Rice MYB transcription factor OsMYB1R1 negatively regulates drought resistance

MYB transcription factors have been demonstrated to play an important role in plant growth, development and abiotic stresses. This study isolated a rice MYB gene, OsMYB1R1(Os04g0583900), and functionally characterized its role in tolerance to drought stress by generating transgenic rice plants with overexpressing (OE) and RNA interference (RNAi) OsMYB1R1. Expression of OsMYB1R1 was down-regulated by drought stress. The tissue-specific expression analysis indicated that OsMYB1R1 was expressed at high level in panicle, but relatively low in the other parts of rice. No difference in germination rate among OsMYB1R1-OE, RNAi and wild-type (WT) seeds under mannitol treatments. No differences in phenotypes, physiological indicators and agronomic traits among WT, OE and RNAi plants were observed under normal grown conditions. Under drought stress, the RNAi plants were more tolerant to drought stress and higher survival rate after re-watering than WT plants. However, the overexpressing plants have found just the opposite. The OsMYB1R1-OE plants exhibited increased relative electrical conductivity (REC), increased malondialdehyde (MDA) content, and decreased proline content compared with the wild type, whereas lower REC and MDA content and higher proline content were found in the RNAi plants. These results suggest that OsMYB1R1 functions, a negative regulator in response to drought stresses, may be used as a candidate gene for molecular breeding of drought-tolerant crop varieties.


Introduction
As one of the most important food crops, rice (Oryza sativa) is consumed by more than 75% of the population in Asia (Fitzgerald et al. 2009). Drought is one of the major stress factors that can seriously affect the plant growth. However, rice is more sensitive to drought stress than other cereals (Huang et al. 2014). According to the statistical data from 1995 to 2007, the loss of rice production caused by drought in North China accounted for 0.13-0.43% of the total rice yield in China (Lin et al. 2013). Therefore, improving the rice resistance to drought stress has been one of the main objectives of agricultural production.
Transcription factors are key players in the regulatory networks underlying plant responses to abiotic stresses (Golldack et al. 2014). The MYB transcription factor family, which is one of the richest transcription factor families in rice, has been well characterized as important regulators in plant tolerance to abiotic stresses and development. Many studies have shown that MYB transcription factor plays an important role in many aspects of plant growth and development in rice. For example, OsMYB1 and OsMYB2P-1 were involved in the regulation of root development and architecture in rice respectively (Dai et al. 2012;Gu et al. 2017). Overexpression of OsMYB1R1-VP64 fusion protein could increase the grain yield in rice by delaying flowering time (Wang et al. 2016). OsMYB103L influences the leaf rolling and mechanical strength in rice (Yang et al. 2014), OsMYB102 delays leaf senescence (Piao et al. 2019). Furthermore, numerous studies have suggested that MYB proteins were also involved in regulating plant responses to abiotic stress. Dehydration, heat and osmotic stress could Communicated by Guosheng Xiong. 1 3 repress the expression of MYBS2. The interaction between MYBS2 and 14-3-3 protein could enhance the plant growth, stress tolerance and total grain weight per plant in rice (Chen et al. 2019). Overexpression of MYB transcription factors, such as OsMYB2 (Yang et al. 2012a, b), OsMYBR1 (Yin et al. 2017) and SiMYB56 (Xu et al. 2020), could significantly enhance the drought resistance of transgenic plants, and have no adverse effect on its normal growth. However, OsMYB91 was also induced by drought stress but transgenic plants over-expressing OsMYB91 exhibited that growth and development was retarded (Zhu et al. 2015). Overexpression of OsMYB55 improved tolerance to high temperature by regulating amino acid metabolism (El-Kereamy et al.2012); OsMYBS3 has a critical role in cold adaptation (Su et al. 2010); the rice high-affinity potassium transporter1;1 which regulated by an MYB-type transcription factor was involved in salt tolerance (Wang et al. 2015). Besides, researchers have also found out that some MYB transcription factors helped to regulate the response to various abiotic stresses. For example, Yang et al. (2012a, b) have reported that OsMYB2 played a regulatory role in determining tolerance to cold, salt, and dehydration.The ectopic overexpression of the rice OsMYB4 gene increased cold and drought tolerance (Vannini et al. 2004(Vannini et al. , 2006Mattana et al. 2005;Pasquali et al. 2008;Park et al. 2010;Baldoni et al. 2013).
OsMYB48-1 played a positive role in salt and drought stress (Xiong et al. 2014). Tang et al. (2019) demonstrated that the increased drought and salinity stress tolerance of the transgenic plants with OsMYB6 was at least partially related to lower MDA content, lower REC, higher proline content and higher CAT and SOD activities. While little is known about the function of rice transcription factor OsMYB1R1 (Os04g0583900) in drought stress response.
This study isolated the OsMYB1R1 gene from rice and tested the expression pattern of OsMYB1R1 in different tissues. The role of the OsMYB1R1 gene in tolerance to drought stress was characterized by generating transgenic rice plants with overexpressing and RNA interference OsMYB1R1 in rice. The effects of OsMYB1R1 on rice seeds germination and agronomic traits of rice were studied. This work not only provides valuable information for exploring the role of the OsMYB1R1 genes in response to abiotic stress in rice, but also provides a candidate gene in molecular breeding to increase crop drought stress resistance.

Generation of transgenic rice plants
The full-length cDNA clones of OsMYB1R1 (AK101209.1) was obtained from the National Institute of Agrobiological Sciences (NIAS, Tsukuba, Japan). To create the overexpression construct of OsMYB1R1, the complete ORFs of OsMYB1R1 was PCR amplified with the primers 5′-CGGGA TCC TCG ATT CAA GTC TGG AGA TGG-3′ (BamHI site underlined) and 5′-GCTCT AGA G CAA GCA TCT ATA GTT GCA GTGAT-3′ (Xba1 site underlined). The full-length cDNA plasmids were used as templates. Then the PCR products were cloned in pCAMBIA1301-Multi (modified from pCAMBIA1301) under the control of the CaMV35S promoter respectively. To make double-strand RNAi construct, the antisense fragment OsMYB1R1-A was PCR amplified with XhoI-KpnI (indcated by underline) linker primers 5′-ATGGA TCC GAT ATC TAC CCG CTT CGC GTCG-3′ and 5′-GGGAA TTC ATT GTT TGC CTC CCT GCT GC-3′, the sense fragment OsMYB1R1-S was PCR amplified with PstI-XbaI (indcated by underline) linker primers 5′-CGGGA TCC TGC AAA CTG TGG C AGA CAT G-3′ (BamHI sites underlined) and 5′-GCC CTC GAG CAT TTC ATC TTC GTT TCT GTT TCC -3′ (Xho1 sites underlined), using the cDNA clone AK101209.1 as the template. The schematic diagram of the recombinant plasmid OsMYB1R1-OE and OsMYB1R1-RNAi were as (Fig. 1a, b). Both of the constructs were transformed into rice (Oryza sativa ssp. japanica var. Nipponbare) by the Agrobacterium-mediated transformation method (Toki et al. 2006).

Drought and mannitol stress treatments
To test the transcription level of OsMYB1R1 in different tissues, we collected tissue samples of roots, stems, leaves, sheaths and spikes from Nipponbare rice plants which grown in the field or climate chamber as described previously (Zou et al. 2009). To check the expression level of OsMYB1R1 gene under drought stress, Nipponbare rice seeds were germinated on 1/2 MS agar for one week and then transplanted to 1/2 MS liquid medium for three weeks. The seedlings at four-leaf stage were treated For estimating the osmotic tolerance of transgenic seeds germination, T2 generation seeds of OsMYB1R1 transgenic lines and the WT were placed on 1/2 MS medium containing 0, 150 and 200 mmol/L mannitol respectively. The germination rate was scored after 7 days, the shoot heights and root length were measured at 10 days after being transferred. And different concentrations of PEG6000 treatment were employed to confirm the results of mannitol treatment. To detect the mannitol stress tolerance of transgenic seedlings, the one-week-old WT and the OsMYB1R1 transgenic seedlings were planted on 1/2 MS agar plates containing 0, 200 and 250 mmol/L mannitol respectively. After 7 days, the root length and shoot height were calculated.
To test drought tolerances of transgenic rice seedlings, T2 generation seeds of OsMYB1R1 transgenic lines were germinated in 1/2 MS medium containing 50 mg/L hygromycin; the WT was placed in normal 1/2 MS medium. Drought tolerance of rice seedlings were evaluated under two droughtstressed conditions. For seedlings vitro drought stress, the three leaf stage rice seedlings were pulled out and washed the medium. After drained of water, we moved them into a growth chamber at 28 °C and 75% relative humidity for 10 h. Then it were transplanted into 1/2 MS medium for culture and re-watered for additional 3 days. The survival rate of transgenic seedlings and the WT seedlings were scored. For seedling natural drought stress, the 1 week-old rice seedlings were grown in plastic pots filled with sandy soil. After two weeks growing in the growth chamber, the seedlings were un-watered for drought treatment. When the WT rice seedlings exhibited the lethal effects of dehydration, watering was resumed. The REC, MDA and proline contents of rice seedlings were measured at the beginning and at the end of natural drought treatment.
Drought testing at the reproductive stage was conducted in plastic pots. After one week germination on 1/2 MS medium, the seedlings were transplanted into field soil under the same growth conditions. Then we moved them into plastic pots when they reached booting stage with flag leaf just pulled out. After two days, drought stress was initiated at the booting stage by discontinuing watering of plants. When soil water contents lower than 20%, the plants were recovered with irrigation. The rate of water loss (RWL), REC, MDA and proline contents in flag leaf of rice at booting stage were measured at the beginning and at the end of drought treatment.
All above experiments were performed three replications. The phenotype of OsMYB1R1-OE, OsMYB1R1-RNAi and WT plants under different treatments was observed and photographed.

Measurements of RWL, REC, MDA and proline content
The leaf RWL was performed according to the method of Xiang et al. (2013). The leaf REC, MDA and proline content were measured at beginning and at the end of drought stress as the method described by Yu et al. (2006), Kuk et al. (2003) and Bates et al. (1973) respectively.

Statistical analysis of agronomic characters
Twenty mature OsMYB1R1-OE, OsMYB1R1-RNAi and WT plants were randomly selected to analyze the tiller number, plant height, flag leaf length, flag leaf width, first stem node length, second stem node length, spike length, 1000 grain weight and seed setting rate.

Statistical analysis
All data were analyzed by analysis of variance with Student's t test using SPSS 19.0 software (IBM Corporation, Chicago, IL, USA), values of P < 0.05 were considered statistically differences.

Expression patterns of OsMYB1R1 in rice
Semi-quantitive RT-PCR analysis was used to examined the spatial and temporal expression level of OsMYB1R1. As presented in Fig. 1, the tissue-specific expression analysis indicated that OsMYB1R1 expression was high in panicle, but relatively low levels in the other parts of rice (Fig. 1a). The expression level of OsMYB1R1 gene at the time points of 0, 0.5, 1, 2, 4, 8 and 24 h after mannitol stress were shown in Fig. 1b. The semi-quantitative PCR analysis of OsMYB1R1 gene expression demonstrated that an decrease in the OsMYB1R1 transcript was observed with the extension of processing time. And the same result was obtained under the PEG6000 treatment (Supplementary information, Fig.S1).

OsMYB1R1 negatively regulates drought tolerance in rice
To test the effect of OsMYB1R1 on drought stress tolerance, OsMYB1R1-OE (Fig. 2a) and RNAi (Fig. 2b) vectors were constructed and were transformed into japonica cultivar Nipponbare separately. The expression level of OsMYB1R1 in transgenic plants was analyzed by semi-quantitative PCR (Fig. 2c, d). Two independent OsMYB1R1-OE (OE4 and OE5) lines and four OsMYB1R1-RNAi lines (Ri1, Ri2, Ri5 and Ri8) were obtained and chosen for drought stress tolerance test. But only images and statistics of OE5 and Ri8 were used in this paper. Because under normal or drought stress condition, there was no significant difference in plant morphology, biochemical index, seed germination and yield between each OsMYB1R1-OE and OsMYB1R1-RNAi line (data not shown). To test the effect of OsMYB1R1 in transgenic rice lines on seed germination under mannitol stress, we counted the germination rate, shoot height and root length of OsMYB1R1-OE, OsMYB1R1-RNAi and WT seeds under different concentrations of mannitol stress (Fig. 3). The results showed that the germination rate and shoot height of WT and transgenic seeds decreased with the increase of mannitol concentration, while the root length increased with the increase of mannitol concentration. However, there was no significant difference among the OsMYB1R1-OE, OsMYB1R1-RNAi and WT plants under the same mannitol concentration. These results indicated that overexpression or RNA interference of OsMYB1R1 did not affect seed germination under normal condition and mannitol stress. And the same result was obtained under the PEG6000 treatment (Supplementary information, Fig. S2). Therefore, these results suggested that the effect of OsMYB1R1 gene on drought tolerance might not reflected in the stage of seed germination. Osmotic stress was further performed at the postgermination stage rice seedling (Fig. 4). The results showed that there were no significant differences in shoot height and root length among the WT, OsMYB1R1-OE and OsMYB1R1-RNAi plants before mannitol stress treatment. After the treatment, the shoot height and root length of WT and transgenic seedlings decreased with the increase of mannitol concentration. Under the same mannitol concentration, the shoot height and root length of OsMYB1R1-OE seedlings were significantly lower than those of the WT, but the OsMYB1R1-RNAi plants were significantly higher than those of the WT. It means that the increase of OsMYB1R1 expression enhanced the sensitivity of rice seedlings to drought stress, while the decrease of OsMYB1R1 expression reduced the sensitivity of plants to drought stress.
Drought tolerance of rice seedlings were evaluated under two drought-stressed conditions. For seedlings vitro drought stress, three leaf stage WT and transgenic plants were withheld water for 10 h and then re-watered for additional 3 days (Fig. 5). After withholding water, the OsMYB1R1-OE plants were discovered to exhibit significant dehydration and wilting, while the leaves of WT seedlings were curled but not completely. However, most of the OsMYB1R1-RNAi plants were still stretched. There were remarkable difference after re-watering. The majority of OsMYB1R1-OE plants leaves showed further withered and the survival rate of seedlings was only 23.33%. While most of the WT plant were recovered and the survival rate of seedlings were 70.00%. However, the OsMYB1R1-RNAi plants were almost restored normal growth and the survival rate of seedlings were 90.33%. The same results were obtained from the natural drought stress treatment 2 week-old plant seedlings (Fig. 6a-c). Next, whether the physiological indicators changed within the period after the drought was investigated (Fig. 6d-f). The contents of REC, MDA and proline showed that there were no significant differences between the transgenic lines and WT plants before the drought stress. After the treatment, the MDA and REC content of OsMYB1R1-OE seedlings were significantly higher than those of the WT and OsMYB1R1-RNAi (Fig. 6c, f). While the lowest proline accumulation was observed in the OsMYB1R1-OE plants (Fig. 6e). The REC and MDA content were lower in the OsMYB1R1-RNAi seedlings than in the WT. While there were no significant differences between the OsMYB1R1-RNAi and WT plants in proline accumulation.
The booting stage is one of the most critical periods in rice growth cycle. Drought stress was also performed at this stage by withholding water for 5 days and then re-watering for additional 7 days (Fig. 7). The results showed that all leaves of WT and transgenic plants became completely rolled after drought treated for 5 days (Fig. 7b, e). After re-recovering with irrigation for 7 days, there were remarkable differences among the OsMYB1R1-OE, OsMYB1R1-RNAi and WT rice plants. Compared with WT, the majority of OsMYB1R1-OE plant leaves turned yellow and withered (Fig. 7c), while the OsMYB1R1-RNAi plants were almost restored normal growth (Fig. 7f). The detached leaves among the OsMYB1R1-OE, OsMYB1R1-RNAi and WT plants were exposed to air to compare the RWL in leaves (Fig. 8a). The results showed that the leaves from OsMYB1R1-RNAi had the lowest RWL, while the leaves from OsMYB1R1-OE plants had higher RWL than that of the WT and RNAi. The difference of drought tolerance of WT and transgenic rice plants means a certain relationship with leaf water loss rate. The REC, MDA content, and proline content as physiological indicators were often used to reflect various abiotic stresses, and they were also very sensitive physiological indexes. The REC, MDA content, and proline content revealed that there were no significant differences between the transgenic lines and WT plants before the drought stress. After the treatment, the MDA and REC content of OsMYB1R1-RNAi plants were significantly lower than those of the WT and OsMYB1R1-OE. While there were no significant differences between the OsMYB1R1-RNAi and WT plants in proline accumulation. The lowest proline accumulation and highest REC content were observed in the OsMYB1R1-OE plants. While there were no significant differences between the OsMYB1R1-OE and WT plants in MDA content (Fig. 8b-d).

Effect of OsMYB1R1 expression on growth and development of rice
The statistical data on the main agronomic traits of WT and transgenic rice plants were shown in Fig. 9. The results showed that there were no significant differences in tiller number, plant height, flag leaf length, flag leaf width, first stem node length, second stem node length, panicle length, 1000 grain weight and seed setting rate among the OsMYB1R1-OE, OsMYB1R1-RNAi and WT rice plants. Our data suggest that over-expression and RNA interference of OsMYB1R1 in rice do not affect the development and yield of transgenic rice.

Discussion
MYB transcription factors contain a large number of family, with 185 members in rice (Dubos et al. 2010). In this study, we isolated a rice MYB gene, OsMYB1R1, and functionally characterized its role in tolerance to drought stress by generating transgenic rice plants with overexpressing and RNA interference OsMYB1R1. Based on the performance of transgenic plants under drought stress and agronomic traits, we conclude that the OsMYB1R1 negatively regulates drought Expression of OsMYB1R1 was detected in all the tissues tested, with the maximum level in panicle, but relatively low levels in the other parts of rice. The previous studies revealed that AtCIR1 and RVE1, as closely related homologous genes of OsMYB1R1, were identified to be related to plant circadian (Zhang et al. 2007;Rawat et al. 2009). However, why OsMYB1R1 was expressed at high level in the young panicles of rice remains to be further studied.
Previous research showed that OsMYB1R1 (Os04g0583900) was strongly induced in rice cultivar 996 under heat stress, and the expression level of OsMYB1R1 was early elevated and then gradually reduced to lower increased level at the time point of 2 h after heat stress . In our study, OsMYB1R1 (Os04g0583900) was greatly down-regulated by drought stress, and the transcript level of OsMYB1R1 was decreased with the extension of processing time. It shows that that abiotic stress could induce the expression of OsMYB1R1 rapidly. OsMYB1R1 might be of great importance for stress tolerance.
Under drought stress, the OsMYB1R1-RNAi plants showed the increased tolerance to drought stress and higher survival rate after re-watering than WT plants, whereas the overexpressing plants were more sensitive to drought stress and lower survival rate after re-watering than WT plants. Previous work showed that drought-tolerant plants had a more perfect defense mechanism to maintain low levels of MDA (Xie et al. 2008). The abundance of REC and MDA can be used as indicators of cell membrane damage, and lower REC and MDA indicates that less membrane damage occurred (Bajji et al. 2002;Marnett et al. 1999). By contrast, proline enrichment as effective indicator of plant stress tolerance is a general response to various abiotic stresses (Akram et al. 2007). Previous works also demonstrated that the increased drought stress tolerance of the transgenic plants with MYB transcription factors was at least partially related to lower MDA content (Tang et al. 2019;Xu et al. 2020), lower REC (Tang et al. 2019) and higher proline content (Yin et al. 2017;Tang et al. 2019). In this study, compared with the wild type plants, the OsMYB1R1-overexpressing plants exhibited increased REC and MDA content and decreased proline content, while RNAi plants showed lower REC and MDA content and higher proline content. And the above results were obtained in the drought treatment experiments at seedling and booting stage. The results of vitro drought experiment and RWL measurement further confirmed the negative regulatory role of OsMYB1R1 gene in plant response to drought stress.
No differences were observed in germination rate among OsMYB1R1-OE, WT and RNAi seeds under mannitol treatments. No differences in phenotypes, physiological indicators and agronomic traits among WT, overexpressing, and RNAi plants were observed when grown under normal conditions. Our results indicate that OsMYB1R1 is a drought stress response gene and OsMYB1R1, overexpressed and RNA interference in rice, does not affect the rice seed germination, development and yield of transgenic rice, but only negatively regulates drought resistance of rice. These results suggest that RNA interference of the OsMYB1R1 gene may have important application value in improving drought resistance of crops.