BACKGROUND
deoxyribonucleases are a large group of enzymes characterized by considerable structural and functional diversity.
in eukaryotic cells they are involved in a range of cellular functions, including dna repair, recombination and genome degradation.
the degradation of nuclear dna, a hallmark of programmed cell death , is a process that occurs both in animals and in plants.
however, despite the many functional similarities between plant and animal pcd, degradation of nuclear dna in representatives of these two kingdoms seems to serve fundamentally different purposes.
dna is an immunogenic molecule for animals and if the dna of apoptotic cells is left undigested it can cause inflammatory responses and auto-immune diseases
<cit> .
in plants, where these processes do not occur, degradation of dna as well as rna is most likely involved in nutrient remobilization, since nucleic acids are a rich source of nitrogen and phosphorus, which belong to the major limiting nutrients for plant growth
<cit> .
the known degradative nucleases involved in plant pcd belong mainly to the s <dig> family, identified as sharing a sequence similarity with the s <dig> nuclease from aspergillus oryzae <cit> .
one of the best characterized members of this family, bfn <dig> endonuclease, is induced during senescence and developmental pcd of arabidopsis <cit> .
direct evidence of nuclease function in pcd has been reported for another member of this family, zen <dig>  which is responsible for dna degradation during xylogenesis
<cit> .
moreover, the expression of other s <dig> type nucleases has been identified in various tissues undergoing pcd
<cit> .
the expression, activity and cellular location of degradative nucleases are strictly controlled due to their potential toxicity to host cells.
in animals apoptotic dna degradation occurs in two systems.
in first, dna endonucleolytical hydrolysis is carried out by caspase-activated dnase , which in non-apoptotic cells resides in the nucleus as an inactive enzyme bound with an inhibitor protein .
the caspase-dependent apoptotic pathway releases cad nuclease, which is further activated by specific chromosomal proteins
<cit> .
moreover, mitochondrial caspase-independent endonuclease g, seems to be also involved in apoptotic dna cleavage
<cit> .
in the next stage, the fragmented dna is digested by lysosomal dnaseii after the dying cells are phagocytosed.
it has been proposed that in plants the degradative nucleases, as well as other hydrolytic enzymes involved in pcd, accumulate in the vacuole of maturing cells.
then, during the final stage of pcd, tonoplast rupture releases them into the cytoplasm resulting in autolysis of the cell contents
<cit> .
although such a mechanism has been observed during tracheary element programmed cell death
<cit> , farage-barhom et al.
<cit>  reported that the senescence-associated bfn <dig> nuclease is initially deposited in filamentous structures spread throughout the cytoplasm but in late senescent cells, it was localized in fragmented nuclei vesicles.
it should be noted that, although the endonucleolytic cleavage of nuclear dna in plant cells undergoing pcd has been well-documented, the explanation of how degraded dna components are exported from the dying cells for further reutilization remains open.
degradative nucleases are also used by some bacteria to utilize exogenous dna molecules as a nutrient source
<cit> .
although most of these enzymes are secretory proteins, the membrane-associated nucleases that have been identified in various mycoplasma species are particularly interesting in the context of this paper.
mycoplasma is an intracellular bacterium which is devoid of enzymes involved in the biosynthesis of nucleotides.
however, mycoplasmas are able to acquire nucleotides from host nucleic acids.
it has been shown that some mycoplasma nucleases can be considered pathogenic determinants because of their ability to induce the apoptotic changes characterized by the internucleosomal fragmentation of host chromatin
<cit> .
furthermore, other observations have revealed that some mycoplasma membrane-associated nucleases are components of an abc transport system involved in the import of nucleotides released from host dna
<cit> .
these mycoplasma nucleases are processed for attachment to the cell membrane through the addition of a lipid moiety.
amino acid sequence analysis revealed that known mycoplasma membrane-associated nucleases possess one of the two types of catalytic domains, i.e.
dnasei-like motif in the case of mnua nuclease of m. pulmonis <cit>  or staphylococcal-like snc domain in mph379
<cit>  and mg_186
<cit> .
the staphylococcal nuclease  domain was first described for the nuc thermonuclease of staphylococcus aureus, in which this protein is secreted to degrade exogenous nucleic acids.
characterized bacterial snase domain containing proteins are small, heat stable, ca2+-dependent nucleases, digesting single-stranded dna and/or double stranded dna
<cit> .
proteins containing staphylococcal nuclease domain have also been identified in other kingdoms of life.
the tudor staphylococcal nucleases , the best characterized multifunctional eukaryotic proteins of this class, are composed of tandem repeats of staphylococcal nuclease-like domains
<cit> .
the next group of plant proteins showing similarity to staphylococcal nuclease is characterized by the presence of a single snase domain.
in the arabidopsis genome, this small family consists of two genes, i.e.
at3g <dig> and at2g <dig>  the literature concerning this protein family is very limited.
isono et al.
<cit>  and guo et al.
<cit>  reported that the bacterially expressed protein products of arabidopsis staphylococcal-like genes, named the can proteins , reveal dnase activity.
moreover, gu et al.
<cit>  observed that the expression of the cucumber homologue of the arabidopsis can nuclease, designated cscan, is ethylene inducible and is probably involved in the primordial anther-specific dna damage of developing female cucumber flowers.
in this paper we present experimental evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization.
despite their sequence similarity, both of these enzymes show different catalytic properties and expression profiles, suggesting that they might exert different biological functions.
since one of them is specifically expressed in tissues undergoing various types of pcd we suspect that it is involved in plant genomic degradation.
we discuss the potential functions of these nucleases in relation to their unique intracellular location.
RESULTS
can nucleases are predicted to be n-myristoylated and palmitoylated proteins with modified snase domains
a blast search of the ncbi protein database revealed that in plants two classes of proteins possess domains homologous to active sites of staphylococcal nucleases.
one of them comprises two genes, at5g <dig> and at5g <dig>  containing four tandem repeats of staphylococcal nuclease-like domains followed by a tudor and c-terminal snc domain.
the second class comprises two genes, at3g <dig> and at2g <dig>  encoding proteins designated in this paper as can <dig> and can <dig>  respectively.
in contrast to tudor motive containing proteins, the can <dig> and can <dig> possess single snase domains.
detailed analyses of both can amino acid sequences revealed that these proteins exhibit complex primary structures consisting of various motifs conserved throughout the different classes of proteins.
one of the most intriguing features of both can proteins is the unique structure of their catalytic domains located near their carboxyl terminal ends.
this domain contains all conserved amino acid residues considered to be functionally critical for the enzymatic activity of the staphylococcal nuclease .
however, our analysis revealed that this domain is divided by a short sequence, including ca.
<dig> amino acids, that does not exhibit any homology to known snase domains.
this insertion separates a highly conserved n-end part of snc domain , containing one of the ca2+ binding aspartate residues, from the c-end part of this domain , containing a second ca2+ binding aspartate and three amino acid residues putatively directly involved in enzyme catalysis, i.e.
two arginines and one glutamate .
both parts of can <dig> and can <dig> snc domains show a most significant similarity to the catalytic domain of bacterial parb nuclease; however, their similarity to well defined staphylococcal nucleases
<cit>  is also significant.
the sequence analysis of the amino acid fragment that divides two parts of snc domain, unexpectedly revealed that this region shares homology with some bacterial cysteinyl-trna synthetases .
what we find particularly interesting is that this can nucleases motive almost exactly corresponds to the trna synthetases domain responsible for the recognition of the trna anticodon loop
<cit> .
furthermore, the five out of the six residues which in trna synthetases either directly interact with anticodon nucleotides or are critical for the stability of the binding cavity are strictly or highly conserved in can proteins.
it should be noted that this insertion is conserved among all plant can homologues including those from evolutionarily ancient plants like physcomitrella and selaginella .
we also examined a potential post-translational modification of can nucleases.
using the myristoylator program we identified the n-terminal myristoylation consensus motifs in both arabidopsis can nucleases.
the glycine residue at position  <dig>  which in myristoylated proteins serves as an acceptor site for myristate
<cit> , is also present in all other plant can homologues.
both can nucleases have been classified by boisson et al.
<cit>  to the list of  <dig> putatively myristoylated arabidopsis proteins , determined on the basis of combined experimental and bioinformatic approaches.
myristoylation can influence the ability of a protein to interact with membrane, but the presence of myristoyl moiety is not sufficient for stable membrane attachment.
it is believed that protein membrane anchorage, induced by myristoylation, is followed by palmitoylation of cysteine residue
<cit> .
application of css-palm software, designed for palmitoylation prediction
<cit> , showed the presence of a putative palmitoylation site directly downstream of the myristoylation motive in can <dig> and can <dig> .
since such an arrangement of myristoylation and palmitoylation sites at the n terminus has been identified in a number of membrane proteins this suggests that this region of can proteins may also be involved in membrane attachment.
the can protein region, located between n-terminal myristoylation/palmitoylation motifs and the snc catalytic domain does not exhibit similarities to any defined domain, neither does it reveal significant homology to any other plant or animal protein sequences.
however, a blast search of this amino acid sequence within the protein databases showed its highest homology to some periplasmic components of bacterial abc-type transporters
<cit> .
although the low sequence similarity existing between can nucleases and abc transporters does not allow any definite conclusions to be drawn about their potentially analogous functions, it might indicate their putative phylogenetic relationships.
can <dig> and can <dig> nucleases transiently expressed in protoplasts vary in their catalytic activities
the nucleotide sequences deposited in public databases have been used to design pcr primers for amplification of full length cdnas encoding both arabidopsis staphylococcal-like nucleases.
the microarray data, discussed later in this article, suggest that can <dig> expression can be induced by developmental and stress cues, including age dependent senescence, and that the activity of the can <dig> gene is essentially constitutive.
consequently, we used reverse transcribed mrna purified from early senescent leaves as a template for pcr amplification of both genes.
the rt-pcr amplified products were cloned into plant expression vectors, which allowed us to obtain transient expression of recombinant native as well as c-terminal ha- and fluorescent protein-tagged fusion proteins.
the published report concerning plant staphylococcal-like nucleases demonstrated the ca2+-dependent activity of bacterially expressed proteins
<cit> .
however, since the activity of plant enzymes may be affected by various post-translation modifications, we decided to examine both members of this family after transient over-expression in arabidopsis protoplasts.
in our study we transformed the protoplasts prepared from root cell cultures and from leaf mesophile and obtained similar results.
to estimate the efficiency of protein synthesis, the protein extracts from transformed protoplasts were subjected to western blot analysis using an antibody against the ha-domain tagged at the c-terminus of the transgenes.
as shown in figure
2a, both transgenes were efficiently expressed in protoplasts prepared from arabidopsis suspension cells.
the molecular mass of the recombinant can <dig> and can <dig> proteins, estimated using western blot analysis, was approximately  <dig> kda and  <dig> kda, roughly the same as predicted from the deduced amino acid sequences:  <dig>  kda and  <dig>  kda, respectively.
to identify the dnase activity of can proteins over-expressed in protoplasts, we used an in-gel nuclease activity assay.
application of this assay in combination with single stranded dna used as a substrate and in the presence of calcium ions allowed us to detect clear bands representing nucleolytic activity at the position corresponding to the molecular mass of can <dig> and can <dig> .
all repeated experiments revealed that, despite the comparable efficiency of expression of both nucleases, the yield of nuclease activity was significantly higher for can <dig>  analogous experiments performed with undenatured dna demonstrated that both nucleases also digest double stranded dna, but in this case the activity of both nucleases is comparable.
an adjustment of the reaction conditions allowed us to estimate that the maximum activity of both proteins was at ph  <dig>  and a ca2+ concentration of about  <dig> mm with a range of  <dig> to  <dig> mm .
moreover, we found that in the presence of edta both nucleases did not reveal any activity.
a pronounced inhibition effect was also observed with zn2+.
moreover, we found that heat denaturation in the presence of 2-mercaptoethanol prior to electrophoresis does not inhibit the activities of either enzyme, suggesting a lack of the functionally important disulphide bonds in their native structure.
in gel rnase assay, conducted with plant total rna as a substrate  revealed that both nucleases also have ability to degrade rna which demonstrate that they can be defined as the sugar-nonspecific nucleases.
such a diverse substrate specificity suggests that the function of can nucleases is associated with degradative processes.
in the present studies we also used can recombinant nucleases fused with ecfp, gfp and rfp.
since the stability of recombinant proteins with a c-end or n-end attached fluorescent marker protein can be altered post-translationally by proteolytic processing we also analyzed them via western blot and in-gel nuclease activity assay.
as shown in figure
2d and e, the electrophoretic mobilities of both nucleases with c-tagged ecfp were retarded as compared with co-electrophoresed untagged proteins, corresponding to their enhanced molecular weight.
furthermore, the nuclease activity of can2-ecfp seems to be unaltered in comparison to untagged can <dig> and is significantly stronger than can1-ecfp.
bioinformatic analysis revealed that plant can nucleases, in addition to snc domains, also contain some conserved domains of unknown function.
in order to understand their relevance for nucleolytic activity, the can <dig> deletion mutants were constructed to remove regions, comprising conserved residues of each domain .
because we intended to use these constructs to study can <dig> nucleolytic activity as well as its cellular location, deletion mutants of untagged can <dig> and cfp-tagged can <dig> were prepared.
despite the identical condition of transformation in all repeated experiments we observed a decreased amount of can <dig> mutant with the deleted myristoylation and palmitoylation motifs, which suggests that this region can be involved in protein stability .
the analysis of the nuclease activity of untagged and cfp-fused can <dig> revealed that mutants lacking the n-snc, c-snc domains and abc protein-like motifs, are completely inactive .
instead, deletion of the amino acid sequence showing a similarity to the anti-codon binding domain decreases but does not eliminate can <dig> dnase activity.
this is particularly interesting, given the fact that this region is located inside the catalytic domain, but apparently is not crucial for can1/ <dig> dnase and rnase activities .
the expression of can <dig> nuclease is induced during xylogenesis and leaf senescence and correlates with host-pathogen interactions, whereas the expression of can <dig> is generally constitutive
in order to establish gene expression profiles for both can nucleases we analyzed microarray experiments reported by genevestigator
<cit> .
this database has collected results of  <dig> microarray experiments  performed by authors exploring various aspects of arabidopsis biology.
since in many cases the same determinant of gene expression was analyzed independently by several research groups, in our opinion their results provide an opportunity to obtain verifiable and objective information about the expression patterns of selected genes.
we began our studies by examining can <dig>  and can <dig>  genes expression during different developmental processes.
in principle all microarray experiments conducted to compare the arabidopsis tissue specific transcription profiles clearly indicate that can <dig> nuclease is preferentially expressed in stems and roots .
on the other hand, low expression of can <dig> mrna was observed in seeds, seedling, young leaves and flowers.
in contrast to can <dig>  the organ specific variability of can <dig> expression was relatively small.
the analysis of other microarrays revealed that transcription of can <dig> is low in the upper part of the stem but significantly increased in the lower part .
since these parts of the stem differ mainly in the degree of xylem development, this suggests that can <dig> nuclease can be involved in this process.
this assumption has been confirmed by experiments conducted to identify the genes involved in development of different parts of the vascular bundle, i.e.
xylem, phloem, cortex, , as well as genes activated in cells induced to transdifferentiate into xylem tracheary elements .
the level of can <dig> expression in young and mature leaves is low but it increases with leaf aging.
this effect was observed in developmental leaf senescence  as well as in leaves that have been induced to senesce by nitrogen deficiency .
figure
3h-i shows that the expression pattern of can <dig> nuclease in senescent leaves is analogous to the at1g <dig> gene encoding the well defined bfn <dig> nuclease involved in leaf senescence
<cit> .
both genes, at1g <dig>  and at3g <dig> , have also been included in the list of about  <dig> genes showing at least a  <dig> fold up regulation during leaf senescence, identified by buchanan-wollaston et al.
<cit> .
numerous microarray studies have demonstrated upregulation of can <dig> expression during environmental stresses.
similar to developmental processes, external stimuli significantly modulate expression of can <dig>  but weakly affect expression of can <dig> gene .
we observed that among all the tested factors those that are related with pathogenesis have the strongest influence on can <dig> expression.
the analysis of the experiments presented in figure
4a-d and additional file
<dig> clearly indicates that upregulation of this gene is stimulated by some bacteria and viruses, while fungi, nematodes and insects do not exert such an effect.
among the ten experiments conducted to study various aspects of pseudomonas sp.
infection, nine revealed an elevated level of can <dig> mrna in relation to controls in tissues attacked by this pathogen .
the transcription of the can <dig> gene was also stimulated by e.coli  and dsdna virus, calcuv .
other experiments have shown that activation of can <dig> does not depend on the virulence of the pseudomonas strain   and also occurs after elicitor treatment.
three of four available experiments investigating the influence of flagellin on gene expression have shown that this elicitor can activate can <dig> transcription .
the particularly strong upregulation of the can <dig> gene was observed in two experiments studying the influence of syringolin a, the virulence factor secreted by p.syringae .
<dig>   microarray study showing specific activation of can <dig> expression in response to bacterial infection.
plant responses to the following microbial and insect pathogens are presented: leaf bacterium , pathogenic leaf fungus , tissue-chewing caterpillars , cell-content-feeding thrips  and phloem-feeding aphids .
three experiments showing the effect of p. syringae  and dna virus   on can <dig> gene expression.
influence of different p. syringae strains on can <dig> expression.
influence of different pathogen-derived elicitors on can <dig> gene expression.
- harpin elicitor,  - necrosis-inducing phytophthora protein  <dig>   – flagellin,  – lipopolysaccharide.
expression of can <dig> gene in plants treated with syringolin a  alone and with a combination of syringolin a and erysiphe cichoracearum infection.
to verify the data obtained from the microarray experiments we examined whether suggested mrna expression profiles of can <dig> and can <dig> genes correspond to any endogenous nucleolytic activity.
we analyzed nuclease activity profiles of developing stem and senescing leaves.
the development of xylem begins in the lower part of the stem and this zone successively moves towards the upper part of the growing stem.
according to the scheme presented by brown et al.
<cit>  we analyzed the protein extracts isolated from different parts of stems obtained from plants aged from  <dig> to  <dig> weeks.
as shown in figure
5a, in-gel nuclease activity assay revealed that the samples prepared from stems contain two nucleases migrating to the same position as over-expressed can <dig> and can <dig> used as a control.
the activity of endogenous nucleases corresponding to can <dig> was essentially constant, which agrees with the observation derived from the microarray data.
in contrast, the activity of the endogenous nuclease migrating in an identical fashion as can <dig> is much more variable, i.e.
it is invisible or weak in the upper part of stems and increases towards the stem base.
to identify the nucleases associated with senescence we analyzed protein extracts obtained from the individual rosette leaves of  <dig> and  <dig> week old plants.
as presented in figure
5b and c, endogenous nuclease activity corresponding to can <dig> appears for the first time in older leaves of a  <dig> week old rosette and becomes more common in the  <dig> week old rosette leaves.
thus, this result supports the role of can <dig> nuclease in age-dependent development.
the activity profile of can <dig> nuclease identified in  <dig> and  <dig> week old leaves as well as in the leaves of younger plants  was much more constant, which supports the hypothesis that this nuclease is characterized by a constitutive expression.
the assumption that observed endogenous nuclease activities refer to can <dig> and can <dig> proteins stems from the facts that corresponding proteins possess exactly the same electrophoretic mobility, catalytic requirements, and unusual resistance to heat denaturation, detergents and 2-mercaptoethanol.
resistance to a reducing agent provides an opportunity to distinguish endogenous plant staphylococcal-like nucleases from plant s <dig> type nucleases, which possess disulphide bonds and in our experience are inhibited by 2-mercapthoethanol .
this allows us to conclude that microarray mrna expression data, confirmed by our protein activity assays, together suggest that expression of can <dig> nuclease is associated with different forms of pcd, such as xylem development or senescence, while can <dig> expression is generally constitutive.
the can <dig> and can <dig> nucleases are anchored to the plasma membrane, probably via n-terminal myristoylation and palmitoylation
in order to determine the cellular distribution of can nucleases, the translational fusion constructs, can1-ecfp and can2-ecfp, were transiently expressed in arabidopsis root protoplasts.
as a negative control, an empty vector plasmid, psat4a-ecfp , was used.
as demonstrated in figure
6a, ecfp alone, used as a control, is typically distributed throughout the cytoplasm and nucleus.
in turn, intensive and sharp fluorescent rings were visible around the protoplasts with over-expressed can <dig> and can <dig> nucleases fused to ecfp .
the same subcellular location of both proteins we observed in transformed leaf protoplasts .
protoplasts transfected with the same constructs but analyzed via fluorescence microscopy displayed uniform fluorescence of the protoplast surfaces .
these fluorescence images suggest that both nucleases are associated with plasma membrane.
to ensure that this unusual location of proteins possessing nuclease activity is not the artificial result of the protoplast preparation procedure we also analyzed the location of both nucleases in plant cells transformed via agrobacterium tumefaciens.
as shown in figure
7a and d, the fluorescence of can1-rfp and can2-gfp fusion proteins appeared as sharp lines along the edges of transformed epidermal cells.
magnifications of selected areas showed that the fluorescence signal forms two parallel lines indicating plasma membranes of two neighboring cells .
moreover, as presented in figure
7e, drought induced plasmolysis caused displacement of the fluorescence signal towards the cell center, which further proves the association of can nucleases with the plasma membrane.
the bioinformatic analysis of can nucleases amino acid sequences did not reveal any pronounced hydrophobic regions that could represent transmembrane domains.
to identify the segments potentially interacting with the plasma membrane we applied deletion mutants of the can <dig> nuclease  for transfection experiments.
as shown in figure
8b-e, the deletion mutations created in the abc-like domain, the anticodon binding-like domain and both fragments of snc domains did not affect the plasma membrane localization of can <dig>  insted, deletion of the n-end fragment containing the myristoylation/palmitylation motifs significantly affected subcellular localization of this nuclease .
the removal of the myristoylation/palmitylation motifs from the can <dig> nuclease resulted in the same effect .
since it is known that n-terminus myristoylation is required for membrane targeting and that palmitoylation of proximal cysteine residue greatly stabilizes membrane association, we concluded that can nucleases are most probably anchored to the plasma membrane via these posttranslational modifications.
2f the length of the white scale bar at the right bottom of each panel corresponds to  <dig> μm.
we took into account that can nucleases could change their location in response to various cellular and exogenous factors related to pcd.
to verify such a possibility the protoplasts containing over-expressed can1-ecfp and can2-ecfp were then subjected to treatment with the following factors, in the ranges given in brackets: h2o <dig> , heat stress , salt stress , flg <dig> peptide , e. coli strain dh5α  and a.tumefaciens strain gv <dig> .
moreover, we compared the cellular location of can nucleases over-expressed in protoplast prepared from the cells of young , mature  and early senescent  rosette leaves.
however, we did not observe any influence of the above treatment on can subcellular location .
moreover, we did not detect over-expressed proteins in the protoplast medium which suggests that under the tested conditions they are not secreted from the cell .
these results show that can nucleases probably do not change their cellular localization under stress conditions that could trigger pcd or plant defense response.
discussion
despite the significant sequence similarity, eukaryotic proteins possessing snase domains appear to fulfill different biochemical and biological functions.
for example, the tudor staphylococcal nuclease  possessing both dnase and rnase activities, specifically interacts with and promotes the cleavage of dsrna substrates.
since this protein is a component of the risc complex, it is expected that it contributes to the rna degradation observed in rnai
<cit> .
the other eukaryotic homologue of staphylococcal nuclease, the parb, acquired a novel function during evolution, acting as a redox enzyme
<cit> .
our attempt to detect the potential nuclease activity of the arabidopsis parb-like protein  which we performed in parallel to the study described in this paper also did not reveal any nuclease activity of this protein .
in this paper, we examined the molecular activity and biological function of enzymes belonging to the arabidopsis two member protein family characterized by the presence of a single snase domain.
transient expression assay using protoplasts, together with biochemical approaches, enabled us to show that can <dig> and can <dig> exhibit calcium dependent deoxyribonuclease activities similar to that of the bacterial snases.
we found that, despite the presence of conserved calcium binding and catalytic amino acid residues in can snase domains, they also contain an additional, conserved motif showing strong homology to the anticodon binding domain of some bacterial trna synthetases.
although both of these domains appear to be of bacterial origin, they do not occur together in such an arrangement in any known prokaryotic protein.
how the putative rna binding motif affects dnase activity of plant nucleases remains unknown.
however, because the deletion of this motif decreases, but does not eliminate dnase and rnase activities, it could be speculated that the anticodon-binding like domain contributes to the enhancement of the can nucleases catalytic activity.
despite their amino acid sequence similarity, can <dig> and can <dig> exhibit some significant differences.
we demonstrate that, while can <dig> nuclease digests the single stranded and double stranded dna with comparable efficiency, can <dig> strongly prefers single-stranded dna as its substrate.
moreover, the genes encoding both nucleases exhibited fundamentally different expression profiles which may suggest that, despite having the same subcellular distribution, they do not cooperate in a common biological process.
it seems that the most remarkable feature of both can nucleases is their unusual cellular localization.
nucleases that have been studied so far were identified in cellular compartments containing nucleic acids, such as the nucleus
<cit> , plastids and mitochondria
<cit>  or in the organelles where they are stored before final application, for example, vacuole
<cit> , lysosome
<cit>  and other intracellular membrane vesicles
<cit> .
to our knowledge, the can enzymes described in this paper are the only eukaryotic nucleases associated with the plasma membrane.
considering the potential function of can nucleases in the context of their unusual location, it might be tempting to speculate that plasma membrane nucleases could be involved in defense response against pathogen invading plant cells.
this assumption could be supported by our finding that expression of can <dig> correlates well with bacterial and viral infection.
however, overall analysis of can <dig> expression patterns displayed that activation of its gene is also associated with developmental processes unrelated to the pathogenic response, such as xylem formation and leaf senescence.
thus, it seems that the can <dig> nuclease is not involved in the pathogen’s genetic material degradation, unless it has more than one function.
similarly, it cannot currently be excluded that a second member of this family, constitutively expressed can <dig> nuclease, is involved in some, as yet uncharacterized mechanism of pathogen dna processing at the cell surface or in the endosomes.
detailed analysis of the can <dig> expression profile revealed that its gene is specifically up-regulated in two developmental processes leading to controlled cell death, i.e.
xylogenesis and senescence.
induction of can <dig> expression in response to bacterial and viral attack may also be related to cell death, because recognition of avirulent pathogens can trigger resistance-associated pcd, a hypersensitive response, which also involves dna degradation.
it should be noted that some hr induced tobacco deoxyribonucleases
<cit> , whose coding sequences have not yet been identified, possess catalytic properties similar to those of can <dig> and can <dig> as presented in this paper.
the assumption that can nucleases can be involved in pcd was also proposed by gu et al.
<cit> , who observed the correlation of increased expression of their cucumber homologue, cscan, with primordial anther-specific dna damage.
moreover, it is worth noting that ability of can nucleases to efficient and nonspecific degradation of dna as well as rna is also characteristic for pcd associated degradation processes.
it is believed that one of the main function of the degradation processes occurring during some types of plant pcd, mainly in the case of senescence, is redistribution of nutrients from dying cells to the expanding or sink tissues.
since nitrogen deficiency is the major nutritional factor limiting plant growth, most research has focused on understanding the reutilization of reduced nitrogen released from proteins, especially from rubisco, which is the most abundant protein in plants.
available evidence shows that this is a multistage process characterized by intra- or intercellular compartmentalization
<cit> .
degradation of rubisco initiated by plastidial and vacuolar endopeptidases is continued into single amino acids and then to ammonium, which is finally reassimilated into glutamate, glutamine and aspartate.
since the concentrations of these amino acids in phloem sap increase during late senescence, it is believed that they are the major forms of long-distance translocatable products of protein degradation
<cit> .
although the initial proteolysis occurs in the senescent mesophill cells, subsequent steps, catalyzed by glutamate dehydrogenase  or glutamine synthetase , occur in cells adjacent to vascular bundles, such as vascular parenchyma or companion cells
<cit> .
the catabolic pathways responsible for the recycling of nucleic acid-derived nutrients are much less understood than those concerning proteins.
nucleotides are the second main source of cellular nitrogen as well as an important reservoir of phosphorus.
however, it is still unknown to what extent nucleic acids are degraded during pcd, and how these degradation products are processed for long-distance transport via the phloem.
nucleic acid degradation seems to be initiated by s1-type enzymes, such as bfn <dig>  zen <dig>  ben <dig>  whose expression correlates with different types of plant pcd.
the nature of the reaction products generated by these nucleases is still not clear but, since these enzymes possess endonucleolytic activity, they probably produce dna fragments of various sizes.
however, the study of phloem sap content during senescence did not reveal any dna fragments or single deoxynucleotides.
instead, pronounced expression of some nucleobase and phosphorus transporters was demonstrated in phloem vessels
<cit>  suggesting that some additional reaction must lead to further degradation of the s <dig> type nuclease derived dna fragments and subsequent nucleotide catabolism.
moreover, assuming that nucleotides, similar to amino acids, are catabolized in cells associated with vascular bundles, some mechanism responsible for intercellular transfer of dna degradation products from dying cells should also be considered.
this, as yet hypothetical, process would be in analogy with those, that enable the mycoplasma to acquire nucleic acid precursors.
since the can <dig> nuclease presented here seems to be associated with pcd and its properties resemble the mycoplasma membrane nucleases, in the future we intend to verify the hypothesis that this enzyme combines dna degradation with transfer of degradation products between the dying cells and the cells that are responsible for further nucleotide catabolic processes.
the current state of knowledge does not allow us to clearly explain at what stage of above described processes the can nucleases may be involved and various hypotheses concerning their function require further studies.
however, the identification of a novel class of nucleases characterized by their plasma membrane location indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for dna degradation.
CONCLUSIONS
one of the main function of plasma membrane is to provide a highly selective transport of molecules important to cell growth.
an intensive export of nutrients released from degraded macromolecules is observed in plant tissues undergoing pcd.
although nucleic acids are a rich source of nitrogen, phosphorus and nucleotide bases little is known about the export of dna and rna degraded products from dying cells.
nucleases responsible for genomic dna degradation during pcd were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application.
in this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization.
the identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for nucleic acid degradation.

 deoxyribonucleases are a large group of enzymes characterized by considerable structural and functional diversity. in eukaryotic cells they are involved in a range of cellular functions, including dna repair, recombination and genome degradation. the degradation of nuclear dna, a hallmark of programmed cell death, is a process that occurs both in animals and in plants. however, despite the many functional similarities between plant and animal pcd, degradation of nuclear dna in representatives of these two kingdoms seems to serve fundamentally different purposes. dna is an immunogenic molecule for animals and if the dna of apoptotic cells is left undigested it can cause inflammatory responses and auto-immune diseases. in plants, where these processes do not occur, degradation of dna as well as rna is most likely involved in nutrient remobilization, since nucleic acids are a rich source of nitrogen and phosphorus, which belong to the major limiting nutrients for plant growth. the known degradative nucleases involved in plant pcd belong mainly to the s family, identified as sharing a sequence similarity with the s nuclease from aspergillus oryzae. one of the best characterized members of this family, bfn endonuclease, is induced during senescence and developmental pcd of arabidopsis. direct evidence of nuclease function in pcd has been reported for another member of this family, zen which is responsible for dna degradation during xylogenesis. moreover, the expression of other s type nucleases has been identified in various tissues undergoing pcd. the expression, activity and cellular location of degradative nucleases are strictly controlled due to their potential toxicity to host cells. in animals apoptotic dna degradation occurs in two systems. in first, dna endonucleolytical hydrolysis is carried out by caspase-activated dnase, which in non-apoptotic cells resides in the nucleus as an inactive enzyme bound with an inhibitor protein. the caspase-dependent apoptotic pathway releases cad nuclease, which is further activated by specific chromosomal proteins. moreover, mitochondrial caspase-independent endonuclease g, seems to be also involved in apoptotic dna cleavage. in the next stage, the fragmented dna is digested by lysosomal dnaseii after the dying cells are phagocytosed. it has been proposed that in plants the degradative nucleases, as well as other hydrolytic enzymes involved in pcd, accumulate in the vacuole of maturing cells. then, during the final stage of pcd, tonoplast rupture releases them into the cytoplasm resulting in autolysis of the cell contents. although such a mechanism has been observed during tracheary element programmed cell death, farage-barhom et al. reported that the senescence-associated bfn nuclease is initially deposited in filamentous structures spread throughout the cytoplasm but in late senescent cells, it was localized in fragmented nuclei vesicles. it should be noted that, although the endonucleolytic cleavage of nuclear dna in plant cells undergoing pcd has been well-documented, the explanation of how degraded dna components are exported from the dying cells for further reutilization remains open. degradative nucleases are also used by some bacteria to utilize exogenous dna molecules as a nutrient source. although most of these enzymes are secretory proteins, the membrane-associated nucleases that have been identified in various mycoplasma species are particularly interesting in the context of this paper. mycoplasma is an intracellular bacterium which is devoid of enzymes involved in the biosynthesis of nucleotides. however, mycoplasmas are able to acquire nucleotides from host nucleic acids. it has been shown that some mycoplasma nucleases can be considered pathogenic determinants because of their ability to induce the apoptotic changes characterized by the internucleosomal fragmentation of host chromatin. furthermore, other observations have revealed that some mycoplasma membrane-associated nucleases are components of an abc transport system involved in the import of nucleotides released from host dna. these mycoplasma nucleases are processed for attachment to the cell membrane through the addition of a lipid moiety. amino acid sequence analysis revealed that known mycoplasma membrane-associated nucleases possess one of the two types of catalytic domains, i.e. dnasei-like motif in the case of mnua nuclease of m. pulmonis or staphylococcal-like snc domain in mph379 and mg_186. the staphylococcal nuclease domain was first described for the nuc thermonuclease of staphylococcus aureus, in which this protein is secreted to degrade exogenous nucleic acids. characterized bacterial snase domain containing proteins are small, heat stable, ca2+-dependent nucleases, digesting single-stranded dna and/or double stranded dna. proteins containing staphylococcal nuclease domain have also been identified in other kingdoms of life. the tudor staphylococcal nucleases, the best characterized multifunctional eukaryotic proteins of this class, are composed of tandem repeats of staphylococcal nuclease-like domains. the next group of plant proteins showing similarity to staphylococcal nuclease is characterized by the presence of a single snase domain. in the arabidopsis genome, this small family consists of two genes, i.e. at3g and at2g the literature concerning this protein family is very limited. isono et al. and guo et al. reported that the bacterially expressed protein products of arabidopsis staphylococcal-like genes, named the can proteins, reveal dnase activity. moreover, gu et al. observed that the expression of the cucumber homologue of the arabidopsis can nuclease, designated cscan, is ethylene inducible and is probably involved in the primordial anther-specific dna damage of developing female cucumber flowers. in this paper we present experimental evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. despite their sequence similarity, both of these enzymes show different catalytic properties and expression profiles, suggesting that they might exert different biological functions. since one of them is specifically expressed in tissues undergoing various types of pcd we suspect that it is involved in plant genomic degradation. we discuss the potential functions of these nucleases in relation to their unique intracellular location.  can nucleases are predicted to be n-myristoylated and palmitoylated proteins with modified snase domains a blast search of the ncbi protein database revealed that in plants two classes of proteins possess domains homologous to active sites of staphylococcal nucleases. one of them comprises two genes, at5g and at5g containing four tandem repeats of staphylococcal nuclease-like domains followed by a tudor and c-terminal snc domain. the second class comprises two genes, at3g and at2g encoding proteins designated in this paper as can and can respectively. in contrast to tudor motive containing proteins, the can and can possess single snase domains. detailed analyses of both can amino acid sequences revealed that these proteins exhibit complex primary structures consisting of various motifs conserved throughout the different classes of proteins. one of the most intriguing features of both can proteins is the unique structure of their catalytic domains located near their carboxyl terminal ends. this domain contains all conserved amino acid residues considered to be functionally critical for the enzymatic activity of the staphylococcal nuclease. however, our analysis revealed that this domain is divided by a short sequence, including ca. amino acids, that does not exhibit any homology to known snase domains. this insertion separates a highly conserved n-end part of snc domain, containing one of the ca2+ binding aspartate residues, from the c-end part of this domain, containing a second ca2+ binding aspartate and three amino acid residues putatively directly involved in enzyme catalysis, i.e. two arginines and one glutamate. both parts of can and can snc domains show a most significant similarity to the catalytic domain of bacterial parb nuclease; however, their similarity to well defined staphylococcal nucleases is also significant. the sequence analysis of the amino acid fragment that divides two parts of snc domain, unexpectedly revealed that this region shares homology with some bacterial cysteinyl-trna synthetases. what we find particularly interesting is that this can nucleases motive almost exactly corresponds to the trna synthetases domain responsible for the recognition of the trna anticodon loop. furthermore, the five out of the six residues which in trna synthetases either directly interact with anticodon nucleotides or are critical for the stability of the binding cavity are strictly or highly conserved in can proteins. it should be noted that this insertion is conserved among all plant can homologues including those from evolutionarily ancient plants like physcomitrella and selaginella. we also examined a potential post-translational modification of can nucleases. using the myristoylator program we identified the n-terminal myristoylation consensus motifs in both arabidopsis can nucleases. the glycine residue at position which in myristoylated proteins serves as an acceptor site for myristate, is also present in all other plant can homologues. both can nucleases have been classified by boisson et al. to the list of putatively myristoylated arabidopsis proteins, determined on the basis of combined experimental and bioinformatic approaches. myristoylation can influence the ability of a protein to interact with membrane, but the presence of myristoyl moiety is not sufficient for stable membrane attachment. it is believed that protein membrane anchorage, induced by myristoylation, is followed by palmitoylation of cysteine residue. application of css-palm software, designed for palmitoylation prediction, showed the presence of a putative palmitoylation site directly downstream of the myristoylation motive in can and can. since such an arrangement of myristoylation and palmitoylation sites at the n terminus has been identified in a number of membrane proteins this suggests that this region of can proteins may also be involved in membrane attachment. the can protein region, located between n-terminal myristoylation/palmitoylation motifs and the snc catalytic domain does not exhibit similarities to any defined domain, neither does it reveal significant homology to any other plant or animal protein sequences. however, a blast search of this amino acid sequence within the protein databases showed its highest homology to some periplasmic components of bacterial abc-type transporters. although the low sequence similarity existing between can nucleases and abc transporters does not allow any definite conclusions to be drawn about their potentially analogous functions, it might indicate their putative phylogenetic relationships. can and can nucleases transiently expressed in protoplasts vary in their catalytic activities the nucleotide sequences deposited in public databases have been used to design pcr primers for amplification of full length cdnas encoding both arabidopsis staphylococcal-like nucleases. the microarray data, discussed later in this article, suggest that can expression can be induced by developmental and stress cues, including age dependent senescence, and that the activity of the can gene is essentially constitutive. consequently, we used reverse transcribed mrna purified from early senescent leaves as a template for pcr amplification of both genes. the rt-pcr amplified products were cloned into plant expression vectors, which allowed us to obtain transient expression of recombinant native as well as c-terminal ha- and fluorescent protein-tagged fusion proteins. the published report concerning plant staphylococcal-like nucleases demonstrated the ca2+-dependent activity of bacterially expressed proteins. however, since the activity of plant enzymes may be affected by various post-translation modifications, we decided to examine both members of this family after transient over-expression in arabidopsis protoplasts. in our study we transformed the protoplasts prepared from root cell cultures and from leaf mesophile and obtained similar results. to estimate the efficiency of protein synthesis, the protein extracts from transformed protoplasts were subjected to western blot analysis using an antibody against the ha-domain tagged at the c-terminus of the transgenes. as shown in figure  2a, both transgenes were efficiently expressed in protoplasts prepared from arabidopsis suspension cells. the molecular mass of the recombinant can and can proteins, estimated using western blot analysis, was approximately kda and kda, roughly the same as predicted from the deduced amino acid sequences: kda and kda, respectively. to identify the dnase activity of can proteins over-expressed in protoplasts, we used an in-gel nuclease activity assay. application of this assay in combination with single stranded dna used as a substrate and in the presence of calcium ions allowed us to detect clear bands representing nucleolytic activity at the position corresponding to the molecular mass of can and can. all repeated experiments revealed that, despite the comparable efficiency of expression of both nucleases, the yield of nuclease activity was significantly higher for can analogous experiments performed with undenatured dna demonstrated that both nucleases also digest double stranded dna, but in this case the activity of both nucleases is comparable. an adjustment of the reaction conditions allowed us to estimate that the maximum activity of both proteins was at ph and a ca2+ concentration of about mm with a range of to mm. moreover, we found that in the presence of edta both nucleases did not reveal any activity. a pronounced inhibition effect was also observed with zn2+. moreover, we found that heat denaturation in the presence of 2-mercaptoethanol prior to electrophoresis does not inhibit the activities of either enzyme, suggesting a lack of the functionally important disulphide bonds in their native structure. in gel rnase assay, conducted with plant total rna as a substrate revealed that both nucleases also have ability to degrade rna which demonstrate that they can be defined as the sugar-nonspecific nucleases. such a diverse substrate specificity suggests that the function of can nucleases is associated with degradative processes. in the present studies we also used can recombinant nucleases fused with ecfp, gfp and rfp. since the stability of recombinant proteins with a c-end or n-end attached fluorescent marker protein can be altered post-translationally by proteolytic processing we also analyzed them via western blot and in-gel nuclease activity assay. as shown in figure  2d and e, the electrophoretic mobilities of both nucleases with c-tagged ecfp were retarded as compared with co-electrophoresed untagged proteins, corresponding to their enhanced molecular weight. furthermore, the nuclease activity of can2-ecfp seems to be unaltered in comparison to untagged can and is significantly stronger than can1-ecfp. bioinformatic analysis revealed that plant can nucleases, in addition to snc domains, also contain some conserved domains of unknown function. in order to understand their relevance for nucleolytic activity, the can deletion mutants were constructed to remove regions, comprising conserved residues of each domain. because we intended to use these constructs to study can nucleolytic activity as well as its cellular location, deletion mutants of untagged can and cfp-tagged can were prepared. despite the identical condition of transformation in all repeated experiments we observed a decreased amount of can mutant with the deleted myristoylation and palmitoylation motifs, which suggests that this region can be involved in protein stability. the analysis of the nuclease activity of untagged and cfp-fused can revealed that mutants lacking the n-snc, c-snc domains and abc protein-like motifs, are completely inactive. instead, deletion of the amino acid sequence showing a similarity to the anti-codon binding domain decreases but does not eliminate can dnase activity. this is particularly interesting, given the fact that this region is located inside the catalytic domain, but apparently is not crucial for can1/ dnase and rnase activities. the expression of can nuclease is induced during xylogenesis and leaf senescence and correlates with host-pathogen interactions, whereas the expression of can is generally constitutive in order to establish gene expression profiles for both can nucleases we analyzed microarray experiments reported by genevestigator. this database has collected results of microarray experiments performed by authors exploring various aspects of arabidopsis biology. since in many cases the same determinant of gene expression was analyzed independently by several research groups, in our opinion their results provide an opportunity to obtain verifiable and objective information about the expression patterns of selected genes. we began our studies by examining can and can genes expression during different developmental processes. in principle all microarray experiments conducted to compare the arabidopsis tissue specific transcription profiles clearly indicate that can nuclease is preferentially expressed in stems and roots. on the other hand, low expression of can mrna was observed in seeds, seedling, young leaves and flowers. in contrast to can the organ specific variability of can expression was relatively small. the analysis of other microarrays revealed that transcription of can is low in the upper part of the stem but significantly increased in the lower part. since these parts of the stem differ mainly in the degree of xylem development, this suggests that can nuclease can be involved in this process. this assumption has been confirmed by experiments conducted to identify the genes involved in development of different parts of the vascular bundle, i.e. xylem, phloem, cortex,, as well as genes activated in cells induced to transdifferentiate into xylem tracheary elements. the level of can expression in young and mature leaves is low but it increases with leaf aging. this effect was observed in developmental leaf senescence as well as in leaves that have been induced to senesce by nitrogen deficiency. figure  3h-i shows that the expression pattern of can nuclease in senescent leaves is analogous to the at1g gene encoding the well defined bfn nuclease involved in leaf senescence. both genes, at1g and at3g, have also been included in the list of about genes showing at least a fold up regulation during leaf senescence, identified by buchanan-wollaston et al.. numerous microarray studies have demonstrated upregulation of can expression during environmental stresses. similar to developmental processes, external stimuli significantly modulate expression of can but weakly affect expression of can gene. we observed that among all the tested factors those that are related with pathogenesis have the strongest influence on can expression. the analysis of the experiments presented in figure  4a-d and additional file clearly indicates that upregulation of this gene is stimulated by some bacteria and viruses, while fungi, nematodes and insects do not exert such an effect. among the ten experiments conducted to study various aspects of pseudomonas sp. infection, nine revealed an elevated level of can mrna in relation to controls in tissues attacked by this pathogen. the transcription of the can gene was also stimulated by e.coli and dsdna virus, calcuv. other experiments have shown that activation of can does not depend on the virulence of the pseudomonas strain and also occurs after elicitor treatment. three of four available experiments investigating the influence of flagellin on gene expression have shown that this elicitor can activate can transcription. the particularly strong upregulation of the can gene was observed in two experiments studying the influence of syringolin a, the virulence factor secreted by p.syringae. microarray study showing specific activation of can expression in response to bacterial infection. plant responses to the following microbial and insect pathogens are presented: leaf bacterium, pathogenic leaf fungus, tissue-chewing caterpillars, cell-content-feeding thrips and phloem-feeding aphids. three experiments showing the effect of p. syringae and dna virus on can gene expression. influence of different p. syringae strains on can expression. influence of different pathogen-derived elicitors on can gene expression. - harpin elicitor, - necrosis-inducing phytophthora protein – flagellin, – lipopolysaccharide. expression of can gene in plants treated with syringolin a alone and with a combination of syringolin a and erysiphe cichoracearum infection. to verify the data obtained from the microarray experiments we examined whether suggested mrna expression profiles of can and can genes correspond to any endogenous nucleolytic activity. we analyzed nuclease activity profiles of developing stem and senescing leaves. the development of xylem begins in the lower part of the stem and this zone successively moves towards the upper part of the growing stem. according to the scheme presented by brown et al. we analyzed the protein extracts isolated from different parts of stems obtained from plants aged from to weeks. as shown in figure  5a, in-gel nuclease activity assay revealed that the samples prepared from stems contain two nucleases migrating to the same position as over-expressed can and can used as a control. the activity of endogenous nucleases corresponding to can was essentially constant, which agrees with the observation derived from the microarray data. in contrast, the activity of the endogenous nuclease migrating in an identical fashion as can is much more variable, i.e. it is invisible or weak in the upper part of stems and increases towards the stem base. to identify the nucleases associated with senescence we analyzed protein extracts obtained from the individual rosette leaves of and week old plants. as presented in figure  5b and c, endogenous nuclease activity corresponding to can appears for the first time in older leaves of a week old rosette and becomes more common in the week old rosette leaves. thus, this result supports the role of can nuclease in age-dependent development. the activity profile of can nuclease identified in and week old leaves as well as in the leaves of younger plants was much more constant, which supports the hypothesis that this nuclease is characterized by a constitutive expression. the assumption that observed endogenous nuclease activities refer to can and can proteins stems from the facts that corresponding proteins possess exactly the same electrophoretic mobility, catalytic requirements, and unusual resistance to heat denaturation, detergents and 2-mercaptoethanol. resistance to a reducing agent provides an opportunity to distinguish endogenous plant staphylococcal-like nucleases from plant s type nucleases, which possess disulphide bonds and in our experience are inhibited by 2-mercapthoethanol. this allows us to conclude that microarray mrna expression data, confirmed by our protein activity assays, together suggest that expression of can nuclease is associated with different forms of pcd, such as xylem development or senescence, while can expression is generally constitutive. the can and can nucleases are anchored to the plasma membrane, probably via n-terminal myristoylation and palmitoylation in order to determine the cellular distribution of can nucleases, the translational fusion constructs, can1-ecfp and can2-ecfp, were transiently expressed in arabidopsis root protoplasts. as a negative control, an empty vector plasmid, psat4a-ecfp, was used. as demonstrated in figure  6a, ecfp alone, used as a control, is typically distributed throughout the cytoplasm and nucleus. in turn, intensive and sharp fluorescent rings were visible around the protoplasts with over-expressed can and can nucleases fused to ecfp. the same subcellular location of both proteins we observed in transformed leaf protoplasts. protoplasts transfected with the same constructs but analyzed via fluorescence microscopy displayed uniform fluorescence of the protoplast surfaces. these fluorescence images suggest that both nucleases are associated with plasma membrane. to ensure that this unusual location of proteins possessing nuclease activity is not the artificial result of the protoplast preparation procedure we also analyzed the location of both nucleases in plant cells transformed via agrobacterium tumefaciens. as shown in figure  7a and d, the fluorescence of can1-rfp and can2-gfp fusion proteins appeared as sharp lines along the edges of transformed epidermal cells. magnifications of selected areas showed that the fluorescence signal forms two parallel lines indicating plasma membranes of two neighboring cells. moreover, as presented in figure  7e, drought induced plasmolysis caused displacement of the fluorescence signal towards the cell center, which further proves the association of can nucleases with the plasma membrane. the bioinformatic analysis of can nucleases amino acid sequences did not reveal any pronounced hydrophobic regions that could represent transmembrane domains. to identify the segments potentially interacting with the plasma membrane we applied deletion mutants of the can nuclease for transfection experiments. as shown in figure  8b-e, the deletion mutations created in the abc-like domain, the anticodon binding-like domain and both fragments of snc domains did not affect the plasma membrane localization of can insted, deletion of the n-end fragment containing the myristoylation/palmitylation motifs significantly affected subcellular localization of this nuclease. the removal of the myristoylation/palmitylation motifs from the can nuclease resulted in the same effect. since it is known that n-terminus myristoylation is required for membrane targeting and that palmitoylation of proximal cysteine residue greatly stabilizes membrane association, we concluded that can nucleases are most probably anchored to the plasma membrane via these posttranslational modifications. 2f the length of the white scale bar at the right bottom of each panel corresponds to μm. we took into account that can nucleases could change their location in response to various cellular and exogenous factors related to pcd. to verify such a possibility the protoplasts containing over-expressed can1-ecfp and can2-ecfp were then subjected to treatment with the following factors, in the ranges given in brackets: h2o, heat stress, salt stress, flg peptide, e. coli strain dh5α and a.tumefaciens strain gv. moreover, we compared the cellular location of can nucleases over-expressed in protoplast prepared from the cells of young, mature and early senescent rosette leaves. however, we did not observe any influence of the above treatment on can subcellular location. moreover, we did not detect over-expressed proteins in the protoplast medium which suggests that under the tested conditions they are not secreted from the cell. these results show that can nucleases probably do not change their cellular localization under stress conditions that could trigger pcd or plant defense response. discussion despite the significant sequence similarity, eukaryotic proteins possessing snase domains appear to fulfill different biochemical and biological functions. for example, the tudor staphylococcal nuclease possessing both dnase and rnase activities, specifically interacts with and promotes the cleavage of dsrna substrates. since this protein is a component of the risc complex, it is expected that it contributes to the rna degradation observed in rnai. the other eukaryotic homologue of staphylococcal nuclease, the parb, acquired a novel function during evolution, acting as a redox enzyme. our attempt to detect the potential nuclease activity of the arabidopsis parb-like protein which we performed in parallel to the study described in this paper also did not reveal any nuclease activity of this protein. in this paper, we examined the molecular activity and biological function of enzymes belonging to the arabidopsis two member protein family characterized by the presence of a single snase domain. transient expression assay using protoplasts, together with biochemical approaches, enabled us to show that can and can exhibit calcium dependent deoxyribonuclease activities similar to that of the bacterial snases. we found that, despite the presence of conserved calcium binding and catalytic amino acid residues in can snase domains, they also contain an additional, conserved motif showing strong homology to the anticodon binding domain of some bacterial trna synthetases. although both of these domains appear to be of bacterial origin, they do not occur together in such an arrangement in any known prokaryotic protein. how the putative rna binding motif affects dnase activity of plant nucleases remains unknown. however, because the deletion of this motif decreases, but does not eliminate dnase and rnase activities, it could be speculated that the anticodon-binding like domain contributes to the enhancement of the can nucleases catalytic activity. despite their amino acid sequence similarity, can and can exhibit some significant differences. we demonstrate that, while can nuclease digests the single stranded and double stranded dna with comparable efficiency, can strongly prefers single-stranded dna as its substrate. moreover, the genes encoding both nucleases exhibited fundamentally different expression profiles which may suggest that, despite having the same subcellular distribution, they do not cooperate in a common biological process. it seems that the most remarkable feature of both can nucleases is their unusual cellular localization. nucleases that have been studied so far were identified in cellular compartments containing nucleic acids, such as the nucleus, plastids and mitochondria or in the organelles where they are stored before final application, for example, vacuole, lysosome and other intracellular membrane vesicles. to our knowledge, the can enzymes described in this paper are the only eukaryotic nucleases associated with the plasma membrane. considering the potential function of can nucleases in the context of their unusual location, it might be tempting to speculate that plasma membrane nucleases could be involved in defense response against pathogen invading plant cells. this assumption could be supported by our finding that expression of can correlates well with bacterial and viral infection. however, overall analysis of can expression patterns displayed that activation of its gene is also associated with developmental processes unrelated to the pathogenic response, such as xylem formation and leaf senescence. thus, it seems that the can nuclease is not involved in the pathogen’s genetic material degradation, unless it has more than one function. similarly, it cannot currently be excluded that a second member of this family, constitutively expressed can nuclease, is involved in some, as yet uncharacterized mechanism of pathogen dna processing at the cell surface or in the endosomes. detailed analysis of the can expression profile revealed that its gene is specifically up-regulated in two developmental processes leading to controlled cell death, i.e. xylogenesis and senescence. induction of can expression in response to bacterial and viral attack may also be related to cell death, because recognition of avirulent pathogens can trigger resistance-associated pcd, a hypersensitive response, which also involves dna degradation. it should be noted that some hr induced tobacco deoxyribonucleases, whose coding sequences have not yet been identified, possess catalytic properties similar to those of can and can as presented in this paper. the assumption that can nucleases can be involved in pcd was also proposed by gu et al., who observed the correlation of increased expression of their cucumber homologue, cscan, with primordial anther-specific dna damage. moreover, it is worth noting that ability of can nucleases to efficient and nonspecific degradation of dna as well as rna is also characteristic for pcd associated degradation processes. it is believed that one of the main function of the degradation processes occurring during some types of plant pcd, mainly in the case of senescence, is redistribution of nutrients from dying cells to the expanding or sink tissues. since nitrogen deficiency is the major nutritional factor limiting plant growth, most research has focused on understanding the reutilization of reduced nitrogen released from proteins, especially from rubisco, which is the most abundant protein in plants. available evidence shows that this is a multistage process characterized by intra- or intercellular compartmentalization. degradation of rubisco initiated by plastidial and vacuolar endopeptidases is continued into single amino acids and then to ammonium, which is finally reassimilated into glutamate, glutamine and aspartate. since the concentrations of these amino acids in phloem sap increase during late senescence, it is believed that they are the major forms of long-distance translocatable products of protein degradation. although the initial proteolysis occurs in the senescent mesophill cells, subsequent steps, catalyzed by glutamate dehydrogenase or glutamine synthetase, occur in cells adjacent to vascular bundles, such as vascular parenchyma or companion cells. the catabolic pathways responsible for the recycling of nucleic acid-derived nutrients are much less understood than those concerning proteins. nucleotides are the second main source of cellular nitrogen as well as an important reservoir of phosphorus. however, it is still unknown to what extent nucleic acids are degraded during pcd, and how these degradation products are processed for long-distance transport via the phloem. nucleic acid degradation seems to be initiated by s1-type enzymes, such as bfn zen ben whose expression correlates with different types of plant pcd. the nature of the reaction products generated by these nucleases is still not clear but, since these enzymes possess endonucleolytic activity, they probably produce dna fragments of various sizes. however, the study of phloem sap content during senescence did not reveal any dna fragments or single deoxynucleotides. instead, pronounced expression of some nucleobase and phosphorus transporters was demonstrated in phloem vessels suggesting that some additional reaction must lead to further degradation of the s type nuclease derived dna fragments and subsequent nucleotide catabolism. moreover, assuming that nucleotides, similar to amino acids, are catabolized in cells associated with vascular bundles, some mechanism responsible for intercellular transfer of dna degradation products from dying cells should also be considered. this, as yet hypothetical, process would be in analogy with those, that enable the mycoplasma to acquire nucleic acid precursors. since the can nuclease presented here seems to be associated with pcd and its properties resemble the mycoplasma membrane nucleases, in the future we intend to verify the hypothesis that this enzyme combines dna degradation with transfer of degradation products between the dying cells and the cells that are responsible for further nucleotide catabolic processes. the current state of knowledge does not allow us to clearly explain at what stage of above described processes the can nucleases may be involved and various hypotheses concerning their function require further studies. however, the identification of a novel class of nucleases characterized by their plasma membrane location indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for dna degradation.  one of the main function of plasma membrane is to provide a highly selective transport of molecules important to cell growth. an intensive export of nutrients released from degraded macromolecules is observed in plant tissues undergoing pcd. although nucleic acids are a rich source of nitrogen, phosphorus and nucleotide bases little is known about the export of dna and rna degraded products from dying cells. nucleases responsible for genomic dna degradation during pcd were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. in this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. the identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for nucleic acid degradation. 
in this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. a study of microarray experiments and endogenous nuclease activity revealed that expression of can gene correlates with different forms of programmed cell death, while the can gene is constitutively expressed. the identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for dna and rna degradation. bioinformatic and experimental data presented in this paper indicate that two arabidopsis staphylococcal-like nucleases, named can and can are anchored to the cell membrane via n-terminal myristoylation and palmitoylation modifications. the activity of degradative nucleases responsible for genomic dna digestion has been observed in all kingdoms of life. plant degradative nucleases that have been studied so far belong mainly to the s1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. the potential functions of these nucleases in relation to their unique intracellular location are discussed. it is believed that the main function of dna degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. however, the explanation of how degraded dna components are exported from the dying cells for further reutilization remains open. staphylococcal-like nucleaseprogrammed cell deathplasma-membrane proteinsenescencemyristoylation/palmitylation motifabc transportertrna synthetase both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and trna synthetase anticodon binding-like motifs.
 the activity of degradative nucleases responsible for genomic dna digestion has been observed in all kingdoms of life. it is believed that the main function of dna degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. plant degradative nucleases that have been studied so far belong mainly to the s1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. however, the explanation of how degraded dna components are exported from the dying cells for further reutilization remains open.  bioinformatic and experimental data presented in this paper indicate that two arabidopsis staphylococcal-like nucleases, named can and can are anchored to the cell membrane via n-terminal myristoylation and palmitoylation modifications. both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and trna synthetase anticodon binding-like motifs. they are neutral, ca2+-dependent nucleaces showing a different specificity toward the ssdna, dsdna and rna substrates. a study of microarray experiments and endogenous nuclease activity revealed that expression of can gene correlates with different forms of programmed cell death, while the can gene is constitutively expressed.  in this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. the identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for dna and rna degradation. the potential functions of these nucleases in relation to their unique intracellular location are discussed. staphylococcal-like nucleaseprogrammed cell deathplasma-membrane proteinsenescencemyristoylation/palmitylation motifabc transportertrna synthetase 