SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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


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

polyethylene glycol

RACE

rapid amplification of cDNA ends

RT-PCRs

reverse transcriptase-polymerase chain reaction

SA

salicylic acid

TFs

transcription factors

WT

wild-type

YFP

yellow fluorescent protein

Y2H

yeast two-hybrid

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Various abiotic stresses including salinity, drought and cold greatly influence plant growth and crop productivity. A variety of genes are induced by abiotic stresses to defend against abiotic stresses in higher plants (Kasuga et al. 2004). Generally, these stress-induced genes are directly or indirectly modulated by regulators that are components of signaling pathway associated with abiotic stresses (Shinozaki and Yamaguchi-Shinozaki 1997). Transcription factors (TFs) are important player in the regulation of gene expression in response to developmental and environmental cues (Jung et al. 2008). Among them, some members of the MYB, ERF, bZIP and WRKY TF families are implicated in plant defense to abiotic stresses (Jung et al. 2008). In Arabidopsis, the MYB family including 163 members is one of the largest TF families (Yanhui et al. 2006).

The first myb gene, i.e. an oncogene v-MYB, is identified from avian myeloblastosis virus (Klempnauer et al. 1982). So far, it is found that MYB genes exist in almost all eukaryotes. The MYB TFs generally contain one to three imperfect helix-turn-helix repeats that are responsible for the specific recognition to target DNA (Yanhui et al. 2006). On the basis of the arrangement of those repeats, MYB TFs are divided into three subfamilies, i.e. R1R2R3, R2R3 and MYB related (which has one MYB-like domain) (Dubos et al. 2010). Different structural arrangements exist in different kingdoms, suggesting specific functions of MYB TFs in plants. Over the past decades, it has been established that many MYB TFs are involved in various plant-specific processes such as secondary metabolism, cell shape determination and cell differentiation (Lippold et al. 2009). Among them, AtMYB75 and AtMYB90 are responsible for the regulation of anthocyanins biosynthesis (Dubos et al. 2010), while AtMYB28 for glucosinolate accumulation in Arabidopsis (Gigolashvili et al. 2007). The differentiation of cells into the non-hair fate is controlled by AtMYB66 (Bernhardt et al. 2005, Dubos et al. 2010). In addition, the anther endothecial cells show defects in secondary wall thickening in mutant myb26 which is male sterile (Yang et al. 2007). The AtMYB46 gene functions to regulate the formation of the second cell wall (Zhong et al. 2007).

In plants, lateral root formation greatly influences root architecture development and is highly influenced by endogenous and environmental stimuli (Fukaki and Tasaka 2009, Seo et al. 2009). Under drought and abiotic stresses, lateral root initiation is suppressed by abscisic acid (ABA) signals (Seo et al. 2009). Auxin is a crucial phytohormone for the initiation of lateral roots by promoting the development of lateral root formation (Seo et al. 2009). Several MYB genes have been identified for their important roles in auxin and lateral root formation. In Arabidopsis, the R2R3-MYB TF MYB77 is involved in the plant response to auxin (Shin et al. 2007). Mutant myb77 generates low transcript level for auxin-inducible genes, while the overexpression of MYB77 activates gene expression, indicating its role in the regulation of lateral root growth and development (Shin et al. 2007, Fukaki and Tasaka 2009).

In addition to their role in lateral root formation, root hairs are significant in causing the roots to become a source of water and nutrients and to serve as storage and/or synthesis sites for important compounds in the plant (Benfey and Scheres 2000). In a previous report, members of the R2R3 MYB family were found to be involved in many different biological processes, including root hair differentiation (Dubos et al. 2010). The CAPRICE (CPC) gene encodes a MYB-related TF that lacks a transactivation domain and is only known as a negative regulator for the differentiation of non-hair epidermal cells (Wada et al. 1997). As a typical R2R3 MYB-type TF, WER regulates epidermal cell fates (Bernhardt et al. 2005). Among bHLH proteins, GL3 and EGL3 redundantly regulate the specification of root epidermal cell fate (Bernhardt et al. 2005).

Some MYB TFs regulate plant responses to biotic and abiotic stress conditions (Dubos et al. 2010). Among them, AtMYB30 activates the hypersensitive cell death program to suppress pathogen attack (Vailleau et al. 2002, Dubos et al. 2010). The ABA signals mediated by AtMYB96 promote salicylic acid (SA) biosynthesis, and therefore induce the pathogen resistance response in Arabidopsis (Seo and Park 2010). AtMYB15 interacts with AtICE1 to inhibit the expression of cold-responsive genes (or CBF regulon) in a manner that is independent from ABA (Agarwal et al. 2006). AtMYB8 is necessary for the basal tolerance to freezing stress, and the Arabidopsis mutant myb8 is hypersensitive to high salinity and increases the expressions of stress-related genes (Zhu et al. 2005, Lippold et al. 2009). AtMYB2 and AtMYC2 bind to the cis-elements of RD22 and upregulate its transcription, which is induced by dehydration and ABA (Abe et al. 2003, Fujita et al. 2006). Transgenic Arabidopsis that contain FLP and AtMYB88 have elevated abiotic stress resistance that is provided by the restricting divisions that occur late in the stomatal cell lineage (Xie et al. 2010).

As perennial plants, fruit trees are exposed to various abiotic stresses for lifetime once they are planted in orchard, which make it worse for tree growth as well as fruit yield and quality under a deteriorating ecological environment (Wang et al. 2012). Adaptation to various abiotic stresses is crucial for fruit trees throughout their lifespan (Wang et al. 2012). There is thus an urgent need for scientists to clarify the molecular mechanisms of how fruit trees respond to and fight against abiotic stresses. Many attempts have been made to elucidating the molecular mechanism of R2R3-MYB roles stresses in the model plant Arabidopsis, rice and other species, however, limited is known about the role of MYB TFs in fruit trees. In this study, an R2R3-type MYB gene MdSIMYB1 was isolated from apple plants on the basis of its salt-induced expression. Its expression was analyzed with semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCRs). MdSIMYB1 was genetically transformed into tobacco and apple to characterize its function in stress tolerance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and treatments

The root, stem leaf, flower and fruit were collected from a 5-year-old ‘Gala’ apple tree. In vitro apple tissue cultures of ‘Gala’ cultivar were subcultured at a 4-week interval on Murashige and Skoog (MS) medium plus 0.5 mg l−1 of 6-benzylaminopurine (6-BA), 0.1 mg l−1 of gibberellins and 0.2 mg l−1 of indoleacetic acid (IAA) at 25°C under a 16-h photoperiod. For root induction, 4-week-old shoot cultures were shifted to MS medium supplemented with 0.1 mg l−1 of IAA. The self-rooted plantlets were grown in pots containing nursery soil for further investigation.

Four-week-old apple tissue cultures were treated with 200 mM NaCl, a low temperature (4°C) and dehydration according to the method described by Yamaguchi-Shinozaki and Shinozaki (1994). For the hormone treatments, solutions containing 100 µM IAA, 100 µM ABA, 150 µM methyl jasmonate (MeJA), 150 µM SA and 150 µM 1-aminocyclopropane-1-carboxylate (ACC, the immediate ethylene precursor) were sprayed on the leaves, respectively, while distilled water was used as control. Tobacco (Nicotiana benthamiana) plants were grown in pots containing nursery soil at 25°C under a 16-h photoperiod.

Semi-quantitative RT-PCRs gene expression analysis

Apple total RNAs were extracted with a hot borate method as described by Yao et al. (2007), while tobacco total RNAs with Trizol reagent (Invitrogen, Carlsbad, CA). After treated with RNase-free DNase, the first-strand cDNAs were synthesized with a PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China).

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

Vector construction

The full-length cDNA and the 3′-UTR cDNA of MdSIMYB1 gene were amplified with RT-PCR, and were used to construct overexpression and suppression vectors, respectively. They were digested with BamHI/SalI and inserted into the vector pBI121 downstream a cauliflower mosaic virus (CaMV) 35S promoter. The primers used were listed in Table S1.

Subcellular localization

To observe the subcellular localization, the full open reading frame (ORF) of MdSIMYB1 was amplified with primers as shown in Table S3. The PCR product was inserted in frame into the pBI121-GFP vector. The resultant construct p35S:MdSIMYB1-GFP and the control vector p35S:GFP were genetically introduced into onion epidermal cells with an Agrobacterium-mediated transformation. Following a pre-incubation at 22°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 with a Gal4-based two-hybrid system according to the manufacturer's instructions (Clontech, Palo Alto, CA). AtGL3 ORF was inserted into vector pGBKT7. The resultant vector pGBKT7-AtGL3 was used as bait. The ORFs of MdSIMYB1 and AtGL1 were cloned into vector pGADT7. The resultant vectors pGADT7-MdSIMYB1 and 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 yeast strain AH109, while pGADT7 was co-transformed with pGBKT7-AtGL3 as the control. SD/-Trp-Leu-His-Ade medium was used to select positive colonies, and β-galactosidase staining was conducted to confirm the positive colonies.

Bimolecular fluorescence complementation assay

Vectors pSPYNE-35S and pSPYCE-35S, as well as co-transformation vector 35S:P19, were used to construct bimolecular fluorescence complementation (BiFC) plasmids. MdSIMYB1 ORF was inserted in frame into vector pSPYNE-35S, while AtGL3 into vector pSPYCE-35S, which contained DNA encoding the N- or C-terminal of yellow fluorescent protein (YFP). The primers used for plasmid construction were noted in Table S5.

Different combinations of those resultant constructs were genetically introduced into onion epidermis cells with an Agrobacterium-mediated infection method as described by Walter et al. (2004). After incubated at 24°C for 48 h, YFP expression in onion epidermis cells was observed with a laser confocal microscope (Zeiss LSM510 Meta, Jena, Germany) with an excitation wavelength of 488 nm and detection at 500–530 nm with a band-path filter for YFP.

Genetic transformation and stress tolerance assay in tobacco

Leaves of wild-type tobacco were surface-sterilized, cut into small discs of explants which were then placed on MS medium plus 3 mg l−1 of 6-BA and 0.2 mg l−1 of NAA for 3 days. The overexpression construct pBI121-MdSIMYB1 was genetically introduced into these explants with an Agrobacterium-mediated transformation. Subsequently, the explants were transferred 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 carbenicillin for regeneration and selection. The regenerated shoots were then grown on MS medium containing 0.1 mg l−1 IAA, 100 mg l−1 kanamycin and 500 mg l−1 carbenicillin. The self-rooted plants were shifted to pots filled with nursery soil.

For salt tolerance assay, 5-week-old tobacco plants were exposed to 200 mM NaCl for 2 weeks. For drought tolerance assay, 5-week-old tobacco plants were stressed by withholding water for 15 days, and then re-watered for 3 days. For cold treatment, 5-week-old tobacco plants were treated with 4°C for 6 days, and then allowed to restore growth at 22°C for another 3 days.

Genetic transformation and stress tolerance assay in apples

Genetic transformation of MdSIMYB1 overexpression and suppression vectors into apple was carried out with an Agrobacterium-mediated transformation using leaf discs of ‘Gala’ apple as explants according to Kotoda et al. (2010). To get self-rooted plantlets, transgenic in vitro shoots were induced on MS medium containing 0.1 mg l−1 IAA. Subsequently, the rooted plantlets were grown in pots containing nursery soil.

Both in vitro shoots and self-rooted plantlets were treated with stresses for tolerance assay. Two-week-old in vitro shoots were treated on MS medium supplemented with 200 mM NaCl and 10% polyethylene glycol (PEG), respectively, for another 14 days. To test their cold tolerance, they were exposed to 4°C for 7 days, and then returned to normal conditions at 22°C for another 4 days.

The self-rooted plantlets were grown in pots containing nursery soil for 2 months acclimation. The uniform plantlets were chosen for tolerance assays. For salt tolerance assay, pot-grown plantlets were treated with 200 mM NaCl solution twice a week. To test their drought tolerance, they were treated with withdrawal of water for 15 days, and then re-watered for another 6 days. For cold tolerance assay, they were treated with 4°C for 7 days, and then got recovery at 22°C for another 10 days. All experiments were repeated for three times.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Molecular cloning and identification of a salt-inducible MdSIMYB1 gene

Many MYB TFs are implicated in signaling pathways associated with abiotic stress (Lippold et al. 2009, Dubos et al. 2010). To isolate salt-induced MYB TF genes, RT-PCR was conducted using cDNA templates prepared from salt-treated ‘Gala’ apple in vitro tissue cultures. The degenerate primers were synthesized according to the conserved MYB regions. Subsequently, the positive clones were sequenced and their expression patterns were analyzed under abiotic stresses treatment. Finally, MdSIMYB1 was chosen for further investigation due to its remarkably high levels, especially under salt treatment when compared with others. According to the 412 bp cDNA of MdSIMYB1 obtained by sequencing, 5′ RACE and 3′ RACE were amplified to isolate the full-length cDNA (data not shown). Sequencing indicated the full-length cDNA being 891 bp in length. The resultant full-length cDNA was called apple salt-induced MYB1, shortened as MdSIMYB1 (MDP0000143487; GenBank accession number KC691248). MdSIMYB1 contained a 714-bp ORF and predicted to encode a protein containing 237 amino acid residues with a predicted molecular mass of 26.93 kDa and a pI of 8.04.

When compared with all the R2R3-MYB TFs in Arabidopsis, the phylogenetic tree showed that MdSIMYB1 formed a close cluster with AtMYB112, AtMYB10, AtMYB72 and AtMYB63, and these genes have not been associated with abiotic stresses (Fig. 1A). The R2R3-MYB conserved domain of MdSIMYB1 was located near the N-terminus (Fig. 1B).

image

Figure 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.

Download figure to PowerPoint

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

To examine the transcription levels, semi-quantitative RT-PCRs were conducted with primers specific to the 5′-UTR region of MdSIMYB1. The result indicated that MdSIMYB1 was expressed in all five types of tissues tested. Among these tissues, the root generated the lowest level of MdSIMYB1 transcripts (Fig. 2A). However, MdSIMYB1 transcripts elevated in roots upon exposed to high salinity and ABA (Fig. 2B).

image

Figure 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 induced by abiotic stresses. (D) The expression of MdSIMYB1 was induced by IAA, ABA, MeJA, SA and ACC treatment. 18S rRNA was used as an internal standard.

Download figure to PowerPoint

To examine the expression pattern of MdSIMYB1 under abiotic stresses, semi-quantitative RT-PCRs were carried out using cDNA templates prepared from in vitro apple shoots treated with 200 mM NaCl, cold (4°C) and osmotic stress (10% PEG), respectively. The result showed that MdSIMYB1 transcripts were markedly induced by all stresses tested, indicating that these stresses are the inducers of the expression of MdSIMYB1 (Fig. 2C). In addition, it was found that the expression patterns of MdSIMYB1 were positively induced by an immediate ethylene precursor ACC, and several hormones including IAA, ABA, MeJA and SA (Fig. 2D).

MdSIMYB1 protein is localized in the nucleus

TFs are generally localized to the nucleus in order to exert their regulatory action (Liu et al. 1999). To observe the subcellular localizations of MdSIMYB1 protein, the ORF of MdSIMYB1 was fused to the N-terminus of GFP in the pBI121 vector, and its expression was driven by a constitutive 35S CaMV promoter. The resultant construct p35S:MdSIMYB1-GFP was transformed into onion epidermal cells with Agrobacterium-mediated infection. The GFP fluorescence was observed only in the nucleus of transformant cells (Fig. 3), indicating that MdSIMYB1 is localized to the nucleus in vivo.

image

Figure 3. Subcellular localization of MdSIMYB1. An MdSIMYB1-GFP fusion was transiently expressed in onion epidermal cells. (A, D) These photographs were 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.

Download figure to PowerPoint

Seed germination is insensitive to ABA and NaCl in MdSIMYB1 transgenic tobacco

To examine the function of MdSIMYB1 in seeds and in response to plant stress, MdSIMYB1 was transformed into tobacco. As a result, eight transgenic tobacco lines were got. They produced MdSIMYB1 transcripts at different levels. Among them, three lines, i.e. M2, M7 and M8, were chosen for functional characterization. Expression analysis indicated that three transgenic lines produced high levels of MdSIMYB1, while the WT control did not at all (Fig. 4A), demonstrating that these transgenic tobacco lines ectopically expressed MdSIMYB1 gene.

image

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

Download figure to PowerPoint

The effect of ABA and NaCl on the germination of T2 homozygous seeds was examined. The result showed that the seeds of three transgenic lines exhibited a germination ratio similar to the WT control on MS medium (Fig. 4B). On MS medium plus exogenous ABA, the seed germination ratios of both WT and three transgenic lines significantly decreased, however, three transgenic lines exhibited higher seed germination ratios than the WT control (Fig. 4C). Under 1.0 µM ABA treatment, most WT seeds failed to germinate, while approximately 80% seeds of three transgenic lines germinated.

It was also found that the seed germination of three transgenic lines is insensitive to high salinity compared with the WT control (Fig. 4D). On MS medium plus 40 mM NaCl, 70–80% seeds of three transgenic lines germinated at day 8, while only 40% WT seeds germinated. When NaCl was elevated to 80 mM, the seed germination of both WT and transgenic lines was completely inhibited at day 2. However, the seed germination ratios of transgenic lines were much higher at day 5 than the WT, indicating that the ectopic expression of MdSIMYB1 in tobacco leads to insensitivity of seed germination to ABA and NaCl treatments.

Ectopic expression of MdSIMYB1 confers enhanced tolerance to abiotic stresses in tobacco

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

image

Figure 5. Abiotic stress tolerance of MdSIMYB1 transgenic tobacco seedlings and stress-responsive gene expression. (A–D) Tolerance of transgenic tobacco 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 normal conditions. NtActin was used as an internal standard.

Download figure to PowerPoint

To examine drought tolerance, plants were imposed to water deficit for 15 days. The result showed that plant growth was inhibited both in WT and transgenic lines. However, three transgenic lines showed less damage than the WT control. The plant growth of three transgenic lines almost completely recovered after water deficit was relieved for 3 days, while the WT plants did not (Fig. 5C). These results indicate that MdSIMYB1 overexpression conferred drought tolerance in transgenic tobacco.

Tobacco plants were exposed to 4°C for 6 days to test the effect of MdSIMYB1 ectopic expression on cold tolerance. The results showed that plant growth was adversely influenced by cold stress both in WT and three transgenic lines. However, WT plants were more seriously damaged than three transgenic lines. The transgenic and WT plants were subsequently transferred to normal conditions for 3 days of recovery. Three transgenic lines partially recovered to grow, while WT plants nearly died, indicating that MdSIMYB1 ectopic expression noticeably enhanced cold tolerance in transgenic tobacco (Fig. 5D).

To understand the molecular mechanisms that underlie MdSIMYB1 function in abiotic stress resistance, the expression levels of three known stress-induced genes were examined with semi-quantitative RT-PCRs in the WT and transgenic plants. The transcript levels of NtDREB1A, NtERD10B and NtERD10C were increased in transgenic tobacco relative to the WT under normal conditions, indicating that MdSIMYB1 enhanced stress tolerance at least partially, if not completely, by regulating stress-responsive gene expression (Fig. 5E).

Ectopic expression of MdSIMYB1 in tobacco promotes root growth and maintains a robust root system under stress conditions

In addition to the increased expression of stress-responsive genes, root growth promotion is believed to enhance abiotic stress tolerance (Shukla et al. 2006). To determine whether the MdSIMYB1 transgene influences root growth, adult tobacco plants were used for this study. The results showed that MdSIMYB1 transgenic plants exhibited similar appearances to the WT controls for the above-ground shoots (Fig. 6A). However, the transgenic plants generated more robust root systems than the controls, as indicated by the root appearances and dry weights (Fig. 6B, C). Auxin is known to play a central role in root development and growth (Hao et al. 2011). The expression of MdSIMYB1 was positively induced by IAA treatment (Fig. 2D), suggesting that it could be involved in the auxin response. Furthermore, MdSIMYB1 was studied for the regulation of auxin-responsive gene expression, including NtIAA4.2, NtIAA4.1 and NtIAA2.5, which have been associated with root growth in tobacco (Dargeviciute et al. 1998, Shukla et al. 2006). The MdSIMYB1 transgenic tobacco plants produced many more transcripts of NtIAA4.2, NtIAA4.1 and NtIAA2.5 genes than the WT control (Fig. 6D), indicating that MdSIMYB1 promotes root growth by regulating the expression of auxin-responsive genes. In addition, Y2H and BiFC assays observed that MdSIMYB1 protein interacted with Arabidopsis AtGL3 which participates in hair and non-hair formation in the root epidermis (Fig. S1, Bernhardt et al. 2005).

image

Figure 6. Abiotic stress tolerance of MdSIMYB1 transgenic adult tobacco and root phenotypes. (A, B) Above-ground and below-ground plant parts from the WT and transgenic tobacco under normal conditions. (C) Root dry weight of WT and transgenic tobacco under normal conditions. The values 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 transgenic tobacco under normal conditions. NtActin was used as an internal standard. (E–H) Drought tolerance of transgenic tobacco for 18 days, with 200 mM NaCl for 20 days, 4°C for 12 days and root phenotypes recorded after these stresses.

Download figure to PowerPoint

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

MdSIMYB1 overexpression enhances tolerance to abiotic stresses in transgenic apple

To examine whether MdSIMYB1 confers tolerance to salt, drought and cold in apples, MdSIMYB1 transgenic apples were obtained. Three overexpressors T1, T2 and T12, as well as one suppressor RT2, were chosen for further investigation. Expression analysis showed that three overexpressor produced much more transcripts, while the suppressor generated fewer transcripts, than the WT control (Fig. 7A). Correspondingly, when transferred to rooting medium, T1, T2 and T12 plantlets generated more robust root systems just like transgenic tobacco did, while RT2 plantlets produced poorer one, than the WT control (Fig. S2). Therefore, MdSIMYB1 promotes root growth both in tobacco and apple.

image

Figure 7. MdSIMYB1 overexpression enhances tolerance to abiotic stresses in apple. (A) Expression levels of MdSIMYB1 in independent transgenic apple 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 days and 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 days with a 6-day recovery and 4°C for 7 days with a 10-day recovery.

Download figure to PowerPoint

To examine the tolerance to salt stress, transgenic in vitro shoots were transferred in medium containing 200 mM NaCl at 2 weeks after subculture, which permitted growth for another 14 days (Fig. 7B). The transgenic self-rooted plantlets were treated twice each week with 200 mM NaCl solution at 2 months after their transfer to pots, and then were permitted to grow for another 14 days (Fig. 7E). The result showed that the leaf color looked normal in MdSIMYB1 overexpression lines, but WT control plants started to be yellow, indicating that the overexpressors got less damage than the WT control. In contrast, the MdSIMYB1 suppressor RT2 got more serious damage than the WT control, indicating that MdSIMYB1 suppression reduced the tolerance to salt in line RT2.

To induce tolerance to drought stress, in vitro shoots 2 weeks after subculture were shifted to MS medium containing 10% PEG and permitted to grow for 14 days (Fig. 7C). The transgenic rooting plantlets 2 months after their transfer to pots were deprived of water for 15 days and re-watered for another 6 days (Fig. 7F). The overexpression lines grew much better than the WT control or the suppression line RT2. More leaves turned yellow, especially those near the medium or the soil surface, and some even died in the WT control and the suppression line RT2, while three overexpression line leaves remained close to normal in color, indicating that MdSIMYB1 overexpression conferred enhanced drought tolerance to transgenic apples.

To test cold tolerance, in vitro shoots 2 weeks after their subculture were exposed to 4°C for 7 days, and were then recovered under normal conditions. Four days later, the WT and suppressor RT2 plants exhibited noticeable damage phenotypes such as yellow and wilted leaves, while three overexpressor looked normal (Fig. 7D). The transgenic rooted plantlets 2 months after being transferred to pots were treated with 4°C for 7 days, and were then permitted growth at 22°C for another 10 days. The overexpressors completely recovered, while the WT and suppression line RT2 plants eventually died (Fig. 7G), indicating MdSIMYB1 overexpression enhanced cold tolerance in transgenic apples.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, a MYB TF gene MdSIMYB1 was cloned from apple tissues. The predicted MdSIMYB1 protein belongs to the R2R3 subfamily and is localized to the nucleus in a subcellular manner, just like the other MYBs in Arabidopsis and rice (Katiyar et al. 2012). Its expression was induced by multiple abiotic stresses and stress-related hormones, suggesting its involvement in the response to and the fight against environmental stresses. It is consistent with the fact that the expressions of many MYB TF genes are induced by various abiotic stresses in model plants (Yanhui et al. 2006).

Increasing evidence has shown that hormone signaling pathways associated with ABA, SA, JA and ethylene play crucial roles in the crosstalk between abiotic and biotic stress signaling (Fujita et al. 2006). Among these important hormones, ABA acts to inhibit seed germination and early seedling development (Guo et al. 2008). ABA production is triggered under drought stress, which subsequently induces stress-responsive gene expression (Abe et al. 2003). MdSIMYB1 expression was induced by dehydration, salt and cold stress, as well as stress-related hormones, suggesting that MdSIMYB1 may be part of the plant response to these abiotic stresses in relation to the signaling pathways of those hormones.

It is possible that different ABA signaling pathways are involved in seed germination and stress tolerance (Dai et al. 2007). In some cases of transgene investigation, seed germination and early seedling development are sensitive to exogenous ABA, despite of enhanced stress tolerance (Hu et al. 2006, Ko et al. 2006). For example, SNAC1-overexpressing rice seedlings were significantly sensitive to ABA treatment but they had improved tolerance to drought and salt (Hu et al. 2006). AtMYC2 and AtMYB2 transgenic Arabidopsis exhibited an ABA-sensitive phenotype, although they increased tolerance to osmotic stress (Abe et al. 2003). In some other cases, an enhanced tolerance to abiotic stress accompanied a decreased sensitivity to ABA for seed germination. For example, OsMYB3R-2 transgenic plants had enhanced tolerance to multiple stresses and decreased sensitivity of seed germination to ABA (Dai et al. 2007). The enhanced tolerance to stresses and decreased ABA sensitivity of seed germination also exist for other genes such as AtHD2C, CaXTH3 and AtTPS1 (Dai et al. 2007). In this study, the seed germination of MdSIMYB1 transgenic tobacco became more insensitive to ABA and NaCl treatments than the control.

In addition to germination insensitivity, the ectopic expression of MdSIMYB1 increased the expression of NtDREB1A, NtERD10B and NtERD10C, which are stress-responsive genes in tobacco (Park et al. 2001, Shukla et al. 2006). The overexpression of genes such as CBF/DREB1 and DREB1A confers stress tolerance to transgenic plants (Kasuga et al. 2004, Ito et al. 2006). The high transcript levels of NtDREB1A, NtERD10B and NtERD10C in transgenic tobacco plants indicate that MdSIMYB1 conferred tolerance to abiotic stresses at least partially, if not all, by upregulating stress-responsive genes such as NtDREB1A, NtERD10B and NtERD10C.

Auxin plays a crucial role in the root initiation and growth of higher plants (Tripathi et al. 2009). Lateral root formation and root structure adaptation are related to biotic and abiotic stress (Peterson 1992). Many genes are at least partially involved in stress tolerance by controlling auxin transport or response to promote root formation and growth. For example, the overexpression of vacuolar H+-pyrophosphatase gene AVP1 in Arabidopsis and tomato increased tolerance to soil water deficits by regulating auxin transport and thereby affected auxin-dependent root growth (Park et al. 2005). A T-DNA insertion into the Arabidopsis CIPK6 gene caused a reduction in gene expression, the emergence of lateral roots and sensitivity to salt stress (Tripathi et al. 2009). In this study, the expression of MdSIMYB1 was positively induced by IAA treatment, suggesting that it could be involved in the auxin response. The ectopic expression of MdSIMYB1 enhanced the transcript level of auxin signaling genes such as NtIAA4.2, NtIAA4.1 and NtIAA2.5, indicating that MdSIMYB1 most likely promotes root growth by upregulating the expression of auxin signaling genes, and therefore maintains a robust root system to enhance tolerance under multiple abiotic stresses.

Root hairs initiate from root surface to facilitate nutrient and water uptake, thereby being essential for plant growth (Gilroy and Jones 2000). The identification of the hair and non-hair cell fate in Arabidopsis root epidermis has been studied extensively. The bHLH TFs GL3 and EGL3 act together with WER to promote the non-hair cell fate, however, they interact with CPC to block the non-hair pathway and lead to a hair cell fate (Bernhardt et al. 2005). Interestingly, it was that MdSIMYB1 protein interacts with AtGL3 which participates in hair and non-hair formation in the root epidermis in Arabidopsis (Fig. S1, Bernhardt et al. 2005). This finding suggests that MdSIMYB1 may regulate cell fate in the epidermis of the root to influence its growth and development by interacting with GL3-like plant proteins. In conclusion, besides direct or indirect regulation of stress-responsive gene expression, MdSIMYB1 is also involved in stress tolerance by promoting and maintaining root growth under stress conditions by regulating auxin-responsive genes and perhaps by interacting with GL3-like proteins.

Taken together, the overexpression of a novel apple R2R3 MYB gene MdSIMYB1 enhanced the tolerance to salt, drought and cold stresses in transgenic tobacco and apples. Therefore, MdSIMYB1 can be used as a target gene for genetic manipulation to improve multiple abiotic stress tolerance to fruit trees and other crops.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
ppl12069-sup-0001-TableS1.docWord document28KTable S1. Primers for gene cloning and vector construction.
ppl12069-sup-0002-TableS2.docWord document30KTable S2. Primers for expression analysis with semi-quantitative RT-PCRs.
ppl12069-sup-0003-TableS3.docWord document24KTable S3. Primers for subcellular localization.
ppl12069-sup-0004-TableS4.docWord document25KTable S4. Primers for yeast two-hybrid assays.
ppl12069-sup-0005-TableS5.docWord document25KTable S5. Primers for BiFC assay.
ppl12069-sup-0006-FigureS1.tifTIFF image5808KFig. S1. Interaction between AtGL3 with MdSIMYB1.
ppl12069-sup-0007-FigureS2.tifTIFF image8299KFig. S2. Rooted apple plantlets of three overexpression lines (T1, T2 and T12) and one suppression line RT2, with the WT as control.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.