Download Nucleases in higher plants and their possible involvement in DNA

Journal of Experimental Botany Advance Access published March 14, 2014
Journal of Experimental Botany
doi:10.1093/jxb/eru091
Review
Nucleases in higher plants and their possible involvement
in DNA degradation during leaf senescence
Wataru Sakamoto* and Tsuneaki Takami
Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710–0046, Japan
* To whom correspondence should be addressed. E-mail: [email protected]
Abstract
During leaf senescence, macromolecules such as proteins and lipids are known to be degraded for redistribution into
upper tissues. Similarly, nucleic acids appear to undergo fragmentation or degradation during senescence, but the
physiological role of nucleic acid degradation, particularly of genomic DNA degradation, remains unclear. To date,
more than a dozen of plant deoxyribonucleases have been reported, whereas it remains to be verified whether any
of them degrade DNA during leaf senescence. This review summarizes current knowledge related to the plant nucleases that are induced developmentally or in a tissue-specific manner and are known to degrade DNA biochemically.
Of these, several endonucleases (BFN1, CAN1, and CAN2) and an exonuclease (DPD1) in Arabidopsis seem to act in
leaf senescence because they were shown to be inducible at the transcript level. This review specifically examines
DPD1, which is dual-targeted to chloroplasts and mitochondria. Results show that, among the exonuclease family to
which DPD1 belongs, DPD1 expression is extraordinary when estimated using a microarray database. DPD1 is the
only example among the nucleases in which DNA degradation has been confirmed in vivo in pollen by mutant analysis. These data imply a significant role of organelle DNA degradation during leaf senescence and implicate DPD1 as
a potential target for deciphering nucleotide salvage in plants.
Key words: Cell death, chloroplasts, DNA degradation, endonuclease, exonuclease, leaf senescence, organelle DNA.
Introduction
Leaf senescence is the final stage of leaf development, which
involves coordinated regulation of transcriptional networks,
hormonal actions, and reactive oxygen species (reviewed
in Guo and Gan, 2005; Lim et al., 2007; Guo, 2013; Woo
et al., 2013). It is a complex developmental process because
the onset and progression of leaf senescence differ among
plant species. They differ even within individual leaves.
Furthermore, external factors such as soil nutrients are
known to influence senescence. Despite these complexities,
recent studies in model plants have advanced the understanding of the key elements that control leaf senescence at the
molecular level. During senescence, nutrients synthesized
in leaves are broken down and redistributed to other tissues
such as upper leaves and reproductive organs to maximize
natural fitness. Accordingly, numerous degradation enzymes
of macromolecules, including proteins, lipids, nucleic acids,
and pigments, appear to be activated. A priori, the activation
of these enzymes is expected to be possible at both transcriptional and post-transcriptional levels, although some of them
are known to be under the control of senescence-specific
transcription factors.
The most important nutrient to be recycled from senescing leaves is nitrogen. Reportedly, more than 70% of total
leaf nitrogen is contained in chloroplasts, more than a
quarter of which is contained in the abundant stromal protein Rubisco (Makino and Osmond, 1991; Gregersen et al.,
2013). It is therefore noteworthy that one of the physiological targets controlled in senescing leaves is the degradation of chloroplast components. Whereas Rubisco and
other proteins are possibly degraded by proteases as leaves
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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Received 14 January 2014; Revised 31 January 2014; Accepted 7 February 2014
Page 2 of 9 | Sakamoto and Takami
General view of deoxyribonucleases
in plants
An accepted context in both animals and plants is that
apoptosis or other cell-death processes give rise to DNA
degradation (Peitsch et al., 1994; Bortner et al., 1995;
Reape and McCabe, 2008; Mason and Cox, 2012). For
example, as a response to pathogen infection, cells undergo
a destruction pathway in which DNA can be detected as
fragmented or degraded. DNA degradation during programmed cell death likely results either from acid hydrolytic digestion of macromolecules in lysosomes or from
DNA hydrolytic enzymes that degrade DNA directly. Given
these backgrounds, numerous genes encoding plant nucleases have been reported as induced during cell-death events
since the 1990s. Those examples include hypersensitive
response against virulent pathogen infection (Mittler and
Lam, 1995), xylem formation during vascular tissue development (Aoyagi et al., 1998; Ito and Fukuda, 2002; Chen
et al., 2012), suspensor development during embryogenesis
(Lombardi et al., 2007), aleurone layer degradation during cereal seed germination (Fath et al., 1999; Young and
Gallie, 1999), cell death induced by salt stress (Muramoto
et al., 1999; Lombardi et al., 2007), flower development (Xu
and Hanson, 2000; Balk et al., 2003), and leaf senescence
(Blank and McKeon, 1989; Wood et al., 1998; Delorme
et al., 2000; Perez-Amador et al., 2000; Lers et al., 2001;
Canetti et al., 2002).
In most cases, nuclease identification has been conducted
using an in-gel nuclease assay, by which protein extracts
from different tissues are first resolved by a SDS-PAGE gel
containing nucleic acids, with subsequent renaturation of
the protein and then DNA staining with specific dyes; consequently, the nuclease activity is detected as a negatively
stained band. These earlier works revealed the presence of
senescence-induced nucleases, of which enzymic properties such as substrate preference (single-stranded or doublestranded), optimum pH, and divalent cation requirement
have been determined (Table 1); however, the direct relation
between DNA degradation and the nuclease was not tested
in vivo by these works. Most endonuclease activities have a
strong preference to single-stranded DNA (ssDNA) or RNA
rather than double-stranded DNA (dsDNA), raising the possibility that they digest RNA rather than genomic DNA in
vivo. Consequently, overall circumstances suggest strongly
that nucleic acid degradation occurs during leaf senescence,
but the involvement of nucleases should be considered with
caution.
Cytological studies of DNA degradation
during leaf senescence
As aforementioned, progression of leaf senescence varies
depending on plant species and external conditions. Because
of such variation, cytological assessment of the sequential
events in senescing leaves often seems to yield inconsistent
results (Nooden, 1988; Krupinska, 2006). Nevertheless,
common structural changes of senescing leaves that take
place at the cellular level have been reported. Similarly,
decline in DNA levels has been examined by staining DNA
with a fluorescent dye such as 4′,6-diamidino-2-phenylindole (DAPI). Intra-organelle structural changes in the
nucleus, chloroplasts, mitochondria, and other organelles
have also been examined using electron microscopy (Inada
et al., 1998a, b).
To detect DNA fragmentation in situ, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end
labelling) is widely used (Gavrieli et al., 1992). Based on the
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senesce (Sakamoto, 2006; Kato and Sakamoto, 2010),
another degradation pathway operates in which some or
all of an organelle is delivered to vacuoles for hydrolytic
degradation through an autophagy-related mechanism
(Ishida and Yoshimoto, 2008; Wada et al., 2009; Wada
and Ishida, 2013). Concomitantly with the protein degradation, chlorophyll is broken down by a series of degradation enzymes that are likely to avoid the cytotoxicity
of degradation intermediates (Tanaka and Tanaka, 2011;
Hörtensteiner, 2013).
Although it has been documented that nucleic acids are
also subjected to degradation, presumably contributing to
the nutrient redistribution in senescing leaves (reviewed in
Buchanan-Wollaston, 1997; Thomas, 2003), the whole process leading to nucleic acid metabolism is far from understood, in contrast to the case in other macromolecules. In
general, nucleic acid metabolism is regarded as a part of
nucleotide salvage when the substrates for de novo nucleotide
synthesis are limited. Although nucleotide salvage reportedly correlates with nutrient starvation (e.g. phosphate),
the relation between nucleotide salvage and leaf senescence
remains unclear to date. It is possible that the degradation
products from nucleic acids are remobilized as nucleotides,
purine or pyrimidine bases, or phosphates. For example,
leaf senescence has been shown to increase RNase activities (Green, 1994). Nucleic acid metabolism in chloroplasts
was shown to be critical for viability when cells were grown
under phosphate-limited conditions (Yehudai-Resheff et al.,
2007). These results imply that multiplied chloroplast DNA
(cpDNA) represents a storage form of phosphate and undergoes degradation in leaf senescence, as do other compounds
in chloroplasts.
Apart from leaf senescence, DNases of different types
have been reported in association with tissue development
and cell-death-related processes in higher plants. This review
summarizes the knowledge related to the nucleases in plants
and assess their types and possible roles in leaf senescence.
Primarily, DNA degradation enzymes are examined; nucleases related to DNA replication and repair per se are not
described. With regard to senescence, particular emphasis is
given to the degradation of cpDNA and a recently discovered
organelle DNase, which has provided new insight into organelle DNA degradation and senescence.
DNA degradation and leaf senescence | Page 3 of 9
Table 1. Examples of nucleases known to digest DNA in higher plants
ND, not determined experimentally.
Nuclease
Endonucleases
BFN1/ENDO1
ENDO2
ENDO3
ENDO4
ENDO5
CAN2
CsCAN
ZEN1
BEN1
BnucI
CELI
DSA6
EuCaN1/CaN2
Exonucleases
WEX
DPD1
Gene
accession
Type
Substrate
Ion requirement*
Subcellular
localization*
Reference
Arabidopsis
thaliana
At1g11190
S1-like
ssDNA, RNA,
dsDNA
Ca2+, Zn2+
Endoplasmic
reticulum
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Cucumis sativus
Zinnia elegans
Hordeum vulgare
Hordeum vulgare
Apium graveolens
Hemerocallis
hybrid
Eucommia
ulmoides
At1g68290
S1-like
Ca2+, Zn2+
ND
At4g21590
S1-like
ssDNA, RNA,
dsDNA
ssDNA, dsDNA
Zn2+
ND
At4g21585
S1-like
ssDNA, dsDNA
Zn2+
ND
At4g21600
S1-like
ssDNA, dsDNA
Zn2+
ND
At3g56170
Staphylococcal-like
ssDNA, dsDNA
Ca2+, Zn2+
At2g40410
Staphylococcal-like
ssDNA, dsDNA
Ca2+, Zn2+
EU144224
AB003131
D83178
AB028448
AF237958
AF082031
Staphylococcal-like
S1-like
S1-like
S1-like
S1-like
S1-like
ssDNA, dsDNA
ssDNA, dsDNA
ND
ssDNA, RNA
dsDNA
ND
Ca2+
Zn2+
ND
ND
Mg2+
ND
Plasma
membrane
Plasma
membrane
ND
Vacuole
ND
ND
ND
ND
Perez-Amador et al. (2000);
Triques et al. (2007);
Lesniewicz et al. (2013)
Triques et al. (2007);
Lesniewicz et al. (2013)
Triques et al. (2007);
Lesniewicz et al. (2013)
Triques et al. (2007);
Lesniewicz et al. (2013)
Triques et al. (2007);
Lesniewicz et al. (2013)
Lesniewicz et al. (2012)
Gu et al. (2011)
Ito and Fukuda (2002)
Aoyagi et al. (1998)
Muramoto et al. (1999)
Yang et al. (2000)
Panavas et al. (1999)
DQ157695/
EU662201
Staphylococcallike
ssDNA
Ca2+
ND
Chen et al. (2012)
At4g13870
RecQ-like
3′-5′ exo
Mg2+
ND
Plchova et al. (2003)
At5g26940
DnaQ-like
dsDNA
Mg2+
Plastid,
mitochondrion
Matsushima et al. (2011)
Arabidopsis
thaliana
Arabidopsis
thaliana
activity of terminal deoxynucleotidyl transferase (TdT),
which adds dUDP to the 3′-hydroxy terminal end of the
DNA, this method enables fragmented DNA to be visualized on tissues undergoing programmed cell death (Reape
and McCabe, 2008). Before the decline of the DAPI signals,
morphological abnormalities of the nucleus and other parts/
structures of the cell become readily apparent, showing
phenomena such as condensation of nucleus and irregular
membrane organization and accumulation of oil body-like
structures. Organelle DNA in chloroplasts and mitochondria is detectable when semi-ultrathin sections (<1 μm)
are stained with DAPI (Inada et al., 1998a, b). Within the
senescing chloroplasts (called ‘gerontoplasts’), dismantling
of grana thylakoids into fragmented membrane structures
is typically observed along with accumulation of plastoglobules (Krupinska, 2006). The decline of chloroplastic
DAPI signals also occurs during leaf senescence. Detailed
observations of chloroplasts of rice coleoptiles suggest that
DNA degradation precedes chloroplast disorganization
estimated by chlorophyll degradation, implicating cpDNA
Lesniewicz et al. (2012)
degradation by enzymic activity (Sodmergen et al., 1991;
Inada et al., 1998b).
Type of nucleases and induction
in senescence
Endonucleases
Table 1 presents a list of the nucleases among which the corresponding genes have been identified and demonstrated
to be inducible during leaf senescence, cell death, or organ
development. These nucleases are classifiable by their enzymic properties as either endo-type or exo-type. It is noteworthy that most nucleases in leaf senescence reported to
date are endo-type (Sugiyama et al., 2000). The identified
endonucleases were categorized initially into different types
with regard to either zinc (type I) or calcium (type II) ions
as a catalytic divalent cation. They can also be categorized
according to their similarity to S1 nuclease or staphylococcal
nuclease.
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CAN1
Species
Page 4 of 9 | Sakamoto and Takami
S1 nuclease-like endonucleases
BFN1
BFN1 shows strong preference for ssDNA rather than
dsDNA as a substrate, with the optimum pH slightly basic
and with high activity with Zn2+ as a co-factor (Lesniewicz
et al., 2013). Purified BFN1 cleaves dsDNA at a mismatched
base pair (Triques et al., 2007). Promoter analysis of BFN1 in
vivo suggests that BFN1 expression is not limited exclusively to
senescing leaves. It is also inducible during formation of vascular tissue and abscission of floral organs, implying that this
nuclease plays a role in various cell-death processes (FarageBarhom et al., 2008). ORE1, a NAC transcription factor that
positively regulates leaf senescence, has been demonstrated
Other S1-like nucleases
In contrast to BFN1/ENDO1, other S1-type nucleases
(ENDO2-ENDO5) do not exhibit increased expression in
leaf senescence at the transcription level (Fig. 1). The biochemical properties of ENDO2–ENDO5 have been characterized recently, and ENDO2 shows nuclease activity similar
to that of ENDO1 (Ko et al., 2012; Lesniewicz et al., 2013).
To characterize the physiological function of these nucleases
further, Arabidopsis mutants might be useful: to date, details
of none of them have been described in the literature. S1-like
endonucleases similar to BFN1 have been reported in other
species, such as CELI from celery (Yang et al., 2000), BEN1,
and Bnuc1 from barley (Aoyagi et al., 1998; Muramoto
et al., 1999), and ZEN1 from Zinnia (Aoyagi et al., 1998; Ito
and Fukuda, 2002) (Table 1). CELI is commonly used for
TILLING analysis where a nucleotide mismatch representing a point mutation can be detected as fragmented DNA.
ZEN1 was isolated as an endonuclease that is highly inducible during tracheary element differentiation in Zinnia elegans
suspension-cultured cells. Tracheary elements in Zinnia are
reminiscent of xylem formation, which requires cell death
and which gives rise to DNA fragmentation, indicative of cell
death. ZEN1 is induced as a 40-kD Zn2+-dependent endonuclease along with other Ca2+/Mg2+-dependent nucleases.
Knockdown of ZEN1 suppresses DNA fragmentation, supporting the idea that ZEN1 is the main endonuclease that
causes DNA digestion (Ito and Fukuda, 2002).
Staphylococcus nuclease-like endonucleases
Staphylococcus nuclease (SNase) is characterized by a conserved domain (included as SNase-like OB-fold domain,
IPR016071). Genes that have sequence similarity to SNase
have been reported (Sugiyama et al., 2000). SNase, which
often shows preference for calcium over other ions, has been
termed as calcium-dependent nuclease (CAN). In Arabidopsis,
two SNase proteins, AtCAN1 and AtCAN2, have been
reported (Isono et al., 2000; Guo et al., 2012; Lesniewicz
et al., 2012). These two proteins indeed show Ca2+-dependent
endonuclease activity, with ssDNA as a preferred substrate
over dsDNA and RNA. Biochemical properties of several
SNases have also been examined using proteins expressed in
Escherichia coli.
Fig. 1. Example of microarray data demonstrating abundant transcripts
of several nuclease genes during leaf senescence. Transcript levels
of nuclease genes were estimated using GENEVESTIGATOR (https://
www.genevestigator.com/gv/) (Hruz et al., 2008). Corresponding data
were extracted from an AT-00088 microarray. Expression values were
transferred to spreadsheet software for display as a graph. Bars indicate
standard error.
CAN1 and CAN2
Gene expression analysis indicated that the genes encoding AtCAN1 and AtCAN2 are induced during leaf senescence and disease infection (also see Fig. 1). A nuclease
similar to Arabidopsis CAN, CsCAN, has been identified in
Cucumis sativus through the screening of genes specifically
expressed in female flowers (Gu et al., 2011). It is interesting that CsCAN is apparently induced by ethylene, which
links the expression of SNase with senescence. In a tree species, Eucommia ulmoides, two Ca2+-dependent nucleases,
EuCAN1 and EuCAN2, reportedly act on secondary xylem
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Nucleases that have similar characteristics to those of bacterial S1 nucleases are present in plants (included in S1/P1
nuclease family, IPR003154). Although biochemical properties of plant S1-like nucleases have been studied, only a few
nucleases with physiological roles have been investigated
intensively. Triques et al. (2007) reported five endonucleases
(ENDO1–ENDO5) that are classifiable as S1-type based
on homology. In advance of this report, ENDO1 has been
reported as Bi-Functional Nuclease 1 (BFN1) with both
DNase and RNase activities. BFN1 has been shown to be
induced during leaf senescence (Perez-Amador et al., 2000).
In silico analysis of Arabidopsis nucleases using online available databases indeed confirmed that BFN1/ENDO1, rather
than other ENDOs, is highly upregulated during leaf senescence (Fig. 1).
to control the expression of BFN1 at the transcriptional level
(Matallana-Ramirez et al., 2013; Woo et al., 2013).
DNA degradation and leaf senescence | Page 5 of 9
formation (Chen et al., 2012). Overall, these observations
indicate that CAN nucleases are induced during various
stages of plant development at the transcription level, just as
S1-like endonucleases are. However, most expression studies
of CAN1 and CAN2 rely only on publicly available databases. Further analysis using their promoters and knockout
lines might be necessary.
Exonucleases
DPD1
DPD1 was first identified through the study in Arabidopsis
pollen (Matsushima et al., 2011). During the maturation of
pollen grains, there is a decline in organelle DNA (both in
plastids and mitochondria) that is cytologically detectable
with DAPI staining (Nagata et al., 1999; Wang et al., 2010).
Screening of Arabidopsis mutants that retain organelle DNA
in pollen grains led to isolation of the mutants, which were
defective in pollen organelle DNAs (dpd) (Matsushima et al.,
2011; Tang et al., 2012). DPD1 encodes a protein that includes
three conserved Exo domains (included in the Exonuclease/
RNase family, IPR013520) and which shows high similarity
to DnaQ, a proofreading subunit of the multimeric DNA
polymerase (pol III) in E. coli. DPD1 fusion protein purified
from E. coli indeed shows exonuclease activity with Mg2+ as
a cofactor (Matsushima et al., 2011). The N-terminal portion of DPD1 contains a targeting signal-like peptide that
is predicted to deliver into chloroplast and mitochondria.
Translational fusion of DPD1-GFP expressed by DPD1 promoter showed that DPD1 specifically accumulates in pollen.
Moreover, DPD1-GFP is exclusively localized both in chloroplasts and mitochondria, demonstrating that DPD1 is a
dual-targeted exonuclease. Importantly, subsequent in silico
analysis revealed that DPD1 transcripts accumulate at high
levels not only in pollen but also in senescing leaves (Tang
and Sakamoto, 2011). DPD1 transcripts are apparently at
a high level, even more than the genes encoding other previously described endonucleases (Fig. 1). Although DPD1
protein accumulation in senescing leaves has not been examined, these results enabled us to consider that organelle DNA
is degraded by DPD1. To verify this possibility, organelle
Subcellular localization of senescenceenhanced nucleases
To consider the role of the nucleases physiologically, it is
noteworthy to mention their subcellular localization. Cellular
localization of endonucleases such as BFN1 and ZEN1 suggests that they are compartmentalized respectively to vacuoles or the endoplasmic reticulum (ER). Subsequently, they
are recruited into the nucleus to have access to chromosomal
DNA (Fig. 3). When BFN1 is overexpressed in protoplasts
as a GFP-fusion protein, it exists as a filament-like structure that is presumably derived from ER (Farage-Barhom
et al., 2011). As protoplasts senesce and undergo cell death,
the filament-like BFN1-containing ER structures surround
nucleus, which is likely to represent an early step for nuclear
DNA fragmentation. CAN1 and CAN2 have been shown
to contain N-terminal sequences that allow myristoylation
and palmitoylation, by which they are perhaps localized to
plasma membranes. N-terminal regions of these proteins are
predicted to be myristoylated and palmitoylated, which may
localize CAN nucleases into plasma membranes; however,
these modifications have not been proven experimentally.
Overexpression of CAN1 and CAN2 in protoplasts as ECFPfusion proteins has verified their localization in plasma membranes (Lesniewicz et al., 2012).
DPD1 exonuclease has been localized exclusively to DNAcontaining organelles: plastids and mitochondria. This dual targeting has been confirmed by the localization of DPD1-GFP
expressed in both protoplasts and transgenic plants (Matsushima
et al., 2011). The dual-targeting of DPD1 is consistent with the
observation that both plastids and mitochondria have decreased
DNA levels in dpd1 pollen, providing the conclusive evidence
that DPD1 degrades organelle DNA in vivo.
Organelle DNA degradation in leaf
senescence: a revisited paradigm?
Despite the implication of organelle DNA degradation originally reported more than two decades ago, its molecular
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Exonuclease activity has been implicated in DNA quality
control, such as DNA replication and repair (Mason and
Cox, 2012). It affects 5′ or 3′ ends or both, generating a single
nucleotide monophosphate to correct mismatched base pairing. Although many proteins with exonuclease activity are
known, only a few have been characterized in plants. WEX
is an Arabidopsis protein that has an exo-domain similar to
that of RecQ-like helicase (included in the 3′–5′ exonuclease family, IPR002562), which plays a role in DNA repair.
Its deficiency causes genetic diseases in humans. WEX has
an exo-domain but lacks the C-terminal helicase domain
present in the RecQ protein (Plchova et al., 2003; Li et al.,
2005; Smith et al., 2013). Although biochemical evidence for
its exonuclease activity has been provided, no relation of this
nuclease to senescence has been reported. DPD1 is the only
exonuclease suggested to be inducible during leaf senescence.
DNA levels in senescing leaves from dpd1 mutants should be
examined.
In Arabidopsis, our search for proteins that are included
in the Exonuclease/RNase family enabled the selection of 16
proteins, including DPD1 (Fig. 2). Subcellular localization of
these proteins, as predicted using the online program Target
P (Emanuelsson et al., 2000), reveals that these potential exonucleases might have subcellular locations other than chloroplasts or mitochondria. It is interesting that the transcripts
of some exonuclease genes (e.g. At2g48100) accumulate
substantially. However, induction of these nucleases during
senescence is unlikely or marginal because adult and juvenile
leaves also accumulate these transcripts. Therefore, this in silico analysis highlighted the fact that the induction of DPD1
transcripts during leaf senescence is extraordinary, supporting the critical role of organelle DNA degradation.
Page 6 of 9 | Sakamoto and Takami
Death
Fig. 3. Senescence-related nucleases and their subcellular localization, showing nucleases known to be inducible during programmed cell death and leaf
senescence. BNF1 (Arabidopsis) and ZEN1 (Zinnia) are S1-like endonucleases localized in the endoplasmic reticulum (ER) and the vacuole, respectively.
Upon the onset of cell death, membrane rupture might allow these endonucleases to be accessible to DNA in the nucleus. CAN1/CAN2 (Arabidopsis) are
staphylococcal nuclease-like endonucleases that are also induced by programmed cell death and biotic stress. CAN nucleases are possibly localized on
the plasma membrane via N-terminal myristoylation and palmitoylation. DPD1 (Arabidopsis) is an exonuclease similar to bacterial DnaQ and dual-targeted
to chloroplast and mitochondrion with its N-terminal targeting peptide. In silico analysis of DPD1 transcripts suggests that DPD1 is highly upregulated
during leaf senescence.
mechanisms and influences on nutrient redistribution
have not been reported. In rice coleoptile, cpDNA measured using DAPI fluorescence has been shown to decline
as senescence proceeds (Sodmergen et al., 1991; Inada
et al., 1998b). Detailed analysis of this process suggests
that cpDNA decline occurs at the initial step of senescence,
before chlorophyll degradation. In a tree species, cpDNA
degradation during leaf senescence has also been reported
recently (Fulgosi et al., 2012). In the unicellular alga
Chlamydomonas, the amount of cpDNA decreases when
cells are grown under conditions limited in nutrients such
as nitrogen and phosphate (Yehudai-Resheff et al., 2007),
suggesting that multiple copies of cpDNA can serve as a
reservoir (Sears and VanWinkle-Swift, 1994). Therefore,
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Fig. 2. Phylogenic tree and in silico expression analysis of the exonuclease family. DPD1 protein contains a conserved exonuclease domain (Exonuclease/
RNase family, IPR013520). Search for Arabidopsis proteins containing this domain, using a domain information search in the SALAD database (http://salad.dna.
affrc.go.jp/salad/; Mihara et al., 2010) yielded 16 exonuclease-family proteins. These exonuclease-family proteins were subjected to multiple alignment using
ClustalX 2.1 with the following parameters: gap open penalty 10; gap extension penalty 0.05; and protein weight matrix BLOSUM. An unrooted phylogenic tree
was constructed based on the multiple alignment using neighbour-joining method with 1000 bootstrap replicates. Subcellular localization of each exonucleasefamily protein (shown in the middle) was predicted using the Target P program (http://www.cbs.dtu.dk/services/TargetP/; Emanuelsson et al., 2000), except for
DPD1, which was demonstrated experimentally to be localized in chloroplasts and mitochondria. C, chloroplast; M, mitochondrion; O, others; S, secretory. In
silico expression analysis of the exonuclease-family proteins in adult, juvenile, and senescent leaves (red, green, and blue, respectively) was conducted as for
Fig. 1. using the AT-00088 microarray. ND, no data available.
DNA degradation and leaf senescence | Page 7 of 9
compartment, raising the possibility that they do not act
specifically on genomic DNA degradation. BFN1/ENDO1
has been well characterized for its biochemical properties
as a possible DNase applicable to detect a DNA mismatch
(Triques et al., 2007), although its precise role in DNA degradation demands further investigation. Mutants lacking these
nucleases should be examined to ascertain their function. In
contrast, a recent discovery of DPD1 exonuclease and its
possible involvement in leaf senescence implies that there is
actual degradation of organelle DNA. Given that chloroplasts are the major physiological target of degradation during leaf senescence, it is reasonable to assume that DPD1
plays a role in cpDNA degradation for relocating nucleotides.
To address that assumption further, dpd1 mutant should be a
potential tool. In addition, future studies must address how
the expression of these nucleases is controlled. BFN1 is under
the control of ORE1 transcription factor, which is located
downstream of ethylene signalling cascade. Whether the
other nucleases are also expressed under the same pathway
must be examined.
Perspectives
Emanuelsson O, Nielsen H, Brunak S, von Heijne G. 2000. Predicting
subcellular localization of proteins based on their N-terminal amino acid
sequence. Journal of Molecular Biology 300, 1005–1016.
Although the nucleases related to cell death and senescence in
plants have been known for long time, the understanding of
DNA degradation and the action of these nucleases remains
limited. Among the well-characterized nucleases, BFN1 and
CAN1/CAN2 appear to be the most promising ones for
action during leaf senescence. However, they have high affinity to ssDNA and RNA and are localized in a specific cellular
Acknowledgements
Part of the work described in this manuscript was supported by the Japan
Society for the Promotion of Science [Grants-in-Aid for Scientific Research
(KAKENHI) grant no. 25291063 to W.S.].
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all circumstances imply that organelle DNA degradation
occurs.
In contrast to the detection of organelle DNA decline with
DAPI staining, no careful measurement of DNA levels with
quantitative PCR during the progression of leaf senescence
has been reported in higher plants. Although Zoschke et al.
(2007) reported no significant levels of cpDNA in old leaves
that was likely to represent natural senescence, the work did
not focus on leaf senescence particularly. Our recent work (T
Takami and W Sakamoto, unpublished data) demonstrated
that cpDNA levels, when measured using quantitative PCR,
indeed decline in dark-induced leaf senescence. It is tempting
to examine whether organelle DNA levels are increased in the
leaf tissues of the dpd1 mutant or not. Our previous study
indeed showed that young seedlings in dpd1 have more organelle DNA than those in the wild type, although not statistically significant (Matsushima et al., 2011). Detailed analysis
of organelle DNA levels in senescent leaves of dpd1 will provide evidence as to whether DPD1 is the nuclease involved in
organelle DNA degradation during leaf senescence.
The question arises as to whether only an exo-type nuclease can degrade organelle DNA sufficiently or whether it
requires cooperation with other endonucleases or other factors. Although the latter case cannot be fully excluded, one
explanation that might resolve this question is that organelle
DNA reportedly exists with various configurations such as linearized and nicked (Oldenburg and Bendich, 2004). Although
this idea of heterogeneous genome composition argues against
the conventionally accepted circular organelle genome, accumulating evidence related to both yeast and plants suggests
that frequent recombination through short repeat sequences
spread within the organelle genomes might acts as an origin of DNA replication (Gerhold et al., 2010; Marechal and
Brisson, 2010). Given that most organelle DNA molecules
have a nick or an end, DPD1 alone might be sufficient for degradation. Overall, DPD1 is the only example for which DNA
degradation has been confirmed through mutant analysis
(Matsushima et al., 2011). Further analysis of DPD1 in leaf
senescence is expected to provide new insight into DNA degradation and a concept of nutrient reservoir in chloroplasts.
Organelle DNA degradation in pollen may be along with this
line, given that pollen is the only tissue that is isolated from the
parental plant. Although the genome is small, organelle DNA
exists as multicopies accounting for >20% of total cellular
DNA (reviewed in Liere and Börner, 2013). In this scenario, it
is important to ascertain how such dispensable DNA is stored
and discarded according to plant development.
Page 8 of 9 | Sakamoto and Takami
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