ISSN : 2287-5174(Online)
Overexpression of OsMLD Encoding MYB-like DNA Binding Domain Increases Tolerance to Salt Stress in Rice (Oryza sativa L.)
Rice is one of the most economically important food crops in many developing and developed countries. Cultivation of this crop has been posed by many environ-mental factors including drought and salinity that caused greatest economic losses. Developing rice plants that can withstand against these stresses are therefore important. With the era of functional genomics, several transcription factor genes have been reported that have tolerance against these stresses. Among them are the MYB transcription factors which were first identified as oncogenes in animals, where their function was linked to control the cell cycle (Ito et al., 2001). MYB proteins are divided into different classes depending on the number of adjacent repeats (R1, R2, R3, and R4) (Dubos et al., 2010). The Oryza sativa MYB-like domain (OsMLD) belonged to the third hetero-geneous class comprised of a single or a partial MYB repeat which evolved from R2R3-MYB genes and was involved in the control of cellular morphogenesis (Pesch and Hulskamp, 2009), and in secondary metabolism (Matsui et al., 2008). Moreover, comparative phylogenetic studies have identified new R2R3-MYB subgroups in other plant species for which there were no representatives in A. thaliana suggesting that these proteins might have specialized roles but were either lost in A. thaliana or were acquired after a divergence from the last common ancestor (Wilkins et al., 2009).
Numerous MYB transcription factors studies have been reported to involve in various developmental and physiological processes, especially in abiotic and biotic stress responses (Dubos et al., 2010). Indeed, overexpression of OsMYB3R-2 increases tolerance to freezing, drought, and salt stresses in transgenic Arabidopsis (Dai et al., 2007). The enhanced tolerance to these stresses was probably due to the in-creased accumulation of proline in the overexpression lines (Ma et al., 2009). Moreover, overexpression of OsMYB4 has been shown to increase the chilling and freezing tolerance of Arabidopsis (Vannini et al., 2004). Furthermore, OsMYB2 led to greater accumulation of compatible osmolytes, such as soluble sugars, free proline, and late embryogenesis abundance (LEA) proteins with suppression in the accu-mulation of malondialdehyde (MDA) and H2O2 under con-ditions of salt stress in rice (Yang et al., 2012). Moreover, in Arabidopsis, AtMYB2 functions in the ABA-mediated drought-stress response (Abe et al., 2003) while AtMYB44 and AtMYB96 increased drought and salinity tolerance (Jung et al., 2008; Seo et al., 2009).
Although the functions of MYB proteins have been in-vestigated in numerous plant species like Arabidopsis, maize, rice (Oryza sativa), petunia (Petunia hybrida), snapdragon (Antirrhinum majus), grapevine (Vitis vinifera L.), poplar (Populus tremuloides) and apple (Malus domestica), using both genetic and molecular analyses (Dubos et al., 2010), little studies are known about the function of MYB- like protein. In this study, we characterized the OsMLD gene at the molecular level and the enhanced tolerance of CaMV35S::OsMLD rice lines to salinity. Moreover, we presented the possible involvement of proline accumulation during salinity stress and the up-regulation of OsMLD mRNA expression during salinity, abscissic acid (ABA), hydrogen peroxide (H2O2) and drought stresses.
MATERIAL AND METHOD
Plant materials and growth
The rice variety, Dongjin, was used as a wild type in the generation of transgenic rice. Regenerated plants in root formation (RF) medium were transplanted in soil with 50% composed and 50% earth soil and acclimatized for two weeks in the greenhouse. Young leave samples were collected for genomic DNA analysis with OsMLD specific primers. Subsequently, confirmed plants with gene insert were harvested and the T1 seeds were used. Transgenic rice together with the wild type Dongjin were sown in a seedling-grown tray and placed in the greenhouse for three weeks. Grown seedlings were transplanted in the experi-mental farm spaced at 30×15 cm with one seedling per hill arranged in randomized complete block design replicated 3 times. The fertilizer N-P2O5-K2O were applied at the rate of 90-45-47 kg/ha. The other cultivation and management were per-formed according to the rice cultivation standards of the experimental farm of Chungbuk National University (Sun et al., 2011). Yield and yield components were collected and other morpho-agronomic data.
For abiotic stress experiments, T1 seeds were prepared and soaked for 2 days and grown in a plastic pot and regularly supplied with Murashige and Skoog composition of nutrient solution. The temperature in the glasshouse was maintained at 30～32℃/ 22～24℃ day/night. The experi-ments were laid out in randomized complete block design with three replications.
Vector construction and plant transformation
The full-length cDNA of OsMLD (Accession No. NM_ 001056541) was amplified from rice with the primers 5’- CCGCTCGAGATGTCATCCTCCTTGCCTATT-3’ (XhoI site underlined) and 5’-CGGGGTACCTCAAGATTCATG CACTCTACG-3’ (KpnI site underlined). The product was ligated into the pART vector (Gleave, 1992). The recom-binant vector carrying OsMLD was constructed under the control of CaMV35S promoter and OCS terminator as shown in Fig.
1. The pART-OsMLD construct was electro-porated into Agrobacterium tumefaciens LBA4404, and then introduced into pre-soaked rice seed using the method of Lee et al. (2011) with some modifications.
Fig. 1 Schematic diagram of the binary Ti plasmid pART containing the OsMLD full-length cDNA from rice.
DNA extraction and PCR analysis
Genomic DNA was extracted as described by Cho et al. (2007) with some modifications. The relative purity and concentration of extracted DNA was estimated using NanoDrop- 1000 spectrophotometer (NanoDrop Technologies, Inc. USA). PCR analysis was performed using NPT-Fw (TGAATGA ACTGCAGGACGAG) and NPT-Rv (AATATCACGGGT AGCCAACG) primers to check the introgression of neomycin phosphotransferase gene (npt) and MLD-Fw (ATGTCAT CCTCCTTGCCTATT) and MLD-Rv (TCAAGATTCATG CACTCTACG-3) to check the introgression of the full- length cDNA of OsMLD gene in T0 and T1 plants.
Treatment of transgenic rice for abiotic stress tolerance
Transgenic rice lines overexpressing OsMLD gene (CaMV 35S::OsMLD) were advanced to T2 generation by PCR screening. Previously grown two-week-old seedlings of both wild-type and OsMLD transgenic rice were submerged into 1/2 MS medium supplemented with 250 mM NaCl, 20% PEG6000, 100 μM ABA, and 3% H2O2 placed in the greenhouse. Leaf samples were collected at 0, 0.5, 1, 3, 6, 12, 24, and 36 hr for molecular and biochemical analyses.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the leaf of each CaMV 35S::OsMLD rice lines and wild type Dongjin plant using the RNeasy Plant Mini Kit (QIAGEN, Maryland, USA). This process was repeated after abiotic stress treatments. The relative purity and concentration of extracted RNA was estimated using NanoDrop-1000 spectrophotometer, and stored at -80℃ freezer. Total RNAs were cleaned using DNaseI, and the first-strand cDNA synthesis was performed by reverse transcription of mRNA using Oligo (dT)20 primer and SuperScriptTMⅢ Reverse Transcriptase. The specific sequences of the primer pairs used in a semiquantitative reverse transcription PCR (RT-PCR) are MLD-rt-Fw (5’- TGGCGATATTTGTCCTGTCA-3’) and MLD-rt-Rv (5’- TCGCTTTCAGGTCCAAAGAC-3’). Actin primers were used as a loading control and also used as an internal control for normalization of quantitative RT-PCR reaction.
For the real-time PCR, the cDNA samples were diluted to 10 ng/μl. Triplicate quantitative assays were performed using 5 μl of each cDNA dilution with the SYBR Green Master mix and the CFX connect real-time system (Bio- rad, USA). The relative quantification method (Delta-Delta CT) was used to evaluate quantitative variation between the replicates. The amplification of actin as an internal control to normalize all data was used.
Measurements of lipid peroxidation, free-proline and chlorophyll contents
Lipid peroxidation was measured as the amount of TBARS determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). Fresh control and treated leaves (0.1 g) were homogenized in 1 ml of 20% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 13,000 rpm for 20 min. For every 0.5 ml of the aliquot of the supernatant, 2 ml of 20% TCA con-taining 0.5% (w/v) TBA was added. The mixture was heated at 95℃ for 30 min and then quickly cooled on ice. The contents were centrifuged at 13,000 rpm for 15 min. and the absorbance was measured at 532 nm. Value for non-specific absorption at 600 nm was subtracted. The con-centration of TBARS was calculated using an extinction coefficient of 155 mM-1 cm-1.
Free-proline content in leaves was determined by adopting the methods of Troll and Lindsley (1955) with modifi-cations. About 100 mg of leaves were homogenized in 1 mL of 3% sulfosalicylic acid, and the homogeneous mix-ture was centrifuged at 13,000 rpm for 15 min 4℃. The extract (200 ml) was transferred to a new microcentrifuge tube with 200 ml acid ninhydrin, (0.1 g ninhydrin dissolved in 2.4 ml glacial acetic acid and 1.6 ml 6-mortho-phosphoric acid), and 200 ml acetic acid. The reaction mixture was boiled in a water bath at 100℃ for 30 min and cooled down at 4℃ for 30 min. Then 400 ml of toluene was added to the leaf extract and thoroughly mixed. Finally, 120 ml of the toluene phase was removed for absorbance measurement at 520 nm using spectrophotometer Optizen Series (Model 2120UV).
Chlorophyll in leaves was extracted using the acetone method as described by Lichtenthaler (1987). The absorbance was read at 644.8 and 661.6 nm with a spectrophotometer. The total contents of chlorophyll a and b were calculated using the extinction coefficients and the equations.
All the inserted sequences were checked using the BLAST program in NCBI sequence database. The open reading frame (ORF) and conserved domain were generated by the NCBI BLASTN program (http://www.ncbi.nlm.nih.gov/). Sequence alignment, ORF translation, molecular weight calculation of the predicted proteins and a structural analysis of the deduced proteins were carried out by DNAStar’s Lasergene sequence analysis software (www.dnastar.com).
Data requiring statistical analysis were computed using the Statistix version 8 (www.statistix.com). Significant P value were further analyzed using the two-sided Dunnett’s multiple comparisons with the wild type Dongjin as check using the same software.
Characterization of OsMLD gene
Analysis of the Oryza sativa MYB-like domain gene (OsMLD, NM_001056541) showed an open reading frame (ORF) of 1,287 bp which encodes a polypeptide of 428 amino acid residues with a calculated mass of 46.7 kDa. BLASTX analysis of the deduced amino acid sequences showed an Myb_CC_LHEQLE super family located in the C-terminal region (Fig. 2A) which has been reported to involved in phosphate starvation signaling both in vascular plants and in unicellular algae (Rubio et al., 2001). OsMLD showed 96% amino acid identity to wheat (Accession No, JF951950), 92% to Hordeum vulgare (Accession No. GQ 337895), 91.5% to Arabidopsis thaliana (Accession No. NM 001202877) and 75.8% in Zea mays (Accession No. JF831533). Phylogenetic tree analysis using the deduced amino acid of OsMLD showed close divergence to wheat (Triticum aestivum) and Hordeum vulgare (Fig. 2B).
Generation and gene expression of OsMLD transgenic rice
A total of 23 transgenic plants in T0 generation were analyzed by PCR. We confirmed the presence of npt gene in all transgenic plants, however only 13 plants were con-firmed for the presence of OsMLD gene (Fig.3A). mRNA transcript analysis of CaMV35S::OsMLD plants showed an enhanced expression of OsMLD gene compared to wild type, Dongjin (Fig. 3B). Though transgenic plants showed overexpression, variability in the degree of expression were observed. Along with this, we selected four uniform lines with strong expression and named it as OsMLD-OX1 to 4. These selected lines were used in all the succeeding experiments.
Gene expression analysis in transgenic rice under abiotic stresses
To characterize the expression patterns of OsMLD trans-genic rice plant at the molecular level, we quantitatively measured the expression of mRNA transcript on plants stressed at 250 mM NaCl, 100 μM ABA, 20% PEG6000, and 3% H2O2. Using 2-week-old seedlings, the mRNA transcript of OsMLD gene under salinity stress was signi-ficantly elevated with several fold times higher compared to the wild type after 10 days of exposure (Fig. 4). The increased in the mRNA transcript was visible starting at 0.5 hr and thereafter increased (Fig. 5A). For the other stresses, the onset of increased in mRNA expression was similar to the salinity stress. However, the peak expression was observed after 3 hr in ABA and drought stresses (Fig. 5B, 5D). Peak mRNA transcript expression of hydrogen peroxide was observed after 24 hr of stress (Fig. 5C).
Physiological response of transgenic plants under salt stress
To understand the physiological response of OsMLD gene, we measured the effects of salinity stress on the accumulation of malondialdehyde (MDA), a lipid peroxidation, in wild-type and transgenic rice. There were marked in-creased in the accumulation of MDA both in wild type and transgenic plants upon exposure to 250 mM sodium chloride. The increased, however, was significantly lesser (P0.05=*) in the OsMLD transgenic plants than those in the wild-type plants. OsMLD-OX2 showed the lowest accumulation while the OsMLD-OX4 was the highest (Fig. 6A).
In parallel to the MDA accumulation, we further measured the accumulation of proline, an osmolyte involved in various abiotic stress tolerance in plants. Except OsMLD-OX1, all transgenic lines showed significant increase in the proline accumulation compared to the wild type.
Highest accu-mulation of proline was observed in OsMLD-OX2 line (Fig. 6B). The decreased in the MDA along with the increased in the proline during salinity stress indicated that OsMLD may be involved in the salinity stress. Interestingly, even with the CaMV35S promoter, no significant differences in the MDA and proline accumulation under normal condition (Fig. 6A and 6B). Moreover, the variability in the MDA and proline accumulation in the transgenic plants may be due to the OsMLD gene expression level.
Under normal growing condition, no differences in the chlorophyll contents of transgenic and wild type plants were observed (data not shown). However, under salinity condition, a decreased in chlorophyll contents were observed as shown in Fig. 6C. The phenotypic expression of OsMLD lines is presented in Fig. 6D where the wild type had clearly shown the dryness of the leaves with more than 1/2 tip burned after 10 days of salinity exposure compared to the transgenic plants. The delay in the tip-burn on the transgenic rice during salinity stress is an indicative that the OsMLD indeed involved in the salinity tolerance along with the other osmolytes.
To determine the phenotypic and agronomic traits of OsMLD gene in the transgenic rice, the yield and yield components were evaluated in the field. No significant changes in the plant height and culm length were observed in all transgenic lines compared to the wild type (Fig. 7). Conversely, panicle length and ripened grain were all in-creased in the transgenic lines. In other traits such as yield, number of tillers, and 1,000 grain weight, variable mea-surements were observed, implying that the variations may be due to the different mRNA expression levels. Less number of tillers was counted in OsMLD-OX1 while the yield was as high as OsMLD-OX4.
Fig. 2 Nucleotide and deduced amino acid sequence of the fulllength cDNA of OsMLD gene. (A) The full-length cDNA of OsMLD was 1,287 bp encoding a polypeptide of 428 amino acids (46.7 kDa). The Myb-CC type transcription factor is underlined (solid line) and the binding domain (dash line). (B) Phylogenetic tree constructed by neighborjoining algorithms. Accession numbers for the other MYB proteins are as follows: Triticum aestivum MYB-related protein (JF951950); Ricinus communis transcription factor (XM 002526513); Lupinus albus LaMYB- CC5 mRNA for MYB-CC transcription factor (AB573724); Arabidopsis thaliana myb family transcription factor (NM 001202877); Zea mays isolate A phosphate starvation protein (PHR1) (JF831533); Hordeum vulgare phosphate starvation regulator protein-like protein (GQ337895); Nicotiana tabacum WERBP-1 (AB017693)
Fig. 3 Genomic and expression analysis of OsMLD overexpressing (OsMLD-OX) plants and Dongjin (wild type). (A) PCR confirmation of transgenes in T0 transgenic plants using MLD and nptII primers in 1% agarose gel. (B) Expression of OsMLD genes in regenerated T1 transgenic rice plants using young leaf. Upper panel shows expression level of introduced OsMLD and lower panel shows internal actin used for loading adjustment. WT, wild type; 1-23, independent ovexpression lines.
Fig. 4 mRNA expression of wild type (Dongjin) and OsMLDOX plants in 250 mM NaCl after 48 hr. Actin was used as an internal control. Data are mean±SD of three replicates.
Fig. 5 Expression of OsMLD gene in transgenic rice plants, Dongjin under abiotic stresses using the leaves. (A) 250 mM NaCl, (B) 100 μM ABA, (C) 3% H2O2, and (D) 20% PEG6000 at different time interval of (0, 0.5, 1, 3, 6, 12, 24, 36 hr). The transcript levels were measured by real-time reverse-transcriptase PCR (Donjin was used as reference). Actin was used as an internal control. Data are mean±SD of three replicates.
Fig. 6 The responses of two-week-old transgenic rice seedlings after 10 days of stress on 250 mM sodium chloride. The accumulations of (A) malondialdehyde, (B) proline, (C)chlorophyll contents, and (D) the phenotypic expression.
Transcription factors (TFs) are important regulators of gene expression that are composed of at least four discrete domains: DNA binding domain, nuclear localization signal (NLS), transcription activation domain, and oligomerization site, which operate together to regulate many physiological and biochemical processes by modulating the rate of tran-scription initiation of target genes (Du et al., 2009; Park et al., 2011). In this study, the OsMLD gene belongs to the DNA binding domain which is the most abundant classes of TFs in plants, and its subfamily plants containing the two-repeat R2R3 (Stracke et al., 2001). The C-terminus of the Myb-CC types is LHEQLE similar to the R2R3 tran-scription regulation (Du et al., 2009) but the binding domain SHLQKYR is unique from the 83 TFs of Arabidopsis which has been to recognize the MBSIIG binding site (Romero et al., 1998) (Fig. 2A). The uniqueness in the binding domain of the OsMLD gene may suggest a new specific role in plant regulation although detailed studies on this should be undertaken.
Numerous R2R3-MYB proteins have been characterized by genetic approaches and found to be involved in the control of plant-specific processes including responses to biotic and abiotic stresses (Dubos et al., 2010). Along with this, our CaMV35S::OsMLD rice lines showed enhanced tolerance to salinity with increasing mRNA transcripts over time (Fig. 4) even at 250 mM. Similar results were also obtained by Jung et al. (2008) when AtMYB44 from Arabidopsis treated with 250 mM under the same promoter. Recently, MYB32 gene increased expression level at 200 mM NaCl in wheat (Zhang et al., 2011). It appears that several MYB genes may be involved in the salinity tolerance even at high concentration. Moreover, when transgenic rice was subjected to abscisic acid (ABA), hydrogen peroxide (H2O2) and polyglycolethylene (PEG) stresses, it showed increasing mRNA transcripts (Fig. 5). These results have been reported in Arabidopsis where R2R3-MYB transcription factor AtMYB2 is transiently induced by dehydration. Further analyses indicate that AtMYB2 functions as transcriptional activator in ABA (abscisic acid)-inducible gene expression during drought (Abe et al., 2003). Moreover, AtMYB68 is modulated by temperature, and loss of AtMYB68 reduces the ability of MYB68 plants to compensate their growth at higher temperatures (Feng et al., 2004). Further studies indicate that the R2R3-MYB genes are involved not only in the signal transduction pathways of drought, low-tempera-ture, and light but also in the signal transduction pathways of nutritional deficiency (Miyaki et al., 2003; Hernadez at al., 2007). The same MYB gene may activate expression of different functional genes to response to different environ-mental factors (Vannini et al., 2004; Maeda et al., 2005). Its mechanism may be that the different functional genes have the same cis-elements in their promoters, which could be bound by the same MYB protein. Hence, over-expression of some MYB TFs could produce transgenic plants carrying resistance to multiple environmental stresses. In addition to this mechanism, we observed an increasing accumulation of proline with decreasing MDA in the OsMLD transgenic plants (Fig. 6). Proline has been reported to act as scavenger of ROS and other toxic substance like MDA. These metabolic changes due to overexpression of OsMLD may induce plants for effective osmoregulation and reduces oxidative damages under salt stress, thus con-ferring tolerance to salt stress. Similar results were also found in transgenic plants with overexpressing OsMYB4 and OsMYB3R-2 under cold stress condition (Pasquali et al., 2008; Ma et al., 2009). Moreover, TaMYB33 over-expression lines have higher abundance of AtP5CS (proline biosynthesis gene) transcripts than the wild type (Qin et al., 2012). The same mechanisms perhaps apply to elevated proline content and less MDA accumulation among the OsMLD-OX plants that resulted to enhanced tolerance to salinity.
Based on these studies, the results are evident about the important roles of MYB transcription factors such as OsMLD in response to abiotic stresses in plants. The accumulation of free proline along with the suppression in the accu-mulation of an oxidative enzyme like MDA under con-ditions of salt stress may help in the development of transgenic plants with increase tolerance to numerous unfavorable environmental factors.
Fig. 7 Agronomic traits of OsMLD-OX rice plants grown in the field under normal condition. (A) Spider plots showing the agronomic characteristics. Data in each transgenic line were pooled from the three replications. (B) Phenotype of OsMLD-OX plants and wild type Dongjin.
This work was supported by the research grant of the Chungbuk National University in 2010.
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