THE EFFECT OF SIMULATED DROUGHT ON HVA1 AND SRG6 GENE EXPRESSION IN SPRING BARLEY

Joanna Romanek¹, Hanna Walczak², Magdalena Wójcik-Jagła², Barbara Jurczyk², Marcin Rapacz^2
1 Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production
² Department of Plant Physiology University of Agriculture in Krakow

ABSTRACT

Drought is one of the most important environmental stresses in agriculture. It significantly reduces both the quality and yield of agricultural crops. Under conditions in Poland, spring barley is more sensitive to drought than winter barley. In plants, the expression of dehydrins increases the ability to absorb and hold water in the cell. One can try to obtain plants with increased resistance to drought, among other things, through the selection of genotypes with increased accumulation of dehydrins.
The aim of our study was to examine differences in the expression levels of SRG6 and HVA1 genes in Polish breeding lines of spring barley. In this experiment measurements of the accumulation levels of HVA1 and SRG6 transcripts by means of real-time PCR were performed in twenty seven barley genotypes. Estimations of mRNA abundance were made before drought and on the 15th day of the experiment at a relative maximum water capacity of 32%. The results of our experiments showed that drought stress caused a significant increase in the expression of HVA1 and SRG6 genes in all genotypes. The 27 studied breeding strains of spring barley from Polish breeding companies were characterized by high variation in the expression of both genes.

Key words: Drought, barley, HVA1, SRG6, water deficit tolerance.

INTRODUCTION

Drought is defined as water deficit in the environment. Droughts in Poland occur in different seasons of the year, but usually in the spring, every few years or even more often. In recent years, more frequent droughts have been linked to global warming and the overall area endangered by droughts has increased. Therefore, basic research on drought tolerance may be crucial for providing people with food in the future [3].

The synthesis of certain proteins is stimulated by drought. Some of these proteins can help in preventing cell dehydration, which facilitate plants in surviving water deficit. Dehydrins are members of the LEA (late embryogenesis abundant) protein family - the most important group of soluble proteins which synthesis is stimulated by water stress and other factors causing dehydration of cells (low temperature, salinity), and also in the presence of abscisic acid (ABA) and / or jasmonic acid (JA) [4,5,7]. The first paper on dehydrins was published by Mundy and Chua in 1988 [10]. The synthesis of these proteins may also be associated with the process of seed maturation and dehydration. The first isolated and characterized dehydrin was found in cotton [2].

Genetic engineering has become a new tool in creating transgenic plants tolerant to drought. If plants develop tolerance to the dehydration of cells this leads to an increased crop yield under drought conditions. In theory, such transgenic plants can be introduced in the cultivation areas that are not suitable for the cultivation of present cultivars due to frequent water deficits. Plant tolerance to drought was enhanced by the transformation with genes that code for a LEA protein. The LE25 gene (4 LEA group) isolated from tomato improved tolerance to salinity and freezing in yeast, while HVA1, (3 LEA group) isolated from barley improved tolerance to water deficit and salinity in transgenic rice. Dehydrin Em isolated from wheat has also demonstrated osmoprotective properties in yeast [13].

HVA1 protein belongs to group 3 of LEA proteins. A characteristic feature of proteins in this group is the presence of repeated sequences of 11-amino acids. Differences among proteins belonging to this group are the result of the different number of the copies of this sequence [1]. For the first time HVA1 was isolated from the aleurone layer of barley seeds. In barley, the expression of the HVA1 gene was induced in the aleurone layer and embryo during the late development of the grain. In young seedlings HVA1 expression is rapidly induced by stress conditions such as dehydration, extreme heat, salinity, or ABA treatment [8]. The HVA1 gene encodes a 22kDa protein, which contains nine imperfect repetitions of 11-amino acids [13].

SRG6 expression was identified using the differential display reverse transcription PCR (DDRT-PCR) technique in barley subjected to drought and ABA. The SRG6 gene is located on chromosome 7H between the ABC455 and salfp76 markers. This region is linked to barley water deficit tolerance. The expression of this gene is induced by stress but its role has not been confirmed yet. The product of SRG6 gene is a hydrophobic protein with a structure similar to the structure of the transcription factors superfamily HLH (helix-loop-helix) [14]. The genes encoding transcription factors and kinases are primarily activated by cascades of signal transduction activated by stress. They play a major role in the tolerance to stress in plant cells, because they regulate the expression of genes encoding proteins directly involved in cell protection (enzymes and other functional proteins). In 2010, Rapacz et al. [12] studied the relationship between the physiological characters associated with the drought tolerance of barley and the different levels of expression of HVA1 and SRG6 genes between malting and feed barley from selected Polish conditions. They showed that the expression of SRG6 in both groups of barley was correlated with the photochemical efficiency of PSII. In malting barleys HVA1 expression was correlated with net photosynthetic rate, transpiration and photochemical quenching. However, in feed-type genotypes, it was only negatively correlated with RWC.

The aim of our study was using the real-time PCR technique to test the differences in the expression levels of HVA1 and SRG6 in Polish breeding lines of spring barley.

MATERIALS AND METHODS

Plants

The real-time PCR analyses were performed on 27 breeding strains of spring barley from two Polish breeding companies: SHR Modzurow (Danko) MOB6562, MOB7890, MOB9609, MOB10654, MOB10740, MOB11558, MOB11723, MOB5735, MOB7009, MOB11728, MOB11803, MOB11990, MOB12055 and HR Strzelce STH754, STH779, STH836, STH917, STH1036, STH1146, STH369, STH828, STH858, STH906, STH915, STH1034, STH1067, STH1112. Altogether 10 seeds of each strain were sown in flowerpots containing a mixture of clay, peat and sand (3:2:1). The conditions of plant growth were described in detail by Rapacz et al. [12]. Before drought induction soil water content was kept at a level of 70% of field water capacity. Samples (0.03g to 0.05g) were collected from the youngest but fully developed leaves twice: before the drought, and after 7 days of drought, when soil water content was 32% of field water capacity. Then samples were frozen in liquid nitrogen and stored at -80°C.

RNA isolation and reverse transcription

The total RNA was isolated using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the attached protocol. Reverse transcriptions were performed using a QuantiTect® Reverse Transcription Kit (Qiagen), according to the manufacturer's protocol. The quantity of extracted cDNA was estimated by Ultraspec 2100, (Amersham Bioscience, Uppsala, Sweden)

Quantitative real-time PCR

Three leaves were collected for each genotype before and during the drought treatment. Further analyses were performed on two samples and the third sample was analysed only when the results of the remaining two were inconsistent.

The HVA1 and SRG6 mRNA expressions were quantified on a 7500 Real-Time PCR System using labelled TaqMan® probes and a TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Relative to the level before drought quantification for each gene was performed with beta-actin gene as an endogenous control. The values of relative expression and standard errors were calculated with 7500 Real-Time PCR System software using the standard curve method. Primers and probes were designed using Primer Express software v. 3.0 (Applied Biosystems) based on the published sequence for the Hordeum vulgare from NCBI:  FJ026804 - HVA1, AJ300144 – SRG6, AY145451 – beta-actin.Primers and probes used in this experiment were the same as those used in Rapacz et al. [12]. In our study, 62.5 ng of cDNA to the sample was added.

The reactions were performed in three repeats and in accordance with the TaqMan® Universal PCR Master Mix protocol: one step at 95°C for 10 min (polymerase activation) and 40 cycles at 95°C for 15 sec (denaturation) and 1 min at 60°C (annealing/extending). The results obtained were analyzed using Sequence Detection System 7500 software v. 2.0.1 (Applied Biosysystems).

RESULTS AND DISCUSSION

The results of our study showed a high variation in the relative expression of both genes studied during the drought treatment of Polish breeding material of barley (Fig. 1, 2, 3, 4). However, the increase in the SRG6 gene transcript was not related to the changes observed in HVA1 gene expression. Among the 27 spring barley genotypes, STH 754 demonstrated a high level of the expression of both genes studied. Contrary to STH 754, in genotype MOB 12055, the level of transcript accumulation for both genes was close to the minimum value.

Fig. 1. HVA1 transcript accumulation observed after 7 days of drought treatment in plants of Polish breeding lines of barley from SHR Modzurow (Danko) relative to the values before drought.

Fig. 2. HVA1 transcript accumulation observed after 7 days of drought treatment in plants of Polish breeding lines of barley from HR Strzelce relative to the values before drought.

Fig. 3. Srg6 transcript accumulation observed after 7 days of drought treatment in plants of Polish breeding lines of barley from SHR Modzurow (Danko) relative to the values before drought.

Fig. 4. Srg6 transcript accumulation observed after 7 days of drought treatment in plants of Polish breeding lines of barley from HR Strzelce relative to the values before drought.

The highest level of HVA1 induction was observed for two genotypes: STH 915 and STH754 (respectively, over 4 and 3 times increase in comparison to unstressed plants). For 18 genotypes of barley the level of transcript accumulation remains almost unchanged. For 7 was increased by a factor of 1.4 to 2.2. Thus a high variation of induction level was observed. The differences between genotypes in SRG6 transcription level were not as large as in the case of HVA1. The highest level of SRG6 transcript accumulation was observed in STH 917 and it was over 4 time larger than before stress induction. Higher level of relative gene expression was also measured for 18 genotypes (increased by a factor of 1.5 to 3.8). For the 8 genotypes changes from the gene expression were hardly observed. Moreover, in 5 genotypes the expression level observed before the drought was higher than after the drought, showing rather gene repression. The lowest value of relative gene expression was measured for  STH 1034. Although in our study only 27 genotypes were examined, a wide spectrum of expression levels was observed. These differences can be used in breeding programmes of cultivars for the areas where droughts occur frequently.

The results of our experiments showed that drought stress caused a significant increase in the expression of HVA1 and SRG6 genes in almost all genotypes (excepted STH 1034, STH836 and MOB 12055/04). The tested strains of barley showed different level of expression. It was in general higher in genotypes more resistant to drought stress [12]. It is suggested that more efficient mechanisms of stress response exist in these plants. In vivo studies carried out on yeast showed that yeast with higher expression of the HVA1 gene demonstrated a higher tolerance to cell dehydration [8]. Other studies showed that rice transformed by the HVA1 gene showed a higher growth rate and for a longer time retained the integrity of cells during drought compared with the control plant [15]. Under control conditions a wild type and a transformed plant did not differ from each other. After the induction of water deficit, transgenic plants showed no symptoms of dehydration of the cells two days longer than the wild type. Even when they already started to lose turgor, this process was not as severe as in non-transformed plants [16].

Other plants transformed by the HVA1 gene with positive effects for drought tolerance were oat [9] and wheat [13]. Oraby's et al. [11] research showed the stability of the transfer of the active HVA1 gene to the next generation, and thus its tolerance to drought.

Up to now only a few experiments have been carried out for SRG6 and the function of this gene is not clearly defined. Tong et al. [14] reported that over expression of the TaSRG6 gene (wheat analogue of SRG6) conferred to the tolerance to drought stress in modified Arabidopsis plants in many aspects such as: survival rate, cell membrane stability and relative water loss. It was suggested that the SRG6 gene may play a major role in enhancing tolerance to water stress, but the exact mechanism of its action was not confirmed. In the same paper the accumulation of fusion protein during transient expression (a protein marker and protein TaSRG6) was observed in the nucleus of onion epidermal cells, which indicates possible SRG6 function as a transcription factor. Also the mechanism of this gene induction in drought remains unknown.

It should also be mentioned that the level of HVA1 and SRG6 transcript accumulation measured in our experiment may not always be an optimum criterion for the selection towards drought tolerance. On the one hand, the effectiveness of this method may depend on the basic genetic pool, while on the other, on environmental conditions for which the selection should be directed. Rapacz et al. [12] showed that it is possible to select genotypes of higher drought tolerance among Polish breeding lines of spring barleys, but this selection should be different for the group of malting and fodder barleys. In the Rapacz et al. paper [12] the increase in water content in leaves which may depend on HVA1 accumulation, was important in the improvement of the drought tolerance of fodder barleys only. In our experiment the increase in transcript accumulation was evaluated under controlled conditions, which simulated only one drought scenario in the field. Cattivelii et al. [3] in their research confirmed the fact that the impact of the environment may be important for HVA1 gene transcript accumulation and final plant tolerance to drought. The effect of the environment-genotype interaction was also examined by Comadran et al. [6].

HVA1 and SRG6 proteins play roles in the tolerance to cells dehydration in plants. The identification of other genes whose expression may affect the accumulation or keeping of water in plant cells could be helpful in gaining a better understanding of plants tolerance to drought. Our results may be useful in developing new genetic markers for the selection of genotypes with improved drought tolerance from among Polish spring barleys. However, to create a viable working system for Polish plant breeding it will be necessary to characterize the genotype-environment interactions on both gene expressions under field conditions in Poland.

ACKNOWLEDGEMENTS

This work was supported by Polish National Research and Development Center [PBZ-MNiSW-2/3/2006/17].

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Accepted for print: 30.09.2011

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Joanna Romanek
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production
Krakowska 1,
32-083 Balice
email: jromanek@izoo.krakow.pl

Hanna Walczak
Department of Plant Physiology
University of Agriculture in Krakow
Podłużna 3
30-239 Krakow, Poland

Magdalena Wójcik-Jagła
Department of Plant Physiology
University of Agriculture in Krakow
Podłużna 3
30-239 Krakow, Poland

Barbara Jurczyk
Department of Plant Physiology
University of Agriculture in Krakow
Podłużna 3
30-239 Krakow, Poland

Marcin Rapacz
Department of Plant Physiology
University of Agriculture in Krakow
Podłużna 3
30-239 Krakow, Poland

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