Overexpression of Hbmbf1a, Encoding Multiprotein Bridging

Plant Molecular Biology (2020) 102:1–17 https://doi.org/10.1007/s11103-019-00926-7 Overexpression of HbMBF1a, encoding multiprotein bridging factor 1 from the halophyte Hordeum brevisubulatum, confers salinity tolerance and ABA insensitivity to transgenic Arabidopsis thaliana Lili Zhang1,2 · Yunxiao Wang1,2 · Qike Zhang3 · Ying Jiang1,2 · Haiwen Zhang1,2 · Ruifen Li1,2 Received: 13 June 2019 / Accepted: 13 October 2019 / Published online: 26 October 2019 © The Author(s) 2019 Abstract Key message HbMBF1a was isolated and characterized in H. brevisubulatum, and overexpressed HbMBF1a could enhance the salt tolerance and ABA insensitivity in Arabidopsis thaliana. The transcript levels of stress-responsive genes were signifcantly increased in the transgenic lines under salt and ABA conditions. Abstract Salinity is an abiotic stress that considerably afects plant growth, yield, and distribution. Hordeum brevisubulatum is a halophyte that evolved to become highly tolerant to salinity. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator and an important regulator of stress tolerance. In this study, we isolated and characterized HbMBF1a based on the transcriptome data of H. brevisubulatum grown under saline conditions. We overexpressed HbMBF1a in Arabidopsis thaliana and compared the phenotypes of the transgenic lines and the wild-type in response to stresses. The results indicated that HbMBF1a expression was induced by salt and ABA treatments during the middle and late stages. The overexpression of HbMBF1a in A. thaliana resulted in enhanced salt tolerance and ABA insensitivity. More specifcally, the enhanced salt tolerance manifested as the increased seed germination and seedling growth and development. Similarly, under ABA treatments, the cotyledon greening rate and seedling root length were higher in the HbMBF1a-overexpressing lines, suggesting the transgenic plants were better adapted to high exogenous ABA levels. Furthermore, the transcript levels of stress-responsive genes were signifcantly increased in the transgenic lines under salt and ABA conditions. Thus, HbMBF1a is a positive regulator of salt and ABA responses, and the corresponding gene may be useful for producing transgenic plants that are salt tolerant and/or ABA insensitive, with few adverse efects. This study involved a comprehensive analysis of HbMBF1a. The results may provide the basis and insight for the application of MBF1 family genes for developing stress-tolerant crops. Keywords HbMBF1a · STEM (short time-series expression miner) · Hordeum brevisubulatum · Transgenic Arabidopsis thaliana · Salt and ABA treatments Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1110 3-019-00926 -7) contains supplementary material, which is available to authorized users. * Ruifen Li Haiwen Zhang [email protected] [email protected] Lili Zhang 1 Beijing Academy of Agriculture and Forestry Sciences, [email protected] No. 9 Shuguang Huayuan Middle Road, Haidian District, Yunxiao Wang Beijing 100097, China [email protected] 2 Beijing Key Laboratory of Agricultural Genetic Resources Qike Zhang and Biotechnology, Beijing Agro-Biotechnology Research [email protected] Center, Beijing 100097, China Ying Jiang 3 College of Life Science, Hebei Normal University, [email protected] Shijiazhuang 050024, China Vol.:(0123456789)1 3 2 Plant Molecular Biology (2020) 102:1–17 Introduction increase in seed yield (Suzuki et al. 2005). These studies suggest that MBF1c likely evolved diferently than MBF1a Salt stress is a serious threat afecting crop production and and MBF1b, and that MBF1c has a non-redundant function. distribution. High salinity, especially during the seedling In addition, The A. thaliana mbf1 triple knock-down mutant stage, dramatically decreases crop yield and quality, and (mbf1 abc-) is hypersensitive to oxidative and osmotic may result in the death of all seedlings. Therefore, the main stresses, and the phenotypes are either partially or fully objective of this study was to functionally characterize salt- restored by AtMBF1c overexpression, implying AtMBF1c responsive genes in the halophyte Hordeum brevisubulatum is the predominant MBF1 subtype responsible for stress tol- and use them for breeding halotolerant crops. Previous stud- erance (Arce et al. 2010). ies have identifed and described the salt-tolerance genes Though the preliminary progress was made in the cloned from model plant species such as Arabidopsis thali- MBF1 genes associated with plant tolerance to abiotic and ana (Kim et al. 2007; Alavilli et al. 2017). biotic stresses, most plant species produce only one type Hordeum brevisubulatum, which is a wild relative of cul- of MBF1 protein (Tsuda and Yamazaki 2004). Moreover, tivated barley in the genus Hordeum in the tribe Triticeae, these MBF1 genes were all cloned from the common plants, is a typical clonal plant of the grass family with a short rhi- but not specifc plants tolerant to stresses. The expression zome. The salient feature of H. brevisubulatum is its high of wheat MBF1 may be induced by salicylic acid and eth- tolerance to multiple abiotic stresses, including salinity, ephon stresses (Zhang et al. 2009). The phosphorylation of alkalinity and drought, enabling it to grow in the saline- StMBF1 is induced in potato cells in response to a fungal alkali grasslands of northern China, where it is used as a treatment (Zanetti et al. 2003). In tobacco, the simultaneous major forage crop for livestock (Li et al. 2012). Although exposure to high temperature and drought stresses can up- previous studies have investigated H. brevisubulatum regard- regulate MBF1 expression (Rizhsky et al. 2002). Tomato ing ion balance and gene functions (Zhang et al. 2015), very mosaic virus mobile proteins and proteins from related little is known about the molecular mechanism underlying viruses can interact with MBF1 to regulate host gene expres- the resistance of H. brevisubulatum to stress. sion (Matsushita et al. 2002). Transcriptional coactivators are important for the expres- To investigate the functional advantages of H. brevisubu- sion of eukaryotic genes because they represent the link latum genetic resources, we performed the transcriptome between transcription factors/other regulatory elements and sequencing and found that it had three subtypes and these the basic transcriptional machinery (Takemaru et al. 1997). three subtypes were diferentially produced in development For example, multiprotein bridging factor 1 (MBF1) is a and various tissues. So the salt stress-responsive HbMBF1a highly conserved transcriptional coactivator that infuences gene was isolated this halophytic grass based on tran- diverse processes. The MBF1 proteins of various organisms scriptome data. Moreover, we functionally characterized are linked to the TATA-binding protein via interactions with HbMBF1a under salt and abscisic acid (ABA) treatments C-Jun, GCN4, ATF1, or other nuclear receptors to activate by gene overexpression and stress-related gene expression gene transcription (Brendel et al. 2002). Plant MBF1s are analyses. The HbMBF1a gene may be useful for the culti- involved in growth and development as well as a variety of vation of new highly salt-tolerant transgenic crops, but the stress responses. The overexpression of MBF1 may regu- underlying mechanism and function of HbMBF1a remain late diverse signal transduction pathways and activate the relatively unclear. Therefore, investigating the MBF1 gene production of multiple defense factors, ultimately increase family is important for revealing its specifc role and the plant resistance to multiple stresses (Arce et al. 2010; Wang related regulatory mechanism associated with plant stress et al. 2017). resistance. The MBF1 gene is likely one of the primary targets of physiological signals in plants. The functions and types of MBF1 have been reported for A. thaliana as a model plant Materials and methods (Tsuda and Yamazaki 2004). A previous study confrmed that MBF1 can control the leaf cell cycle and leaf expansion Hordeum brevisubulatum material and stress in A. thaliana, and that has been divided three subtypes, treatments namely AtMBF1a, AtMBF1b and AtMBF1c. AtMBF1c is a positive regulator of seed germination and plant growth Hordeum brevisubulatum plants were collected from the (Hommel et al. 2008). Other investigations indicated there saline grassland region of Hohhot, which is in the Inner are no growth rate differences between AtMBF1a-over- Mongolia Autonomous Region of China. The collected expressing and wild-type (WT) lines (Kim et al. 2007), plants were screened for salt tolerance. The seeds were ver- whereas the overexpression of AtMBF1c results in a 20% nalized by immersing in water for 2 days at 4 °C, after which 1 3 Plant Molecular Biology (2020) 102:1–17 3 they were incubated at 22–25 °C under a 16-h light/8-h dark soil and vermiculite (1:1). When the seedlings reached the photoperiod for 2–3 days to promote germination. When six-leaf stage, they were treated twice with 300 mM NaCl the resulting seedlings grew to about 1.0 cm long, they for a total of 23 days. After resuming normal watering for were transferred to a 250-ml beaker containing Hoagland’s an additional 7 days, the plant survival rate was calculated nutrient solution. The seedlings were then cultured for and plants were photographed. approximately 2 weeks until the plants reached the two-leaf To investigate stress-related gene expression, the and one-heart stage, at which

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