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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.38 No.3 pp.190-195

Screening of Multiple Abiotic Stress-Induced Genes in Italian Ryegrass leaves

Sang-Hoon Lee1, Md. Atikur Rahman2, Kwan-Woo Kim1, Jin-Wook Lee1, Hee Chung Ji2, Gi Jun Choi2, Yowook Song2, Ki-Won Lee2*
1Animal Genetic Resources Center, National Institute of Animal Science, Rural Development Administration, Namwon 55717, Republic of Korea
2Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan, 31094, Republic of Korea
Corresponding author : Ki-Won Lee, Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31094, Republic of Korea. Tel: +82-41-580-6757, E-mail:
August 29, 2018 September 13, 2018 September 13, 2018


Cold, salt and heat are the most critical factors that restrict full genetic potential, growth and development of crops globally. However, clarification of genes expression and regulation is a fundamental approach to understanding the adaptive response of plants under unfavorable environments. In this study, we applied an annealing control primer (ACP) based on the GeneFishing approach to identify differentially expressed genes (DEGs) in Italian ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42°C) treatments for six hours. A total 8 differentially expressed genes were isolated from ryegrass leaves. These genes were sequenced then identified and validated using the National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. However, this study provides new insight regarding molecular information about several genes in response to multiple abiotic stresses. Additionally, these genes may be useful for enhancement of abiotic stress tolerance in fodder crops as well a crop improvement under unfavorable environmental conditions.


    Rural Development Administration


    Abiotic stresses mainly cold, salt, heat, drought and heavy metals are the major limitations of crop productivity which restrict full genetic potential, growth and productivity of crops globally (Tuteja et al., 2011). Cold stress induces several philological constrains through i) generating of reaction oxygen species (ROS), ii) reducing the photochemical efficiency (PSII), and that inhibits photosynthesis in plants (Paredes and Quiles, 2015). Salt stress negatively affects to cells by disturbing the ionic and osmotic equilibrium, whereas, heat stress leads to overproduction of free radicles, induce oxidative stress and subsequent non-reversible damage plant growth (Chalanika et al., 2017). In near future, it is expected that the initiation of heat weave with higher stress intensity will more severe due to global warming (Wang et al., 2016). Abiotic stresses induce a series of physiological, biochemical, molecular and morphological changes in crops including forages (Staniak et al., 2015; Rahman et al., 2015, 2016a). In addition, crop performance and yield are the consequence of genotypic expression that is modulated by crop interaction with environment. Thus, screening and functional characterization of genetic resources under abiotic stress would be useful for better understanding the adaptive response of plants to multiple abiotic stresses.

    Italian ryegrass (Lolium multiflorum) is the winter forage crop mostly cultivated in Southern Brazil and other South American countries (Pavinato et al., 2014). Recently, it has been extensively cultivated in Korea. Ryegrass is economically most important forage species, considered as excellent source of high productivity with high nutritive value (Gerdes et al. 2005). Despite of its many superior properties of ryegrass, some species are known to be sensitive to abiotic stresses, thus limiting its cultivation in certain area (Hulke et al. 2008). Plants have evolved some strategies to avoid and/or tolerate several abiotic stresses, and molecular breeding approach using suitable candidate genes provides excellent support for forage crop improvement against multiple abiotic stresses (Alam et al., 2010; Singer et al., 2017).

    Numerous molecular candidates have been extensively disclosed in several forages legume and grass species including perennial ryegrass (Hulke et al. 2008), alfalfa (Rahman et al., 2015, 2016 a,b), tall fescue (Lou et al., 2017), and Siberian wild rye (Xie et al., 2017), teff grass (Lee et al., 2018) under abiotic stresses. Advanced molecular tools give the new opportunities for understanding of global gene expression in plants. Regulation of gene expression, and functional responses towards abiotic stress tolerance have been demonstrated in forage and turf grass (Kopecký and Studer, 2014), Arabidopsis (Azevedo et al., 2016), and alfalfa (Li et al., 2017). Despite of great progresses in diverse plants, still needs to be elusive in Italian ryegrass (cv. Kowinearly).

    Sequence-based several molecular approaches present a better alternative to identify and analyze gene expression. For instance, the serial analysis of gene expression (SAGE) was considered as suitable approach for gene expression profiling (Velculescu et al., 1995), followed by massively parallel signature sequencing (MPSS) (Brenner et al., 2000), suppression subtractive hybridization (SSH) (Sahu and Shaw, 2009), and next-generation sequencing (NGS) technologies (Jain, 2012) which were used to target range of transcript and low abundant genes in plants. In recent years, a GeneFishing approach is being successfully used to identify the differentially expressed genes (DEGs) in plants (Rahman et al., 2016b; Lee et al., 2018). Hence, the benefit of this suitable approach is that it increases the annealing specificity to the template and able to amplify only the genuine gene products. In this study, we applied annealing control primer (ACP) based gene fishing technique to identify cold, salt and heat-induced DEGs in Italian ryegrass (cv. Kowinearly), subsequently would be target genes to develop abiotic stress tolerant forage crops.


    1. Plant materials, growth conditions and application of abiotic stresses

    Italian ryegrass (Lolium multiflorum cv. Kowinearly) seeds were collected from the Division of Grassland and Forages, National Institute of Animal Science (NIAS). Italian ryegrass seeds were sterilized using 70% ethanol for 2 minutes and washed three times by Mlli-q, followed by the seeds were treated by 30% sodium hypochlorite (NaOCl) solution for 30 min. Finally, surface sterilized seeds were moved to germination medium containing 3% sucrose and half MS (Murashige & Skoog). The culture room environment was maintained in the room temperature as 25 ±1°C with of 400 μmol m-2 s-1 light and 14 h light/8 h dark period. Two-week old ryegrass seedlings were exposed to cold (4 °C), salt (200 mM NaCl) or heat (42 °C) treatments for 6 h, respectively. The control plants were maintained at 25 ±1°C temperature and/or irrigated by only water. After 6 h of all stress treatments the plants leaves were cut from shoot and leaf transition zone, washed properly using Milli-q water for three times, quickly frozen in liquid N2 for further molecular analyses.

    2. Isolation of total RNA and cDNA synthesis

    Total RNA was extracted from abiotic stresses treated and non-treated (control) ryegrass leaves using TRIzolreagent (Qiagen, CA, USA). These RNA samples were used for the first stand cDNAs synthesis by reverse transcriptase. The reserve transcription reaction was carried out as described earlier by Lee et al. (2009). After completing of first-strand cDNAs synthesis, samples were diluted at by adding 80 μl of one fold dilution of Milli-q water, and prepared for the GeneFishingTM approach.

    3. GeneFishing by annealing control primer (ACP)-based approach

    DEGs using control primer (ACP)-based approach was carried out. For this molecular technique, Gene Fishing-kit (Seegene, Inc., Republic of Korea) was used. Subsequently, synthesis of the second strand cDNA was performed according to traditional thermal cycler’s protocol and experimental methods of Lee et al. (2011). Finally, the amplified products were collected and separated by using 1% agarose gel containing RedSafe™ (5μL/100 mL solution; ABC Scientific, USA).

    4. Cloning and identification of DEGs

    Abiotic stress-induced DEGs were isolated from the gel by using the GENCLEAN II Kit (Q-BIO gene, Carlsbad, CA, USA), and subsequently cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA) based the manufacturer’s protocol. Followed by the sequencing of cloned plasmids were performed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Finally, the sequences of specific genes were validated and confirmed by comparing to corresponding homologs in NCBI database (


    We successfully screened and identified abiotic stress-induced several DEGs including light harvesting chlorophyll a/b binding protein (named as DEG 1), alpha-glactosidase b (DEG 2), chromosome 3B (DEG 3), elongation factor 1-alpha (EF1a; DEG 4), FLbaf106f03 (DEG 5), Lolium multiflorum plastid, complete genome (DEG 6), translation initiation factor SUI1 (DEG7), nd glyceraldehyde-3-phosphate dehydrogenase (DEG 8) in Italian ryegrass leaves (Figs. 1-3; Table 1). In the following sections, we have clarified the potential role of these identified candidate genes by evaluating previous genomics, abiotic stress responses, and biological importance in plant system. Table 2, Table 3

    A light complex is comprised of several subunit proteins which are supercomplex of photosystem, the functional unit of photosynthesis. We have identified cold stress-induced a gene encoding light harvesting chlorophyll a/b binding protein (LHCB; DEG 1). The LHCB gene is known as key apoprotein of the light-harvesting complex of photosystem II (PSII), this proteins is known to be associated in guard cell signaling. The up-regulation of LSCB in response to cold stress suggests that it is possibly inv LHCB protein gene containing important stress responsive element, showed similarities among dark, heat, salinity and drought response at developmental stage in barley (Qin et al., 2017). In our study, we identified cold-induced LHCB, therefore we hypothesized that this LHCB gene has pivotal role in photosynthesis and developmental stages in Italian ryegrass leaves.

    Salt-induced DEG2 expression is enhanced by salt in Italian ryegrass leaves and identified as alpha-glactosidase b (α-Gal b; Table 1, Fig. 1). The α-Gals are member of the glycoside hydrolase family, which have pivotal role in leaf elongation during plant development (Chrost et al., 2007). Moreover, it was documented that α-Gal showed tolerance in plants against freezing (Pennycooke et al., 2003). In this study, α-Gal gene was found to be highly induced response to salinity, which indicates α-Gal might be also involved in salt tolerance in Italian ryegrass. Therefore, this gene would be used as suitable biomarker in abiotic stress response as well as development of abiotic stress tolerant forage crops. DEG3 is identified as encoded chromosome 3B, Choulet et al. (2014), produced a reference sequence of 1 gigabase (gb) chromosome 3B in Triticum aestivum and distributed its structural and functional features. Moreover, they clarified the relationship, gene expression function and chromosome location under15 conditions, also revealed that the several distal regions were enriched wherein some are responded to abiotic stimulus. However, the gene response in Italian ryegrass to abiotic stress such as NaCl in our study, was supported by this observation.

    Cold-induced DEG4 identified as elongation factor 1 alpha (EF-1 alpha) in ryegrass leaves. LeEF-1 alpha led to enhance

    protein synthesis in tomato plants (Pokalsky et al., 1989). In addition, they also found that the increased expression of LeEF-1 alpha mRNA was correlated with higher levels of protein synthesis in developing tissues of tomato. Therefore, up-regulation of EF-1 alpha in our study would be recommend as a candidate for enhancement of protein synthesis in forage leaves, as our main deal with fodder improvement specially for livestock. In this study, we identified some genes in ryegrass leaves, and those specific function still unknown and uncharacterized. For example, the combined cold-heat induced FLbaf106f03 (DEG 5), and a salt-induced Lolium multiflorum plastid, complete genome (DEG6). However, further exploration concerning abiotic stress-induced functional regulation of these genes is needed to develop our understanding.

    Salt and heat-induced DEG7 was identified as translation initiation factor Shortened Uppermost Internode1 (SUI1). The member of SUI genes family has significant importance in the development of plant aerial part (Yin et al., 2013). In our study, we hypothesized that the up-regulation of SUI1gene might be involved in the response of several abiotic stress regulations, as it induced significantly by salt and heat in ryegrass leaves. Moreover, Yin et al. (2013) found that overexpression of SUI1 and SUI2 led to enhance growth of internodes at the stages of vegetative development in rice. However, these above observations provide the role of SUI1 in abiotic stress response along with plants development.

    We also identified the glyceraldehyde-3-phosphatedehydrogenase (GAPDH, DEG8) enzyme in ryegrass leaves; this is a ubiquitous enzyme that plays important role in the glycolysis process. GAPDH is known to be participated in metabolic processes as well as abiotic stress tolerance in plants. Zeng et al. (2016) observed that the expression of several GAPDH genes, which were modulated by abiotic stimuli and associated with enhanced abiotic stress tolerance in wheat. However, the up-regulation of GAPDH in rye grass suggests that it would be potential candidate for conferring stress tolerance in forage crops.


    In this study, several DEGs including light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase were identified using ACP-based GeneFishing approach. These genes were significantly up-regulated by various abiotic stresses (cold, salt and heat) in Italian ryegrass leaves. These identified genes were involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance. The identification of these genes in ryegrass leaves would be effective as promising candidates for the fodder crops improvement under multiple abiotic stresses.



    This study was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01193504)” and Postdoctoral Fellowship Program of 2018, National Institute of Animal Science, Rural Development Administration, Republic of Korea.



    Cold, salt, and heat-induced genes in Italian ryegrass leaves. Agarose gel image shows the differentially expressed genes (DEGs) induced by cold (4 °C), salt (NaCl; 200 mM), and heat (42 °C). Arrows on gel image indicate the stress specific DEGs. MW, molecular weight size marker; N, non-treatment; C, cold; S; salt (NaCl); H, heat; ACP, annealing control primer


    Cold-induced DEGs in Italian ryegrass leaves.

    Salt–induced DEGs in Italian ryegrass leaves.

    Cold, heat, and salt-induced DEGs in Italian ryegrass leaves.


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