- Email: [email protected]
NOX or not? Evidence for algal NADPH oxidases
Update Letters
NOX or not? Evidence for algal NADPH oxidases Alexander Anderson1, John H. Bothwell2, Anuphon Laohavisit1, Alison G. Smith1 and Julia M. Davies1 1 2
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
NADPH oxidases (NOX) are highly conserved plasma- and endo-membrane enzymes present in animals, plants and fungi. They catalyse the production of superoxide anion (O2 ) [1,2] by translocating an electron from NADPH across the membrane, via flavin adenine dinucleotide (FAD), to react with oxygen. The resulting O2 is readily converted to other reactive oxygen species that, in plants, participate in signalling, growth and defence [1,2]. In a recent paper, Ron Mittler and colleagues [2] proposed that plant NOX (also termed RBOH; Respiratory Burst Oxidase Homologues) evolved in early land plants to supplement the system of regulatory antioxidant enzymes shared by plants and algae, and adduced the apparent absence of algal NOX to support this theory [2]. Algae, however, do produce extracellular O2 in signalling, growth and defence, which raises the question of their generation. This question is of more than simple evolutionary interest, as it offers insights into how commercially grown algal crops may be protected, and environmentally harmful algal blooms may be controlled [3]. There is, it transpires, substantial evidence to show that there are indeed algal NOXlike proteins that could be responsible for moving electrons out of the cell to produce O2 . In algae, superoxide production has been recorded in unicellular representatives from the chlorophyte, heterokont (stramenopile), haptophyte and alveolate lineages [4]. Extracellular O2 production sensitive to the NOX inhibitor diphenyleneiodonium (DPI) has also been reported for their multicellular counterparts in the red (Chondrus crispus and Gracilaria conferta), brown heterokont (Laminaria digitata and Fucus serratus) and green chlorophyte (Dasycladus vermicularis and Ulva compressa) seaweeds [5–9]. To date, the resultant ROS have been shown to be involved in osmotic and oxidative stress signalling, polar growth and defensive oxidative bursts [4– 10]. With extracellular O2 production evident in the green algal sisters of land plants, and their red/brown algal cousins, are credible NOX candidates present in their genomes? The red macroalga C. crispus does indeed express a NOX-like transcript that responds to stress and hormonal treatments [11]. The predicted Chondrus protein has 21% overall similarity to the canonical animal NOX gp91phox, rising to 40% similarity in the C-terminal FAD-binding site, 24% in the NADPH-adenine binding site and 43% in the NADPH-ribose binding site ([11]; Figure 1a and b). The NOX histidine residues in the Corresponding author: Davies, J.M. ([email protected]).
third and fifth transmembrane (TM) domains (required for haem binding and electron transport) are conserved in Chondrus ([11]; Figure 1b). As haem is a DPI target, this is structurally consistent with DPI-inhibited O2 production by C. crispus [5]. An additional four TM domains are predicted between the NADPH binding sites. The N-terminal EF hands characteristic of plant NOX (enabling stimulation by Ca2+) are missing ([11]; Figure 1a). An algal EST database search revealed that these ten predicted TM domains were not only present in NOX homologues in Cyanidioschyzon merolae (unicellular red) and Porphyra yezoensis (multicellular red), but also in the heterokonts Phaeodactylum tricornutum and Thalassiosira pseudonana (both diatoms) ([11]; Figure 1a and b). Supporting this observation of NOX homologues in diatom genomes, the related heterokont Chattonella marina, one of the most harmful of the ‘red tide’-forming during phytoplankton, produces extracellular O2 growth and contains a 91-kDa protein (of similar mass to those predicted in [11]) that cross-reacts with antigp91phox antibody [3]. In the green algae, the unicellular Chlamydomonas reinhardtii can generate extracellular superoxide [10] and contains a six TM domain candidate NOX (Figure 1a; [12]). Previously, this RBOL1 (Respiratory Burst Oxidase-Like 1) was investigated as a ferric reductase (FRE) homologue but, in contrast to FREs, its expression was unaffected by iron deficiency [12]. Its predicted mass of 62 kDa is closer to animal NOX (gp91phox 65 kDa) than the red algal NOX or plant RBOHs (e.g. AtRBOHA is 103 kDa). A CrRBOL2 is also evident; its expression is similarly unaffected by iron deficiency [12], but it contains only four predicted TM domains (Figure 1a) and it may be that variability in the number of TM domains influences the functions of specific algal NOX [11]. Both CrRBOL/NOX have the conserved TM histidine residues and FAD/NADPH binding domains required for NOX function in animals and plants although, in common with other algal NOX, there are no EF hands, which suggests indirect (if any) regulation by Ca2+ (Figure 1a). Unresolved questions do remain concerning algal NOX. For example, the oxidative burst of Laminaria digitata is DPI-sensitive [8], but no highly conserved NOX sequences are apparent [11]. Clearly, more work is needed to determine unequivocally whether or not the candidate NOX proteins identified to date really do operate as NOX, but it appears premature to dismiss the algae as an entirely NOX-free zone. 579
Update
Trends in Plant Science November 2011, Vol. 16, No. 11
(a)
TM domains
TM domains FAD
EF hands
NADPH-a
NADPH-r
Human gp91phox N-
1
2
3
4
5
6
-C
Arabidopsis RBOH N-
1
2
3
4
5
6
-C
C. reinhardtii RBOL 1 N-
1
2
3
4
5
6
-C
1
2
3
4
2
3
4
5
C. reinhardtii RBOL 2 N-
1
Red Algae NOX / Diatom NOX N-
-C
7
6
Human gp91phox
Arabidopsis RBOH
C. reinhardtii RBOL
A C B F E 1 2
C.crispus NOX C.merolae NOX
1 2
P.yezoensis NOX P. tricornutum NOX T. pseudonana NOX
1 2 1 2
9
10
-C
TM domain β
TM domain α
(b)
8
205 525 534 480 568 546 160 174 183 191 265 178 159 159 285 312
97 423 430 377 465 444 89 103 111 112 186 106 76 76 222 249
TRENDS in Plant Science phox
Figure 1. Algal NOX homologues. (a) Comparison of Human gp91 [11], Arabidopsis thaliana RBOH proteins (representative of all isoforms; [2]), Ch. reinhardtii RBOL/ NOX [12] and the red algae/diatom NOX (representative of isoforms in C. crispus, Cy. merolae, Ph. tricornutum, Po. yezoensis and T. pseudonana [11]) and showing positions of TM domains (green boxes). Corresponding TM domains are aligned vertically. Arrows indicate TM domains containing conserved haem-binding histidine (H) residues. NADPH-r, NADPH-ribose binding site; NADPH-a, NADPH-adenine binding site; FAD, flavin adenine dinucleotide-binding site. (b) Alignment of NOX/NOX homologue TM domains containing putative haem-binding sites in Human gp91phox, Arabidopsis RBOH (A–E), Ch. reinhardtii RBOL1 (NCBI accession number XP_001891855) and RBOL2 (NCBI accession number XP_001691856), C. crispus, Cy. merolae (NOX1 and 2; termed RBOH 1 and 2 in [11]), Po. yezoensis, Ph. tricornutum (NOX 1 and 2) and T. pseudonana (NOX 1 and 2; termed RBOH1 and 2 in [11]). TM domain a is equivalent to TM3 in gp91phox, AtRBOH, CrRBOL1, red algae/diatom NOX, and TM2 in CrRBOL2. TM domain b represents TM5 in gp91phox, AtRBOH, CrRBOL1, red algae/diatom NOX, and TM4 in CrRBOL2. Conserved histidine (H) residues are marked with arrows. Numbers on the left represent the position of the first amino acid residue in each sequence. Dashes represent gaps in the amino acid alignment. Sequences were aligned in Jalview (http://www.jalview.org/) and coloured according to the ClustalX classification. Both C. reinhardtii RBOL sequences are annotated as putative NOX on the PLAZA server [13] used previously [2] in inter-genomic analyses of NOX (protein identifier 145439 (CrRBOL1) and 206678 (CrRBOL2)).
Acknowledgements We thank the UK EPSRC for funding and apologise to colleagues whose work could not be cited owing to space limitations.
References 1 Aguirre, J. and Lambeth, J.D. (2010) Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals. Free Radic. Biol. Med. 15, 1342–1353 2 Mittler, R. et al. (2011) ROS signalling: the new wave? Trends Plant Sci. 16, 300–309 3 Kim, D. et al. (2007) Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata. J. Plankton Res. 29, 241–247 4 Marshall, J-A. et al. (2005) Superoxide production by marine microalgae. Mar. Biol. 147, 533–540
580
5 Potin, P. (2005) Oxidative burst and related responses in biotic interactions of algae. In Algal Chemical Ecology (Amsler, C.D., ed.), pp. 245–271, Springer 6 Kustka, A.B. et al. (2005) Extracellular production of superoxide by marine diatoms: Contrasting effects on iron redox chemistry and bioavailability. Limnol. Oceanogr. 50, 1172–1180 7 Coelho, S.M.B. et al. (2008) A tip-high, Ca2+-interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes. Planta 227, 1037–1046 8 Ku¨pper, F.C. et al. (2001) Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiol. 125, 278–291 9 Gonzalez, A.J. et al. (2010) Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess. Plant Cell Environ. 33, 1627–1640
Update
Trends in Plant Science November 2011, Vol. 16, No. 11
10 Hema, R. et al. (2007) Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes. Planta 226, 655–670 11 Herve´, C. et al. (2006) NADPH oxidases in Eukaryotes: red algae provide new hints! Curr. Genet. 49, 190–204 12 Allen, M.D. et al. (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are
expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryot. Cell 6, 1841–1852 13 Proost, S. et al. (2009) PLAZA: A comparative genomics resource to study gene and genome evolution in plants. Plant Cell 21, 3718–3731 1360-1385/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2011.09.003 Trends in Plant Science, November 2011, Vol. 16, No. 11
Plant Science Conferences in 2012 Keystone Symposia: Nuclear Events in Plant Gene Expression and Signaling 6 – 11 March, 2012 Taos, New Mexico http://www.keystonesymposia.org/meetings The Biology of Plants 30 May – 4 June, 2012 Cold Spring Harbor, USA http://meetings.cshl.edu/meetings.html 23rd International Conference on Arabidopsis research (ICAR) 3 – 7 July, 2012 Vienna, Austria http://www.icar2012.org/ -----------------------------------------------------------------------------------------------------------------------------------Suggest a conference Please use the form at http://www.cell.com/conferences/SuggestConference to suggest a conference for Cell Press the Conference Calendar
581
NOX or not? Evidence for algal NADPH oxidases Alexander Anderson1, John H. Bothwell2, Anuphon Laohavisit1, Alison G. Smith1 and Julia M. Davies1 1 2
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
NADPH oxidases (NOX) are highly conserved plasma- and endo-membrane enzymes present in animals, plants and fungi. They catalyse the production of superoxide anion (O2 ) [1,2] by translocating an electron from NADPH across the membrane, via flavin adenine dinucleotide (FAD), to react with oxygen. The resulting O2 is readily converted to other reactive oxygen species that, in plants, participate in signalling, growth and defence [1,2]. In a recent paper, Ron Mittler and colleagues [2] proposed that plant NOX (also termed RBOH; Respiratory Burst Oxidase Homologues) evolved in early land plants to supplement the system of regulatory antioxidant enzymes shared by plants and algae, and adduced the apparent absence of algal NOX to support this theory [2]. Algae, however, do produce extracellular O2 in signalling, growth and defence, which raises the question of their generation. This question is of more than simple evolutionary interest, as it offers insights into how commercially grown algal crops may be protected, and environmentally harmful algal blooms may be controlled [3]. There is, it transpires, substantial evidence to show that there are indeed algal NOXlike proteins that could be responsible for moving electrons out of the cell to produce O2 . In algae, superoxide production has been recorded in unicellular representatives from the chlorophyte, heterokont (stramenopile), haptophyte and alveolate lineages [4]. Extracellular O2 production sensitive to the NOX inhibitor diphenyleneiodonium (DPI) has also been reported for their multicellular counterparts in the red (Chondrus crispus and Gracilaria conferta), brown heterokont (Laminaria digitata and Fucus serratus) and green chlorophyte (Dasycladus vermicularis and Ulva compressa) seaweeds [5–9]. To date, the resultant ROS have been shown to be involved in osmotic and oxidative stress signalling, polar growth and defensive oxidative bursts [4– 10]. With extracellular O2 production evident in the green algal sisters of land plants, and their red/brown algal cousins, are credible NOX candidates present in their genomes? The red macroalga C. crispus does indeed express a NOX-like transcript that responds to stress and hormonal treatments [11]. The predicted Chondrus protein has 21% overall similarity to the canonical animal NOX gp91phox, rising to 40% similarity in the C-terminal FAD-binding site, 24% in the NADPH-adenine binding site and 43% in the NADPH-ribose binding site ([11]; Figure 1a and b). The NOX histidine residues in the Corresponding author: Davies, J.M. ([email protected]).
third and fifth transmembrane (TM) domains (required for haem binding and electron transport) are conserved in Chondrus ([11]; Figure 1b). As haem is a DPI target, this is structurally consistent with DPI-inhibited O2 production by C. crispus [5]. An additional four TM domains are predicted between the NADPH binding sites. The N-terminal EF hands characteristic of plant NOX (enabling stimulation by Ca2+) are missing ([11]; Figure 1a). An algal EST database search revealed that these ten predicted TM domains were not only present in NOX homologues in Cyanidioschyzon merolae (unicellular red) and Porphyra yezoensis (multicellular red), but also in the heterokonts Phaeodactylum tricornutum and Thalassiosira pseudonana (both diatoms) ([11]; Figure 1a and b). Supporting this observation of NOX homologues in diatom genomes, the related heterokont Chattonella marina, one of the most harmful of the ‘red tide’-forming during phytoplankton, produces extracellular O2 growth and contains a 91-kDa protein (of similar mass to those predicted in [11]) that cross-reacts with antigp91phox antibody [3]. In the green algae, the unicellular Chlamydomonas reinhardtii can generate extracellular superoxide [10] and contains a six TM domain candidate NOX (Figure 1a; [12]). Previously, this RBOL1 (Respiratory Burst Oxidase-Like 1) was investigated as a ferric reductase (FRE) homologue but, in contrast to FREs, its expression was unaffected by iron deficiency [12]. Its predicted mass of 62 kDa is closer to animal NOX (gp91phox 65 kDa) than the red algal NOX or plant RBOHs (e.g. AtRBOHA is 103 kDa). A CrRBOL2 is also evident; its expression is similarly unaffected by iron deficiency [12], but it contains only four predicted TM domains (Figure 1a) and it may be that variability in the number of TM domains influences the functions of specific algal NOX [11]. Both CrRBOL/NOX have the conserved TM histidine residues and FAD/NADPH binding domains required for NOX function in animals and plants although, in common with other algal NOX, there are no EF hands, which suggests indirect (if any) regulation by Ca2+ (Figure 1a). Unresolved questions do remain concerning algal NOX. For example, the oxidative burst of Laminaria digitata is DPI-sensitive [8], but no highly conserved NOX sequences are apparent [11]. Clearly, more work is needed to determine unequivocally whether or not the candidate NOX proteins identified to date really do operate as NOX, but it appears premature to dismiss the algae as an entirely NOX-free zone. 579
Update
Trends in Plant Science November 2011, Vol. 16, No. 11
(a)
TM domains
TM domains FAD
EF hands
NADPH-a
NADPH-r
Human gp91phox N-
1
2
3
4
5
6
-C
Arabidopsis RBOH N-
1
2
3
4
5
6
-C
C. reinhardtii RBOL 1 N-
1
2
3
4
5
6
-C
1
2
3
4
2
3
4
5
C. reinhardtii RBOL 2 N-
1
Red Algae NOX / Diatom NOX N-
-C
7
6
Human gp91phox
Arabidopsis RBOH
C. reinhardtii RBOL
A C B F E 1 2
C.crispus NOX C.merolae NOX
1 2
P.yezoensis NOX P. tricornutum NOX T. pseudonana NOX
1 2 1 2
9
10
-C
TM domain β
TM domain α
(b)
8
205 525 534 480 568 546 160 174 183 191 265 178 159 159 285 312
97 423 430 377 465 444 89 103 111 112 186 106 76 76 222 249
TRENDS in Plant Science phox
Figure 1. Algal NOX homologues. (a) Comparison of Human gp91 [11], Arabidopsis thaliana RBOH proteins (representative of all isoforms; [2]), Ch. reinhardtii RBOL/ NOX [12] and the red algae/diatom NOX (representative of isoforms in C. crispus, Cy. merolae, Ph. tricornutum, Po. yezoensis and T. pseudonana [11]) and showing positions of TM domains (green boxes). Corresponding TM domains are aligned vertically. Arrows indicate TM domains containing conserved haem-binding histidine (H) residues. NADPH-r, NADPH-ribose binding site; NADPH-a, NADPH-adenine binding site; FAD, flavin adenine dinucleotide-binding site. (b) Alignment of NOX/NOX homologue TM domains containing putative haem-binding sites in Human gp91phox, Arabidopsis RBOH (A–E), Ch. reinhardtii RBOL1 (NCBI accession number XP_001891855) and RBOL2 (NCBI accession number XP_001691856), C. crispus, Cy. merolae (NOX1 and 2; termed RBOH 1 and 2 in [11]), Po. yezoensis, Ph. tricornutum (NOX 1 and 2) and T. pseudonana (NOX 1 and 2; termed RBOH1 and 2 in [11]). TM domain a is equivalent to TM3 in gp91phox, AtRBOH, CrRBOL1, red algae/diatom NOX, and TM2 in CrRBOL2. TM domain b represents TM5 in gp91phox, AtRBOH, CrRBOL1, red algae/diatom NOX, and TM4 in CrRBOL2. Conserved histidine (H) residues are marked with arrows. Numbers on the left represent the position of the first amino acid residue in each sequence. Dashes represent gaps in the amino acid alignment. Sequences were aligned in Jalview (http://www.jalview.org/) and coloured according to the ClustalX classification. Both C. reinhardtii RBOL sequences are annotated as putative NOX on the PLAZA server [13] used previously [2] in inter-genomic analyses of NOX (protein identifier 145439 (CrRBOL1) and 206678 (CrRBOL2)).
Acknowledgements We thank the UK EPSRC for funding and apologise to colleagues whose work could not be cited owing to space limitations.
References 1 Aguirre, J. and Lambeth, J.D. (2010) Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals. Free Radic. Biol. Med. 15, 1342–1353 2 Mittler, R. et al. (2011) ROS signalling: the new wave? Trends Plant Sci. 16, 300–309 3 Kim, D. et al. (2007) Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata. J. Plankton Res. 29, 241–247 4 Marshall, J-A. et al. (2005) Superoxide production by marine microalgae. Mar. Biol. 147, 533–540
580
5 Potin, P. (2005) Oxidative burst and related responses in biotic interactions of algae. In Algal Chemical Ecology (Amsler, C.D., ed.), pp. 245–271, Springer 6 Kustka, A.B. et al. (2005) Extracellular production of superoxide by marine diatoms: Contrasting effects on iron redox chemistry and bioavailability. Limnol. Oceanogr. 50, 1172–1180 7 Coelho, S.M.B. et al. (2008) A tip-high, Ca2+-interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes. Planta 227, 1037–1046 8 Ku¨pper, F.C. et al. (2001) Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiol. 125, 278–291 9 Gonzalez, A.J. et al. (2010) Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess. Plant Cell Environ. 33, 1627–1640
Update
Trends in Plant Science November 2011, Vol. 16, No. 11
10 Hema, R. et al. (2007) Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes. Planta 226, 655–670 11 Herve´, C. et al. (2006) NADPH oxidases in Eukaryotes: red algae provide new hints! Curr. Genet. 49, 190–204 12 Allen, M.D. et al. (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are
expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryot. Cell 6, 1841–1852 13 Proost, S. et al. (2009) PLAZA: A comparative genomics resource to study gene and genome evolution in plants. Plant Cell 21, 3718–3731 1360-1385/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2011.09.003 Trends in Plant Science, November 2011, Vol. 16, No. 11
Plant Science Conferences in 2012 Keystone Symposia: Nuclear Events in Plant Gene Expression and Signaling 6 – 11 March, 2012 Taos, New Mexico http://www.keystonesymposia.org/meetings The Biology of Plants 30 May – 4 June, 2012 Cold Spring Harbor, USA http://meetings.cshl.edu/meetings.html 23rd International Conference on Arabidopsis research (ICAR) 3 – 7 July, 2012 Vienna, Austria http://www.icar2012.org/ -----------------------------------------------------------------------------------------------------------------------------------Suggest a conference Please use the form at http://www.cell.com/conferences/SuggestConference to suggest a conference for Cell Press the Conference Calendar
581