JIA-2019-11

2458 XU Bing-qin et al. Journal of Integrative Agriculture 2019, 18(11): 2457–2471 responses because of its improved water-use efficiency and short life cycle (Nations 2011; Lata and Prasad 2013). Compared with other model plants such as Arabidopsis and rice, foxtail millet has evolved notable morphological and physiological innovations to sustain itself under limited moisture supply rather than succumbing to senescence or death (Chaves et al . 2003). Some genotypes of foxtail millet have evolved an extensive root system to actively supply water and a relatively small leaf area and thick leaf cuticle to reduce transpiration as forms of drought avoidance mechanisms (Vetriventhan et al . 2016). Meanwhile, to adapt to osmotic stress, foxtail millet organizes an array of biochemical and physiological interventions by activating the expression of numerous stress-associated genes, decreasing photosynthetic rate, closing stomata, synthesizing compatible osmoles and antioxidants, and other characteristic responses to counteract the reduced water availability (Anjum et al . 2011). In previous studies, many attempts have been made at the transcriptome level to dissect the molecular mechanisms of foxtail millet response to drought. Before the release of the S . italica genome sequence, the transcriptome of S . italica was investigated mainly by cDNA libraries or subtractive hybridization analysis (Zhang 2007; Lata et al . 2010; Puranik et al . 2011). Zhang (2007) found that tissue- specific expression exists in S . italica . Puranik (2011) demonstrated the existence of gene sets in foxtail millet which circumvent drought stress. Following the release of the entire S . italica genome (Bennetzen et al . 2012), many stress-responsive genes encoding for transcription factors, signaling molecules and enzymes have been identified and characterized (Lata et al . 2014; Muthamilarasan et al . 2014; Zhu et al . 2014). Qi et al . (2013) analyzed the transcriptome of foxtail millet seedlings by deep sequencing, and found that genes encoding late embryogenesis abundant protein (LEA), dehydrin, heat shock protein (HSP), aquaporin, and phosphatase 2C (PP2C) were the most abundant among the up-regulated genes. In the study of Puranik et al . (2013), 50 NAC domain-encoding genes were identified by systematic sequence analysis in response to dehydration, salinity, cold, and phytohormone treatments during early and late durations of treatments in foxtail millet. Plant drought stress responses and resistance are complex biological processes. Previous transcriptomic studies of foxtail millet mainly focused on determinants for stress tolerance, such as the miRNAs and transcription factors, so the perspective was narrow and the duration of drought was too short (1 to 10 h drought-treated) to allow the full response mechanisms to develop. The comprehensive transcriptomic analysis combined with physiological tests of foxtail millet under osmotic stress from 0 to 72 h, during which the seedlings exhibit an apparent wilt phenomenon, has not been investigated thus far. Therefore, this study of transcriptomic analysis of foxtail millet under osmotic stress can provide fundamental information on gene expression regulation and subsequent cell metabolic regulation pathways, but also can be used to characterize resistance mechanisms and screen for drought tolerance candidate genes. These tools can provide for further developing rational breeding and transgenic strategies to improve the drought tolerance of foxtail millet, and may serve as reliable gene resources for studying drought tolerance in other crops. 2. Materials and methods 2.1. Plant materials and growth conditions To validate the drought screening of genotypes, two cultivars widely grown in the semi-arid region of northwestern China, drought-tolerant S . italica cultivar Damaomao (DM; number: 00002348; drought resistance index (DRI) in 2016 regional trial, DRI: 1.08) and drought-sensitiveHongnian (HN; number: 00000183; DRI: 0.03), were used as the plant materials. One hundred seeds of each cultivar were surface-sterilized in 10% (v/v) NaOCl for 5 min and 70% ethanol for 5 min, rinsed five times, and then germinated at 28°C in darkness. When one leaf was visible, the uniform seedlings were transferred into plastic seedling-raising plates containing 2 L of hydroponic nutrient solution. Plants were grown in a climate-controlled incubator with a day/night temperature of 23–25°C/16–18°C, 75–85% relative humidity, a 14-h photoperiod, and a light intensity of 200 μmol m –2 s –1 . Half strength Hoagland’s nutrient solution for hydroculture (Hoagland 1983) was supplied for foxtail millet growth. The nutrient medium was resupplied every other day and an air compressor was used to supply aeration. Moderate osmotic stress was induced by adding 20% (g mL –1 ) polyethylene glycol (PEG 6000) to the 1/2 Hoagland solution, which corresponded to –0.8 MPa water stress, measured by Wescor Psypro (Wescor, USA), at the three-leaf stage of foxtail millet seedlings. The leaf samples were randomly selected after drought treatment at 0, 24, 48, and 72 h and numbered DM-1, DM-2, DM-3, DM-4, and HN-1, HN-2, HN-3, and HN-4 respectively for the two cultivars. Then leaf samples were frozen with liquid nitrogen and stored at –80°C for the extraction of proline, malondialdehyde (MDA), and abscisic acid (ABA). Fresh leaves were used for RWC and chlorophyll content determination, and RNA extraction. The experimental treatments were assigned at random using randomized complete block design with three replicates to ascertain the data reproducibility.