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NAP-Related Protein 1 (Atnrp1) Overexpression Increases the Heat Tolerance of Arabidopsis Cells/Plantlets | OMICS International
ISSN: 2329-9029
Journal of Plant Biochemistry & Physiology
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NAP-Related Protein 1 (Atnrp1) Overexpression Increases the Heat Tolerance of Arabidopsis Cells/Plantlets

Bíró J, Domoki M and Fehér A*
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt 62, H-6726 Szeged, Hungary
Corresponding Author : Fehér A
Institute of Plant Biology
Biological Research Center
Hungarian Academy of Sciences
Temesvári krt. 62, H-6726 Szeged, Hungary
Tel: +36 62599701
E-mail: [email protected]
Received April 26, 2013; Accepted July 10, 2013; Published July 17, 2013
Citation: Bíró J, Domoki M, Fehér A (2013) NAP-Related Protein 1 (Atnrp1) Overexpression Increases the Heat Tolerance of Arabidopsis Cells/Plantlets. J Plant Biochem Physiol 1:115. doi:10.4172/2329-9029.1000115
Copyright: © 2013 Bíró J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Keywords
Nucleosome assembly protein-related protein; Elevated temperature; Survival; Transgenic; Cellular localization
Introduction
Nucleosome-assembly protein-related proteins (NRPs) are multifunctional proteins having histone chaperone [1] and histone phosphatase inhibitor [2] properties and being implicated in root meristem maintenance [1], mitotic regulation [1], chromatinmediated gene silencing [1], genotoxic sensitivity [1] and homologous recombination [3]. The drosophila homologue of plant NRPs, the SET protein, has been shown to accumulate at heat-shock gene loci in response to increased temperature where it co-localized with Ser10-phosphorylated histoneH3 [4]. It was therefore hypothesized that the drosophila SET protein prevents phosphatase2A (PP2A)- mediated histoneH3 dephosphorylation and contributes to chromatin modifications facilitating heat shock protein gene transcription [4].
 
Based on the structural and functional homology of SET and NRP proteins [2], we investigated whether the altered expression of the plant At NRP1 protein interferes with the heat sensitivity of Arabidopsis cells/seedlings.
Experimental
Plant cultures and heat treatment
Experiments were carried out with the wild-type Columbia ecotype of Arabidopsis thaliana (L.) Heynh and it’s At NRP1 overexpressor genetic transformant (NRP OX; kindly provided by Valerie Frankard, CropDesign N.V., Ghent, Belgium). In these plants, the Arabidopsis nrp1-1 cDNA clone was cloned after the Helianthus annuus Gubb1 promoter for constitutive expression. T4 generation homozygous transgenic (OX) and wild type (WT) segregants of the same transformation event were used in the experiments.
Seeds of Arabidopsis thaliana were surface-sterilized by soaking in 70% ethanol (v/v) for 1 minute and in 30% bleach (v/v) for 10 minutes and then rinsed five times with sterile distilled water, and plated on solid Murashige and Skoog (MS) medium (Duchefa Biochemie, B.V., Haarlem, The Netherlands) containing 3% (w/v) sucrose, 0.6% (w/v) agar, with pH adjusted to 5.8. The seeds were germinated at 24ºC using an 8 hours light/16 hours dark light regime. Experiments were carried out at least in triplicates with 7-8-days-old seedlings grown in glass petri dishes on MS medium [2]. Heat shock treatment (45ºC for 45-60 minutes as indicated) was applied in a water bath (50 seedlings/dish; 4 dishes/line). Survival rates of plants (%) were visually determined one week after the treatment as the ratio of seedlings staying green and growing six weeks after the treatment. In the case of leaf protoplastderived cells (for isolation and culture, [5]) dead cells were stained by Evans blue (0, 5% w/v). Altogether app. 1000 cells were investigated per line in three repetitions.
Western blotting
The antibodies used and the immunoblotting protocol is described elsewhere in details [2].
Gene expression analysis by real-time quantitative PCR (RTQPCR)
RNA isolation, cDNA synthesis and RT-QPCR analysis by an ABI Prism 7700 sequence detection system have been described earlier [2]. DNA damage and repair-associated (PARP2, At4g02390; RAD51, At5g20850; AtMYB, At5g03780; RNR2, At3g27060) as well as the control unresponsive (UBC18, At5g42990) genes were selected based on the work of Chen et al. [6] (Table 1).
Results and Discussion
Using a specific antibody recognizing Arabidopsis NRP proteins [2] it was verified that NRP proteins accumulated in the AtNRP1 overproducing transgenic plants (Figure 1). 8-day-old seedlings of the wild type (WT) and NRP overexpressing (OX) plants were subjected to heat shock at 45ºC. In a series of experiments the NRP overexpressing seedlings exhibited increased survival rate one week after the treatment in comparison to controls (Figure 2A and 2B).
In order to test whether this difference in heat sensitivity of seedlings is also exhibited at the cellular level, leaf protoplasts have been isolated from WT and OX plants and were similarly subjected to a one hour 45ºC heat shock. Cell viability was determined using Evans blue staining dead cells. Similarly, as was observed in the case of seedlings, an app. 20% difference in cell viability could be detected in favor of NRP overexpressing cells (Figure 2C).
Basal thermo tolerance of plants depends on several factors, including hormonal regulation (ethylene, salicylic acid), production and scavenge of reactive oxygen species and the expression of various genes including heat shock transcription factors and heat shock proteins [7,8], several of which play also role during acquired thermotolerance. However, we have previously shown that NRPs are dispensable for the heat-induced expression of heat shock protein genes in Arabidopsis; despite the fact that they act as potent histoneH3 phosphatase inhibitors in vitro [2].
Strong heat causes DNA strand breaks what if not repaired can finally result in cell death. NRPs has recently been implicated in the maintenance of genome stability [3]. Therefore we investigated whether NRP overexpression may contribute to increased DNA repair and in this way can improve cellular survival. However, we could not detect any difference in the expression of selected DNA repair-associated (PARP2, RAD51) or genotoxic stress-upregulated (AtMYB and RNR2) genes (Figure 3). Therefore at present it is not known via what mechanism NRPs contribute to cellular heat tolerance in Arabidopsis. However, based on the presently known biochemical functions of plant NRPs, it might be related to their capability to influence chromatin structure and/or phosphatase activity [1,2].
Several functions, such as gene silencing, mitosis regulation and homologous recombination [1,3] of NRPs are associated with nuclear functions, and especially with the organization of the chromatin. Interestingly, when NRP proteins were detected by Western blotting in control cells and in cells heat shocked for one hour at 45ºC, it was observed that while in untreated cells the proteins were in the cytosolic, in heat shocked cells they were in the nuclear protein fractions (Figure 4). Therefore one can suppose that these proteins shuttle between the cytosol and the nucleus in response to heat shock. However, the detection of NRP proteins in the cytosol of WT plants is contradictory to a previous report describing the nuclear localization of green fluorescent protein-NRP fusions [1].
The animal homologues of NRPs are also nuclear proteins having, however, cytoplasmic functions as well [9]. Therefore, further studies are required to determine the dynamic cellular location of plant NRPs and its role in the high temperature response of plant cells.
Acknowledgements
The presented work was supported by the NKTH-OTKA grant K68051.
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