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Byline: Ki-Won Lee, Md. Atikur Rahman, Gi Jun Choi, Ki-Yong Kim, Hee Chung Ji, Tae Young Hwang and Sang-Hoon Lee


Heat shock proteins (Hsps) are key components contribute to cellular homeostasis under stressful conditions. They function as molecular chaperone involved in protein folding, plant development, signal transduction and protecting plants against several stresses. In this study, we developed transgenic alfalfa by Agrobacterium-mediated genetic transformation, and integration and expression of MsHsp23was confirmed by polymerase chain reaction (PCR), Southern and Northern blot analyses. Transgenic (Tg1, Tg3, Tg4 andTg7) and non-transgenic (NT) alfalfa plants were exposed to heat (42 degC)treatment for 24 h. Compared to NT control plants, Tg plants showed less wiltingthat maintained leaves color compared to NT plants. The expression of MsHsp23gene and the ascorbate peroxidase (APX) levels were higher in MsHsp23alfalfa lines relative to NT plants. The result indicates the chaperone activity of MsHsp23genethat allows for the induction of APX in transgenic alfalfa.

We conclude that high level of MsHsp23withAPX activity enhanced tolerance to heat stress in transgenic alfalfa plants.

Keywords: MsHsp23, Transgenic, Alfalfa, Heat stress, Tolerance.


Increasing temperature is a great challenge for agricultural crops production all over the world. Field study and mathematical modelling of crop plants reveal that changing temperatures have significant effect on crop productivity (Wang et al., 2015). Using light energy, plants accumulate biomass by altering inorganic carbon to carbohydrate this process is known as photosynthesis. This is one of the most heat-sensitive processes in crop plants (Sulvucci, 2008).Heat stress negatively affects on plants survival, growth and agricultural yield (Duncan and Carrow, 2001). However, series of molecular responses including genes, proteins and their expressions are found to be changed by abiotic stresses including heat, salinity, drought, and extreme temperature in plants (Li et al., 2013; Rahman et al., 2015: Rahman et al.,2016).Long-term abiotic stresses greatly affect on plant metabolism, inhibits cellular function, reduce plant growth that leads to plants die.

The effects of abiotic stresses overburden the cells, affecting the ability of the cells to function. These stresses reduce the growth of the plants and, in extreme conditions, cause the death of the plants. Therefore, plant biotechnological approach is necessary for improvement of crop plants against environmental conditions. Molecular breeding technique would be effective tool for abiotic stress tolerant crop production (Bowler et al., 1992; Noctor and Foyer, 1998).

Alfalfa (Medicago sativa L.) is a forage legume, cultivated as high feed value with high yield potentials globally (Kechang et al., 2009). In livestock industry, alfalfa plays an important role as good source of feed nutrient for animals (Hill et al., 1991). Moreover, alfalfa is excellent source of biological nitrogen fixation that increases soil fertility for crop production(Deak et al.,1986). Generally, alfalfa is able to drought tolerance but suffers from extended period of heat with drought stress. Heat stress reduces plant growth, leaf water potential, photosynthesis, and carbohydrate accumulation. Moreover, heat stress induced oxidative stress subsequently generates reactive oxygen species (ROS), resulting cellular damage and lipid peroxidation in plants (Wang et al., 2015).Oxidative stress leads to inhibits photosynthesis, respiration process as well as plant growth.

Plants have evolved an antioxidant system where overproduced ROS are scavenged by several antioxidant enzymes including ascorbate peroxidase (APX) and superoxide dismutase (SOD). APX utilizes ascorbate as a source of electron donor to decrease H2O2 level. However, the function of scavenging enzyme can be interrupted by heat stress that leads to lipid peroxidation, cellular injury, and membrane damage in plants.

In this study, the objective was to develop heat tolerant transgenic alfalfa using Agrobacterium-mediated transgenic approach. We generated transgenic alfalfa plants over expressing small heat shock protein 23 (MsHsp23) under control ofCaMV35S promoter. We observed thatMsHsp23 alfalfa plant enhanced tolerance to heat stress.


Plant materials and culture explants: Alfalfa (Medicagosativa cv. Xinjiang Daye) seeds were collected from the National Institute of Animal Science, Rural Development Administration (RDA), Republic of Korea. Seeds were rinsed with 70% ethanol for 1-2minutes. The seeds were then washed three times with distilled water then surface sterilized for 30 minutes using of 30% (w/v) sodium hypochlorite and 1% Tween-20.For the removal of surfactant, the sterilized seeds were rinsed properly with sterile deionized water. The sterilized seeds were germinated on hormone-free, half-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 2% (w/v) sucrose and 0.3% (w/v) Gelrite. The seeds were allowed to culture in a growth chamber at 25degC with 14 h photoperiod. Hypocotyl segments of alfalfa plants were used as explants.

Vector construction: The alfalfa small heat shock proteins (MsHsp23) cDNA fragment was ligated into the translation ligation initiation codon within the 5'untranslated sequence of the constitutively expressed cauliflower mosaic virus promoter (35S). The resultant chimeric gene cassette was then inserted into the Kpnl/Zbal site of the pCAMBIA1300 binary vector (Fig. 1). The recombinant pCAM-MsHsp23 was introduced into Agrobacterium tumefaciens strain EHA105, which was used for genetic transformation.

Genetic transformation: Seven-day-old hypocotyls were used as explants for transformation. Hypocotyls were carefully excised (1-3 mminlong) from the seedlings without meristematic axillary buds. Hypocotyls were gently shaken in Agrobacterium suspension for 30 min then blotted on a sterile filter paper. The hypocotyls segments were transferred to the solid co-cultivation medium and maintained up to5 days. The hypocotyls segments were transferred onto shoot-inducing medium (SIM) containing 250mg/L hygromycin, and every three weeks, followed by sub-cultured into fresh SIM medium. The green healthy shoots were screened by MS medium containing 20mg/L hygromycin (Hg). The seedlings were maintained in selection medium up to rooting. Putative transgenic seedlings were transferred in to pot containing nursery medium (soil: perlite; 1: 1), and grown in green house.

Molecular analysis of alfalfa plants: Genomic DNA was isolated from the leaves of the wild type and putative transgenic plants using the CTAB method.

The MsHsp23alfalfaplants were identified by PCR screening. The amplification of MsHsp23 and HPT genes were performed by PCR. The following genes primers were used for PCR analysis: 5'-TCTAGAATGGCGTCTGTTGCT-3'and5'-TTCTCAGCTATTTAGGAGCTC-3'forMsHsp23,and the primers; 5'-CCTGAACTCACGACG-3'and 5'-AAGACCAAGGAGCATAT-3' were designed for the HPT gene, respectively. The conditions of PCR amplification was performed according to Lee et al.(2012a).Integration of the MsHsp23gene into the alfalfa plant genomes was further confirmed with Southern blot analysis. Twenty micrograms of genomic DNA was digested with EcoR1 and separated electrophoretically on a 1.0%agarose gel. Following the each sample was transferred to a nylon membrane and hybridized with the (alpha-32P)-dCTP-labeled MsHsp23 probe by PCR-based labeling using pCAM-MsHsp23 plasmids as a template. Southern blot and Northern blot hybridizations were performed as described previously by Lee et al. (2000).

Heat tolerance assay: The MsHsp23transgenicand wild-type plants were kept at control environment for 24 h prior to heat treatments. Plants were placed in a heat chamber where temperatures of total system were well controlled. Three independent alfalfa lines (Tg1, Tg3, Tg4, and Tg7)were exposed to 42 degC for 24h. Control plants were maintained at normal temperature. Following treatment leaves samples were harvested form MsHsp23plants and not-transgenic (NT) plants. The leaves samples were immediately frozen in liquid nitrogen then kept at -90 degC until used.

Analysis of ascorbate peroxidase (APX): To determine the ascorbate peroxidase (APX) activity, the total soluble proteins were extracted from the leaves of alfalfa plants using extraction buffer. The composition of the protein extraction buffer was 50mMHEPES (pH7.0) and 0.1mM EDTA. The concentration of the protein was estimated with a Bio-Rad protein assay kit. The oxidation of ascorbate was initiated by H2O2, and the decrease to 290nm was monitored for 1.5 minutes. The reaction mixture contained 40mMHEPES (pH 7.0), 0.1mMEDTA, 0.03mM ascorbic acid (AsA), 0.1mM H2O2, and enzyme samples. One unit of APX activity was defined as the amount of enzyme that caused the oxidation of 1mole of AsA. The APX activity was determined by estimating the rate of ascorbate oxidation (extinction coefficient: 2.8mM-1cm-1) as described previously by Nakano and Asada (1981).


Confirmation of MsHsp23gene integration to plants genome: Seven independent primary transformants were obtained by plant selection marker hygromycin.

Out of these 4 lines (Tg1, Tg3, Tg4 and Tg7) have showed the presence of genes by PCR screening using gene specific primers for bothMsHsp23 and hpt genes (Figs. 2A,B).Southern analysis of 4 lines confirmed that genes of interest were successfully integrated into the alfalfa genome. Therefore, it was conformed that alfalfa plant contains at least one copy of transgenes and the transgenic events were truly independent (Fig. 3A). High expressions of mRNA as the genes were driven by a constitutively expressing 35S promoter. These results indicated that transgenes were successfully expressed transcriptionally and translationally in transgenic alfalfa plants. A numerous studies have been revealed that several heat shock proteins (Hsps) are most widely distributed which responded to different abiotic stresses (Sun et al., 2002). These Hsps were contributed in plant development, and stresstolerance in plant under stressful conditions (Dafny-Yelin et al., 2008; Chauhan et al., 2012).

The functional characteristics of different subcellular localized small Hsps and mitochondrial small Hsps are largely unknown. According to previous report, chaperone property of small Hsps prevents the denaturation of heat-injured proteins (Siddique et al., 2008).In our study, MsHsp23 alfalfa plants enhanced tolerance to heat stress. Previously, we discussed how alfalfa mitochondrial small heat shock proteingenesareoverexpresed in tall fescue and tobacco plants subsequently has been proven the biological function ofthis genefor abiotic stresstolerance (Lee et al., 2012a,b).

Over expression of MsHsp23 transgenic alfalfa plants: We confirmed the expression of four lines (Tg1, Tg3, Tg4 and Tg7) by Northern blot analysis. The Tg4 and Tg7 were highly expressed compared to Tg1 and Tg3 (Fig. 3b). We found the MsHsp23 gene was highly expressed in transgenic plants during heat (42degC) stress. It has been well documented that plants respond to heat stress often with induction of HSPs (Fujimoto and Nakai, 2010; Hartlet al.,2011).The up-regulation of MsHsp23 by heat stress suggested that this gene may play some roles related to heat stress response.

MsHsp23 alfalfa plants enhanced tolerance to heat stress: As a consequence of molecular results we try to find out the phenotypic difference between transgenic and NT plants at whole plant level. As shown in Fig. 4,the significant differences were not observed phenotypically before heat treatment while the phenotypic variations were found after heat treatments. The Tg7 line was less affected by heat stress compared to NT plants (Fig. 4A, B).Transgenic Tg7linerepresentsless number of wilted leaves while the NT plants became largely wilted and changed the leaves color. This result indicates that over expressed theMsHsp23may involve to protect stress-induced damages in transgenic plants. It is well known thatthe extreme heat treatment regulates the inactivation of diverse functional proteins and subcellular components in the cells.

Moreover, changing of leaf color, reduction of photosynthesis, breakdown of the chlorophyll from the photo-oxidative damage caused by the heat stress (Feierabend and Winkelhusener, 1982; Zlatev and Yordanow, 2004; Erge et al., 2008). Cytosolic and chloroplastic small Hsps have been investigated in terms of tolerance to heat and drought in plants (Sun et al., 2002; Chauhan et al., 2012). However, the molecular and phonotypical observations of transgenic alfalfa plants indicated that over expression of MsHsp23gene enhances tolerance to heat stress.

Ascorbate peroxidase (APX) activity increased inMsHsp23alfalfa plants: We analyzed the ascorbate peroxidase (APX) activity inoverexpressingMsHsp23 alfalfa plants response to heat treatment at 42degC for 48h. As shown in Fig. 5, APX activity was increased in MsHsp23transgenic line (Tg7) compared to NT plants.

The high accumulation APX activity in transgenic plants indicated thatitregulated by heat stress. Plants have two systems for scavenging ROS: enzymatic system including APX,POD,SOD and CAT; non enzymatic including ascorbic acid (AsA). Possibly, ROS was regulated by enzymatic system including APX. So, the high APX likely dependent on their transcription level. However, antioxidant enzymes APXsare widely distributed in different subcellular organelles including chloroplast lumen, stomata, peroxisomes, cytosol, and inner membrane space of mitochondria that effectively scavenges hydrogen peroxide (Miller et al., 2010).The overexpression of small Hsp 16.45 showed enhance tolerance to abiotic stress in Arabidopsis, whereas the superoxide dismutase (SOD) and catalase (CAT) activities were highly induced (Mu et al., 2013). Together these above discussion, we anticipate that over expression of MsHsp23gene with high level of APX activity enhanced tolerance to heat stress in transgenic alfalfa plants.

Conclusion: We developed MsHsp23 transgenicalfalfa plant using Agrobacterium mediated genetic transformation approach. The MsHsp23gene was driven by a constitutively over expression CaMV 35S promoter. The molecular analyses indicated MsHsp23genes were integrated successfully to alfalfa plants genome. In our study, we found MsHsp23transgenic plants exhibited tolerance in alfalfa plants to heat stress. In addition, the antioxidant enzyme APX that was highly induced in transgenic alfalfa plants. These results suggest that transgene MsHsp23 with high level of APX enhanced heat tolerance in transgenic alfalfa

Acknowledgements: This study was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01193504) and the 2017Postdoctoral Course Program of the National Institute of Animal Science, Rural Development Administration, Republic of Korea.


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Publication:Journal of Animal and Plant Sciences
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Date:Aug 31, 2017

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