Arabidopsis Ca2+-dependent nuclease AtCaN2 plays a negative role in plant responses to salt stress
Introduction
Nucleases are a large group of enzymes, characterized by considerable structural and functional diversity. In eukaryotic cells, they are involved in several processes, including DNA restriction digestion, repair, recombination, transposition, and programmed cell death (PCD) [[1], [2], [3]].
Nucleases are classified into endo- and exo-types, according to their enzymatic properties. The identified endonucleases are usually classified into two major classes, according to their metal ion cofactors and optimum pH [1]. The first class includes Zn2+-dependent endonucleases, characterized by the requirement for Zn2+ and an optimum activity at acidic pH [1]. The second class includes Ca2+-dependent endonucleases, characterized by the requirement for Ca2+, an optimum activity at neutral pH, and an active site similar to that of staphylococcal nucleases (SNases or SNcs) [4]. Some Ca2+-dependent nucleases can also be activated by Mg2+ [5], Mn2+ [6], or Co2+ [7].
Numerous Zn2+-dependent nucleases (also termed S1-type nucleases) have been described in plants. Triques et al. [8] have reported five endonucleases (ENDO1–ENDO5) in Arabidopsis thaliana. ENDO1 has also been reported as bifunctional nuclease 1 (BFN1), with both DNase and RNase activity [9], a strong preference for single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) as a substrate, an optimum activity at slightly basic pH, and a strong activation by Zn2+ [10]. ENDO2 shows nuclease activity similar to that of BFN1 [10,11]. Other S1-like endonucleases, similar to BFN1, have been reported in other species, such as celery mismatch nuclease I (CELI) [12], barley endonuclease 1 BEN1 [13], and Zinnia endonucleases 1 (ZEN1) [13,14]. In the Zinnia culture system, ZEN1, with an optimum pH of 5.5, is localized in the vacuole [13,14]. Only after the collapse of the tonoplast, ZEN1 protein is released and then genomic DNA degenerates during the differentiation of tracheary element (TE) [14]. The degradation of nuclear DNA is a key characteristic of plant PCD. Therefore, ZEN1 is known as a key enzyme involved in PCD during the development of plants.
In addition to Zn2+-dependent nucleases, strong and complex activities of Ca2+-dependent nucleases are observed in plant tissues. Recently, in A. thaliana, two SNase-like proteins, AtCaN1 and AtCaN2, have been reported as Ca2+-dependent nucleases that use ssDNA, dsDNA, and RNA as substrates [4,15,16]. Gene expression analysis from microarray data has indicated that AtCaN1 and AtCaN2 are induced during leaf senescence and infectious diseases [17]. A nuclease similar to these AtCaNs, CsCaN, has been identified in cucumber (Cucumis sativus) by screening of genes specifically expressed in female flowers. CsCaN exhibits Ca2+-dependent nuclease activity, and is involved in the primordial anther-specific DNA damage of developing female cucumber flowers [18]. In the tree species Eucommia ulmoides Oliv., two Ca2+-dependent nucleases, EuCaN1 and EuCaN2, have been shown to digest both ssDNA and dsDNA and be involved in PCD during secondary xylem development [19].
In summary, in plants, the roles of nucleases have been mostly studied in PCD during the process of plant development, whereas only limited information is available on nuclease involvement in response to various abiotic stress factors. In our previous study, we cloned a Ca2+-dependent nuclease, AtCaN2, in Arabidopsis, purified its recombinant protein, and its nuclease activity was primarily examined using λDNA and RNA as substrates [16]. In the present study, we further comprehensively identified the model of AtCaN2 nuclease activity, and characterized its expression pattern, and involvement in plant responses to salt stress.
Section snippets
Phylogenetic analysis
AtCaN2 homologs were identified by BLASTP searches against the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) database. The sequences were aligned using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/2), and the tree was constructed using MEGA 6.06 (http://www.megasoftware.net/), with the maximum likelihood method based on the Jones–Taylor–Thornton model. The reliability of branches was assessed by 1000 bootstrap replications.
Construction of mutated His-AtCaN2 fusion expression plasmids
The fragment of AtCaN2 (R223S) was
Comparison of AtCaN2 with related nucleases
The phylogenetic analysis of the AtCaN1 and AtCaN2 sequences and similar CaN sequences from Arabidopsis, rice, maize, tobacco, cucumber, and Staphylococcus aureus revealed that AtCaN1 and AtCaN2 shared the highest identity with dicot SNc sequences (Fig. 1a). The amino acid sequence alignment (Fig. 1b) showed that these proteins had a similar structure and contained two highly conserved SNc domains, with two Ca2+ binding aspartic acid residues and one arginine residue, which is probably involved
Discussion
Until date, only a few genes encoding nucleases have been identified and characterized [4,13,14,16,18,19]. Among them, studies on ZEN1 [13,14], CsCaN [18], EuCaN1, and EuCaN2 [19] concluded that nucleases could be involved in PCD during plant development. In another report, a Ca2+/Mg2+ nuclease localizes in the nucleus of the wheat grain nucellar cells undergoing PCD, and nuclear extracts from the cells trigger DNA fragmentation and other apoptotic morphology in nuclei from different plant
Conflict of interest
The authors declare no conflicts of interest.
Author contribution statement
XXZ conceived the idea for the study. XXZ and WTS designed experiments. WTS, KYG, and LL performed the experiments. SKL and TT gave precious comments on the experiments. XXZ wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study was supported by a grant to Shenkui Liu from Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT_17R99) and by two grants to Xinxin Zhang from New Century Excellent Talents in University (NECT-10-0314) and the Fundamental Research Funds for the Central Universities (2572014DA06).
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