Ⅰ. INTRODUCTION
Abiotic stresses such as soil salinity, chemical pollutants, toxic metals, water stress and temperature extremes negatively affect growth and yield in crops (Wang et al., 2003). These stresses induce cellular damage and secondary stresses such as the oxidative and osmotic ones, which lead to protein dysfunctions (Wang et al., 2003). Plants respond to extreme temperatures by inducing the synthesis of a group of heat shock proteins (HSP) (Vierling, 1991). Among HSPs, low-molecular weight ‘small’ HSPs (sHSP) are the most prevalent in plants. These sHSPs are located at distinct cellular compartments such as the cytosol, endoplasmic reticulum, chloroplast, peroxisome, nucleus and mitochondria (Waters et al., 1995;Waters and Vierling, 2020). sHSPs contain a ~90 amino acidslong basic signature α-crystallin domain (ACD), which has a β -sandwich structure (Waters et al., 1995;Waters and Vierling, 2020). The sHSPs bind to non-native proteins and consequently stabilize and prevent aggregation (Ehrnsperger et al., 1997). This facilitates their subsequent refolding by chaperones such as the HSP70/DnaK or ClpB/HSP100 (Mogk et al. 2003;Haslbeck and Vierling, 2015). However, the mechanism of this process is not fully characterized.
Globally, abiotic stress is the major cause of crop damages, which accounts for average yield reduction by more than 50% (Wang and Luthe, 2003). Understanding various mechanisms of plant stress response is of basic importance. Numerous studies revealed that plant sHSPs are expressed not only under heat but also to a wide range of other environmental insults, such as drought, osmotic and salinity, cold and oxidative stress (Boston et al., 1996;Vierling, 1991). Thus, the role of sHSPs in plant survival to stress conditions are obvious. Mitochondrial sHSPs and chloroplast sHSPs share high similarity in their C-terminal region but differs in the N-terminus (Waters et al., 1996). Hamilton and Heckathorn (2001) reported that complex I electron transport system is protected by mitochondrial sHsp (along with antioxidants), but complex II is protected only by cellular osmoprotectants. These evidences suggests that protection of complex I through mitochondrial sHsps is associated with an antioxidant mechanism (Hamilton and Heckathorn, 2001).
To date, mitochondrial sHsps have been characterized from alfalfa (Lee et al., 2012a), maize (Lund et al., 1998), Arabidopsis (Willett et al., 1996), soybean (LaFayette et al., 1996), Chenopodium rubrum (Debel et al., 1995), pea (Lenne et al., 1995) and cotton (Ma et al., 2018). Overexpression of an alfalfa mitochondrial Hsp24 provides enhanced tolerance to arsenic (Lee et al., 2012b). However, characterized sequence information and function of rice mito-chondrial sHPS protein is rather limited. Our previous proteomic analysis revealed the novel accumulation of a putative mitochondria- localized sHsp in rice leaves under heat stress suggesting potential role in heat stress response mechanism (Lee et al., 2007). We report here the isolation and functional characterization of rice mitochondrial sHSP gene, which is highly expressed in the rice seedlings subjected to heat stress.
Ⅱ. MATERIALS AND METHODS
1. Plant growth and stress treatment
The rice (O. sativa L. cv Dongjin) seedlings were grown in a growth chamber at 25°C, under fluorescent light, with a 16 h photoperiod. Three-week-old seedlings were exposed to heat treatment by increasing the temperature up to 50°C. After each treatment, samples were harvested and immediately frozen in liquid nitrogen, and kept at -80°C until use. For other stress treatments, rice leaves were detached and the basal parts were dipped in methyl viologen (MV, 1 mM), H2O2 (10 mM), NaCl (250 mM), cadmium (200 μM) or copper (200 μM) solutions. Plants were treated with 0.2 ppm ozone, which was generated with a silent electrical discharge in dry oxygen in an open-top chamber.
2. DNA and RNA gel blot analysis
For DNA gel blot analysis, rice genomic DNA (15 μg) was digested with BamHI, EcoRI or HindIII, electrophoretically separated in 0.8% agarose gel and blotted to nylon membranes (Hybond N+, Amersham. For RNA gel blot analysis, 15 μg of total RNA was analyzed on a 1.2% formaldehyde agarose gel. The membranes were hybridized with [α-32P] dCTP-labelled fulllength Oshsp24.1 cDNA probe.
3. Cloning of Oshsp24.1 full-length cDNA by RACE-PCR
Total RNA was isolated from rice leaves and mRNA was purified from it using Oligotex mRNA kit (Quiagen, CA, USA) according to manufacturer's instruction. The purified mRNA was reverse transcribed with oligo-dT primer using reverse transcriptase. RACE-PCR was performed using 5’- or 3’-universal primer with internal Oshsp24.1-specific primers using SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). The purified RACE-PCR product was inserted into pGEM-T Easy vector, and was sequenced in both directions.
4. Expression of OsHSP24.1 recombinant protein in E. coli
The coding sequence of Oshsp24.1 cDNA was amplified with forward (5’- GACATATGGCTTCCATCGTGGCA-3’) and reverse primers (5’- GGGAATTCCTACTCGACGTTGAC-3’), and ligated into NdeI-EcoRI digested pET28a vector (Novagen, Merck, Germany). E. coli BL21 (DE3) cells harboring 6×His-tagged OsHSP24.1- pET28a vector was cultured in LB medium and expression of recombinant fusion protein was induced by IPTG. After 4 h incubation, E. coli cells were pelleted by centrifugation and disrupted by sonication. His-tagged fusion protein were purified by nickel metal-affinity chromatography. The recombinant OsHSP24.1 protein was analyzed by SDS-PAGE.
5. Cross-linking and immunobloting
For cross-linking, 3 mM sulfo-ethylene glycol bis (succinimidyl succinate) (sulfo-EGS) was added to 1.5 μg of purified protein. After incubation for 20 min, the reaction was quenched by the adding 1 M Tris buffer (pH7.4). Cross-linked OsHSP24.1 protein was electrophoresed through 10% SDS-PAGE and transferred to nitrocellulose membranes using a semidry electroblotter (C.B.S. scientific, Del Mar, CA, USA). Peptide (GRGYDTRRPTRDAT) corresponding to amino acid 63 to 76 of the deduced OsHSP24.1 sequence was synthesized and used to polyclonal antibody production in rabbits (Peptron, Daejeon, Korea). The membranes were blocked and incubated with an anti-OsHSP24.1 antibody for 1 h. After washing, the membranes incubated with a secondary goat anti-rabbit IgG antibody conjugated with HRP. The signals on membranes were detected by ECL (Biological industries, Israel) and visualized on X-ray films (Fuji Medical X-ray film, Japan)
Ⅲ. Results and Discussion
1. Cloning of a Oshsp24.1 cDNA from rice
Our previous proteomic study revealed the novel accumulation of a putative mitochondria-targeted low molecular weight sHSP in rice seedlings when exposed to heat for 24 or 48 h (Lee et al., 2007). Subsequently, here we amplified a cDNA fragment from the heat-treated rice leaves by RACE-PCR. Sequencing of this fragment revealed that it is a rice sHSP (Oshsp24.1). The nucleotide sequence has been submitted to the NCBI GenBank and has been allocated the accession number JF710844.
2. Characterization of the Oshsp24.1 sequence
The Oshsp24.1 cDNA clone was 880 bp long. It contains a 61 bp 5'-untranslated region (UTR), a 156 bp 3' UTR, and a 663 bp-long open reading frame (ORF). These shows that the Oshsp24.1 encodes for a protein consists of 220 amino acids with a calculated molecular mass is 24.1 kDa. Sequence analysis revealed a conserved domain of ~90 amino acid-long toward the C-terminus of the protein. This is the signature α-crystallin domain found in all plant sHsps (Fig. 1A). Comparison of OsHSP24.1 amino acid sequence with the Pea mitochondrial HSP22 (Lenne et al., 1995) suggests that OsHSP24.1 contains 44 amino acid-long mitochondria transit peptide. The mature peptide of OsHSP24.1 has 176 amino acids. Comparative analysis showed that OsHSP24.1 shares a high degree of homology with sHsps from other species including maize and sorghum (Fig. 1). OsHSP24.1 showed the highest identity with the other rice mitochondrial sHSP isoform, Oshsp24.1A (XP_025878696) and millet PmHSP24.1B by 99%. All these bioinformatics analysis of Oshsp24 reveals that this cDNA encodes a rice 24 kDa mitochondrial sHSP protein.
3. Genomic organization of the Oshsp24.1 gene
To analyze the copy number of Oshsp24.1 gene in rice and their organization in the genome, DNA gel blot analysis was performed. Genomic DNA digested with restriction enzymes were probed with the full-length cDNA (Fig. 2). Two-hybridized bands were visible in EcoRI and HindIII-digested lane suggesting that Oshsp24.1 is most likely to exist as more than one copy in the rice genome.
4. Expression of Oshsp24.1 in response to abiotic stresses
The cloning and identification of Oshsp24.1 cDNA was based on a newly induced protein by heat stress in rice (Lee et al. 2007), indicating that the Oshsp24.1 transcript might also be expressed in response to heat stress. Therefore, we incubated rice leaves with different level of heat stresses. The Oshsp24.1 transcript is not detectable when rice plants are grown at normal temperatures (25°C), but began to appear at 30°C and peaked between 40°C and 45°C. However, no transcript was detectable in RNA gel blots above 50°C (Fig. 3A). The transcript accumulation was highest in leaves compared to leaf sheaths, while very small amount of transcripts were detected in roots (Fig. 3B). To elucidate the temporal expression, plants subjected to heat for various duration ranging from 5 to 180 min. The transcript began to appear as early as 5 min after heat exposure and reached to the maximum level at 30 min. However, the level of the transcript decreased gradually from 120 min under the stress. Some Oshsp24.1 transcripts were seen after 60 min of recovery. After a recovery of 2 h, no detectable transcript was found. Oshsp24.1 gene is very sensitive to heat exposure.
It is widely known that abiotic stresses causes overproduction of reactive oxygen species (ROS) that leads to oxidative damage and consequent cell death (Shrma et al., 2012). Therefore, we studied the expression pattern of Oshsp24.1 transcript caused by several other abiotic stress including methyl viologen, H2O2, ozone, NaCl and toxic heavy metals including cadmium and copper. Transcript levels were checked at different time point ranging from 30 min to 48 h of treatments. Among the oxidative stress inducers, MV induced accumulation of Oshsp24.1 transcript as early as 3 h of treatment. Consistently, H2O2 lead to an accumulation of transcript much earlier than MV. Ozone, NaCl and copper also showed rapid and large induction of transcripts as short as 30 min. These results suggest that the generation of ROS may induce Oshsp24.1 expression. As shown in Fig. 4, other heavy metal stress, cadmium, also induced Oshsp24.1 expression but, relatively lately. In plants and other eukaryotes, it has been reported that the rate of HSP synthesis is directly proportional to the exposed temperatures (Lindquist and Craig 1988;Chen et al 1990). Appearance and disappearance of mitochondria-localized sHSP in plants is synchronous to the heat acclimatization and de-acclimatization events. Moreover, it protects the electron transport of NADH: ubiquinone oxidoreductase on the membrane of mitochondria during the heat shock (Downs and Heckathorn 1998). Considering these, it is clear that Oshsp24.1 might be involved in multiple stresses. However, rapid and sensitive expression during heat stress suggests its special role in heat stress.
5. Biochemical characterization of recombinant OsHSP24.1 protein
SDS-PAGE gel revealed that the purified recombinant Os HSP24.1 protein has a molecular weight of about 24 kDa. The crosslink experiments with sulfo-EGS were performed to analyze the oligomeric structure of OsHSP24.1. As shown in Fig. 5, in absence of a cross-linker (sulfo EGS), His-OsHSP24.1 protein gave the band corresponding to monomer in SDS-PAGE gel. By contrast, bands with higher molecular weights than monomer observed in silver stained and immuno-blotted gels. These results suggest that OsHSP24.1 could form oligomers, which is basic characteristics as a molecular chaperone. Narberhaus (2002) reported that the significant variations in the sizes of the oligomers is due to the large variability in the length of the N-terminal regions. Thus, N-terminal regions of OsHSP24.1 is need for the interaction with substrate. It also has a role in stabilizing the oligomeric form. However, since we used His-tagged precursor OsHSP24.1 protein for cross-linking experiment, there is a possibility that these extra N-terminal residue could affect oligomerization reaction. Further experiments are required to verify this possibility. It has been reported that mutant studies showed that unstable oligomer formation reduced chaperone activity in vitro and decrease thermotolerance of Synechocystis (Giese and Vierling, 2002). Thus, in vitro oligomer formation of the OsHSP24.1 provides the experimental evidence of molecular chaperone function.
In conclusion, our present results demonstrated that Oshsp24.1 is involved in heat and oxidative stress in rice. High homology with other mitochondrial sHSP sequences, presence of α-crystallin domain and transit peptide suggest that Oshsp24.1 is a nuclear encoded mitochondrial sHsp. Biochemical characterization suggests that it is of 24 kDa size and can form oligomer in vitro indicating the high possibility of chaperone function. Meanwhile, Oshsp24.1 gene would be a desirable target gene in genetic engineering for developing stress-tolerant crop plants.
