A MYB-related transcription factor from sheepgrass, LcMYB2, promotes seed germination and root growth under drought stress

Background Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. Results In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. Conclusions Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.


Results
LcMYB2 expression pattern analysis Based on 454 high throughput sequencing and expression profile analyses of sheepgrass under drought stress, we found 15 MYB and MYB-related transcription factors that were responsive to water changes [32,35]. Contig41859, which was up-regulated by drought stress and named LcMYB2, was a MYB-related transcription factor with unknown function that attracted our attention (Additional file 1: Table S1).
LcMYB2 is highly induced by 300mM mannitol at the 8 th hour after treatment (Fig. 1a), whereas it is relatively slower responding to salt and cold stress (24 h; Fig. 1b,c).
However, it is quickly upregulated by ABA treatment (Fig. 1d). The maximal level of mRNA accumulation under mannitol treatment is higher than under salt, cold and ABA treatments, indicating that LcMYB2 mainly functions in response to osmotic stress.
Furthermore, the expression level of LcMYB2 in different organs is also detected under normal growth conditions. The results show that LcMYB2 has the highest transcript level in roots (Fig. 1e). Based on these combined results, we predict that LcMYB2 is mainly responsible for the osmotic stress response in roots, which may benefit plants under drought stress.

Isolation and sequence analysis of the LcMYB2
Putative full-length LcMYB2 was isolated from sheepgrass by Rapid-Amplification of cDNA  Fig. 2b, c), which is a predicted protein. Therefore, if LcMYB2 is involved in seed germination and root growth under drought stress, it will be a good candidate for the genetic improvement of sheepgrass, barley and wheat to promote drought resistance.

Subcellular localization and transcription activity assay of LcMYB2
To determine the subcellular localization of LcMYB2, the ORF of LcMYB2 (without the TGA stop codon) was fused to a GFP reporter gene under the control of the CaMV 35S promoter (Fig. 3a). Recombinant CaMV35S::LcMYB2-GFP and CaMV35S::GFP were transformed into Arabidopsis separately by the floral dip method. Confocal microscopy showed that the GFP protein was localized throughout the whole cell, whereas the LcMYB2-GFP fusion protein was present only in the nucleus (Fig. 3b), suggesting that LcMYB2 is a nuclear-localized protein.
The transcriptional activation of LcMYB2 was tested using a yeast one-hybrid assay system. The LcMYB2 ORF was inserted at the 3'-end of GAL-BD under the control of P ADH1 to form a BD-LcMYB2 fusion gene (Fig. 3c). The yeast strain AH109, harboring BD-LcMYB2 or BD-WRKY15 (positive controls), grew normally on SD/-His-Trp medium, whereas AH109 harboring only BD (negative control) did not grow. In β-galactosidase activity assays on Whatman filter paper, blue signal appeared in the regions where BD-LcMYB2 or BD-WRKY15-containing yeast were growing (Fig. 3d). Therefore, we suggest that LcMYB2 serves as a transcription activator and functions in the nucleus. were no significant differences between transgenic and wild-type seeds in germination rate, cotyledon greening rate or root length (Fig. 4a,d,g,c,f,i; Fig. 5a,d,g,h,i). Under treatment with 300 mmol/L mannitol, there were significant differences in germination rate (p < 0.01; Fig. 4b,c), and the cotyledon greening rate and root length had very significant differences (p < 0.001) between transgenic and wild-type seeds (Fig. 4e,f,h,i).
Under treatment with 0.25 µmol/L ABA, the germination rate (p < 0.01), cotyledon greening rate (p < 0.001) and root length (p < 0.001) were significantly different between transgenic and wild-type seeds, and similar results were obtained with 0.5 µmol/L ABA treatment (Fig. 5b,c,e,f,g,h,i). Taken together, these data indicate that LcMYB2 can promote seed germination and root growth under osmotic stress and possibly via the ABA signaling pathway. In addition, the transgenic plants maintained green leaves longer under natural drought stress conditions and had a higher refresh rate after rewatering than did wild-type (Fig. 6).
To investigate the physiological responses of transgenic and wild-type A. thaliana under osmotic stress, we irrigated 4-week-old seedlings with 300 mmol/L mannitol. Two days later, the malondialdehyde (MDA), Superoxide dismutase (SOD), soluble sugars and proline contents were measured. The results showed that the two transgenic lines overexpressing LcMYB2 accumulated greater amounts of SOD (p < 0.01), soluble sugars (p < 0.05/0.01) and proline (p < 0.001) than wild-type lines under 300 mmol/L mannitol treatment, whereas they had lower MDA content ( Fig. 7a

CHIP analysis
It has been shown that MYB proteins can recognize the motifs A/TAACCA and C/TAACG/TG [43]. Therefore, we analyzed the promoter sequences of AtLEA14, AtP5CS1, AtDREB2A and LcDREB2 using the sequences ~1500-2000 bp upstream of the predicted transcription start sites (TSSs), and several possible motifs were found in the putative promoter regions (Additional file 3: Table S2). We further confirmed our prediction with CHIP experiments, and the results of qPCR and universal PCR showed that the LcMYB2 does bind the DNA sequences of the promoters of these genes (Fig. 8).

Discussion
MYB and MYB-related transcription factors constitute a large family in plants and are involved in many biological processes, for example, secondary metabolism and responses to environmental factors [44][45][46]. Isolating and characterizing the functions of these genes provide a way to learn about plant-specific events at the transcriptional level. AtMYB2, a MYB-related protein, is induced by drought and by ABA; whereas BcMYB1 is rapidly and strongly induced by drought but only slightly by exogenous ABA, indicating that MYB proteins respond to environmental changes through both ABA-dependent and ABAindependent pathways [47][48][49]. LcMYB2 is induced to its maximal level at one hour after treatment with exogenous ABA and eight hours after treatment with exogenous mannitol (Fig. 1a, d). However, the extent of LcMYB2 induction by mannitol is greater than by ABA, suggesting that LcMYB2 functions mainly in an ABA-independent pathway.
When facing drought stress, plants always adopt avoidance or resistance strategies to mitigate the negative effects of the stress. Rooting deeply is one of the avoidance strategies. For example, the introduction of the DEEPER ROOTING 1 (DRO1) gene into a shallow-rooting rice cultivar increased the downward growth of roots, and the resulting transgenic lines had higher yields under drought conditions [3]. Our results demonstrate that LcMYB2 is induced by mannitol and promotes root elongation at the germination stage and during seedling growth under osmotic stress and ABA treatment ( Fig. 4 and Fig.   5). Therefore, LcMYB2 has the potential to enhance plant root growth to avoid drought stress.
Osmotic adjustment is usually thought to be one of the main mechanisms of resistance to drought or salt stress. Compatible osmolytes, such as proline, soluble sugars and LEA proteins, are often measured as critical physiological criteria to evaluate the tolerance of plants to abiotic stresses. Proline biosynthesis is catalyzed by P5CS1/2 and P5CR, and it is thought to protect subcellular structures and macromolecules under osmotic stress [50,51]. Petunias overexpressing AtP5CS or OsP5CS accumulate more proline and appear to have drought tolerance [52]. In addition, soluble sugars, especially sucrose or trehalose, are correlated with the acquisition of desiccation tolerance and are thought to stabilize the membrane structure in dry environments [53,54]. LEA proteins, first found in cotton, were shown to be up-regulated by drought stress in many species and function as compatible solutes to maintain the cellular structure in severe dehydration conditions [54][55][56]. Overexpression of some LEA protein-encoding genes confers enhanced drought tolerance in transgenic plants [57,58]. Here, we found that LcMYB2 was induced by osmotic stress in sheepgrass (300mMmannitol); and A. thaliana overexpressing LcMYB2 accumulated more soluble sugars and free proline and expressed higher levels of AtLEA14, AtP5CS1 and AtDREB2A than wild-type A. thaliana seedlings under mannitol treatment ( Fig. 7). In sheepgrass, many LEA protein-encoding genes and two P5CS-encoding genes were induced significantly by drought stress 35). Here, we demonstrate that LcMYB2 can bind to the promoter regions of AtLEA14 and AtP5CS1 (Fig. 8a, b, e, f). Therefore, we suggest that LcMYB2 functions in sheepgrass by elevating the content of osmoprotectants.
DREB proteins have been extensively studied and have been shown to improve drought tolerance in transgenic plants. AtDREB2A and AtDREB2B are strongly induced by dehydration stress in roots and stems, and constitutive expression of AtDREB2A results in significant drought stress tolerance [21,59]. In addition, OsDREB2B is markedly induced by various stresses, and overexpressing OsDREB2B in A. thaliana or rice increases the expression of DREB2A target genes and improves transgenic plant drought stress tolerance [60,61]. Previous studies have proposed that DREB proteins, such as DREB1 and DREB2, regulate low-temperature and drought-responsive genes by binding to the DRE/CTR elements through ABA-independent pathway [62,63].
In sheepgrass, the highest transcript level of LcDREB2a occurs at the 12 th hour under 20% PEG6000 treatment [42], whereas LcMYB2 transcript accumulation reaches the highest point at 8 hours after 300Mm mannitol treatment (Fig. 1a). Expression profile sequence analysis showed that both contig62249 (LcDREB2C/LcDREB2B/LcDREB2A) and contig41859 (LcMYB2) were up-regulated by drought stress and returned to basal levels after rewatering; however, the fold change of contig41859 was larger than that of contig62249 in response to drought stress (Additional file 1: Table S1). These results indicate that LcMYB2 is a possible transcription regulator upstream of LcDREB2. Therefore, we cloned the promoter sequence of LcDREB2 (~1500 bp upstream of the predicted transcription start site, Additional file 2)and assayed the binding of LcMYB2 protein to this promoter region using CHIP analysis. Universal PCR and qPCR enrichment of CHIP DNA revealed that LcMYB2 can bind to the promoter regions of both AtDREB2A and LcDREB2 (Fig. 8c, d, g,h). Therefore, we propose that LcMYB2 improves drought tolerance by activating LcDREB2 in sheepgrass. Also, we find that LcMYB2 can bind to the promoters of sheepgrass LcDREB2 and Arabidopsis AtDREB2A, maybe they all have MYB binding elements in their promoters, which shows that the mechanism of response to stress in plants is conservative.
With the development of the sheepgrass industry (artificial cultivation, natural grassland improvement, and artificial grassland establishment), the drought resistance of sheepgrass during the seed germination and seedling establishment stages in waterdeficient areas is critical for propagation and is necessary for reaping economic and ecological benefits. Therefore, LcMYB2 is an important candidate for improving plant drought stress tolerance through genetic engineering.

Conclusion
In conclusion, we showed that a drought and osmotic-inducible transcription factor, LcMYB2, improves the drought and osmotic tolerance of plants by bingding the elements of promoters from the AtDREB2, AtLEA14, AtP5CS1 and LcDREB2 and regulating the transcription of drought-responsive genes to increase the accumulation of osmoprotectants (Fig. 9). These may be among the reasons underlying the tolerance of sheepgrass to drought-prone environments. Others, some the cis-elements of stress response genes from the monocotyledons and dicotyledonous plants are conserved in evolution, they both can be bound by the same trans-factors.

Plant materials and treatments
Sheepgrass (National certified variety Zhongke 1 from Institute of Botany, the Chinese Academy of Sciences, Beijing, China) was used for this experiment. The seedlings were grown in the greenhouse at 27/23 ℃, 16 h light/8 h dark for 8 weeks before treatments.
Abiotic stresses were performed as follows: the seedlings were irrigated with 300mM mannitol for osmotic stress, 400 mmol/L NaCl for salt stress and 100 µmol/L for ABA treatment. Seedlings were transferred to a growth chamber at 4℃ for cold stress. The seedlings were sampled at 0, 1, 3, 8, 12 or 24 h after stress treatments, immediately frozen in liquid nitrogen and stored at -80℃ for RNA isolation. Stem, leaf, root, bud, panicle and rhizomes were also collected from 2-year-old sheepgrass seedlings for tissuespecific analysis.
Arabidopsis thaliana (A. thaliana; Columbia ecotype) seeds were surface-sterilized with 10% NaClO for 10 min, and then washed 5 times with sterile water. The sterilized seeds were planted on MS solid media (pH 5.8) for germination.

Subcellular localization and transcriptional activity assay of LcMYB2
The ORF of LcMYB2 was ligated into pCAMBIA1302 to form the LcMYB2-GFP fusion protein.
The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105 using a freeze-thaw method and further transformed into A. thaliana using the floral dip method To test the role of LcMYB2 at the seedling stage under drought conditions, 4-day seedlings were transplanted and grown in a growth chamber at 22 ℃ with a 16 h / 8 h, light/dark photoperiod for 1 week with sufficient water, then started to natural drought stress without water for the following 42 days. During the drought process, the soil water content was monitored every day in each pot. On the 42 nd day, the seedlings were irrigated again, and survival rates were statistically analyzed after three days. There are 60 seedlings for drought stress experiment in each line (WT, L1, L2), respectively.
Measurement of lipid peroxidation and of proline and soluble sugar content Four-week-old transgenic and WT A. thaliana seedlings were irrigated with 300 mmol/L mannitol. The leaves were sampled at 0 h and 9 h after treatment for gene expression analysis. Two days later, the leaves of transgenic and wild-type lines were harvested for physiological measurements. The level of MDAwas determined by a revised method described by Kramer et al. [67]. Superoxide dismutase SOD activity was measured with the nitro-blue tetrazolium (NBT) reduction method as previously described [68]. The contents of proline and soluble sugars were determined according to the protocols previously described by Shan et al [69] and Bailey [70], respectively. Three replicates were carried out for each assay, and the variability was indicated with the standard error (SE).