupregulating the expression of auxin signaling genes,and therefore maintains a robust root system to enhancetolerance under multiple abiotic stresses.

Root hairs initiate from root surface to facilitatenutrient and water uptake, thereby being essential forplant growth (Gilroy and Jones 2000). The identificationof the hair and non-hair cell fate in Arabidopsis rootepidermis has been studied extensively. The bHLH TFsGL3 and EGL3 act together with WER to promotethe non-hair cell fate, however, they interact withCPC to block the non-hair pathway and lead to ahair cell fate (Bernhardt et al. 2005). Interestingly,it was that MdSIMYB1 protein interacts with AtGL3which participates in hair and non-hair formation inthe root epidermis in Arabidopsis (Fig. S1, Bernhardtet al. 2005). This finding suggests that MdSIMYB1 mayregulate cell fate in the epidermis of the root to influenceits growth and development by interacting with GL3-like plant proteins. In conclusion, besides direct orindirect regulation of stress-responsive gene expression,MdSIMYB1 is also involved in stress tolerance bypromoting and maintaining root growth under stressconditions by regulating auxin-responsive genes andperhaps by interacting with GL3-like proteins.

Taken together, the overexpression of a novel appleR2R3 MYB gene MdSIMYB1 enhanced the toleranceto salt, drought and cold stresses in transgenic tobaccoand apples. Therefore, MdSIMYB1 can be used as atarget gene for genetic manipulation to improve multipleabiotic stress tolerance to fruit trees and other crops.


Acknowledgements – This work was supported by NSFC(30971969), National High Technology Research andDevelopment Program of China (2011AA100204), 948Project from Ministry of Agriculture (2011-G21) andProgram for Changjiang Scholars and Innovative ResearchTeam in University (IRT1155).

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1. Primers for gene cloning and vectorconstruction.

Table S2. Primers for expression analysis with semi-quantitative RT-PCRs.

Table S3. Primers for subcellular localization.

Table S4. Primers for yeast two-hybrid assays.

Table S5. Primers for BiFC assay.

Fig. S1. Interaction between AtGL3 with MdSIMYB1.

Fig. S2. Rooted apple plantlets of three overexpressionlines (T1, T2 and T12) and one suppression line RT2,with the WT as control.

Edited by M. Uemura


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