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. For permissions, please email: [email protected]
Downloaded from http://jxb.oxfordjournals.org/ at Washington University in St. Louis on June 9, 2014
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
Downloaded from http://jxb.oxfordjournals.org/ at Washington University in St. Louis on June 9, 2014
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 (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.
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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. For permissions, please email: [email protected]
Downloaded from http://jxb.oxfordjournals.org/ at Washington University in St. Louis on June 9, 2014
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
Downloaded from http://jxb.oxfordjournals.org/ at Washington University in St. Louis on June 9, 2014
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 (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.
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