MINI-REVIEW Plant Signaling & Behavior 10:11, e1057366; November 2015; © 2015 Taylor & Francis Group, LLC
Enlightenment on the aequorin-based platform for screening Arabidopsis stress sensory channels related to calcium signaling Zhiming Yu1, Jemma L Taylor2, Yue He1, and Jun Ni1,* 1
College of Life and Environmental Sciences; Hangzhou Normal University; Hangzhou, China; 2School of Life Sciences; Gibbet Hill Campus; University of Warwick;
Coventry, United Kingdom
ree calcium ions (Ca2C) are an important signal molecule in response to a large array of external stimuli encountered by plants. Using the aequorin-based Ca2C recording system, tremendous progress has been made in understanding the Ca2C responses to biotic or abiotic stresses in dicotyledonous Arabidopsis. However, due to the lack of a similar detection system, little information has been obtained from the monocotyledonous rice (Oryza sativa). Recombinant aequorin has been introduced into rice, and the Ca2C responses to NaCl and H2O2 in rice roots were characterized. Although rice calcium signal sensor research has just started, the transgenic rice expressing aequorin provides a good platform to study rice adapted to different environmental conditions.
F
Keywords: aequorin, Arabidopsis, calcium signaling, sensor, Oryza sativa *Correspondence to: Jun Ni; Email: [email protected]. cn Submitted: 04/27/2015 Revised: 05/23/2015 Accepted: 05/27/2015 http://dx.doi.org/10.1080/15592324.2015.1057366 www.tandfonline.com
Plants live in a constantly changing environment from which they cannot physically escape. Plants therefore need signaling and response mechanisms to adapt to new local conditions. The primary messages which the plant responds to comprise of extracellular substances, including some hydrophilic molecules, so they cannot cross the bilayer cell membrane to initiate changes within the cell directly.1 This functional limitation necessitates the cell to devise signal transduction mechanisms to transduce primary into secondary messengers, so that the extracellular signal may be propagated intracellularly.1,2 Ca2C is one of the most important secondary messenger molecules. The movement of Ca2C into and out of the cytoplasm functions as a signal for many cellular processes.1,2 Ca2C permeable ion channels can transduce Plant Signaling & Behavior
environmental stimuli into Ca2C-encoded messages which regulate downstream gene expression. Sensory channels are transducers of primary messages to secondary messages. Using these sensors, plants set up a communication bridge between environmental stress and cellular signal cascades. In animals, Ca2C-permeable transient receptor potential (TRP) channels act as sensors for temperature, osmotic potential, and other environmental conditions.3 Although stress-gated Ca2C-permeable channels like TRPs in animals are not found in land plants, several families of proteins, including glutamate receptor-like (GLR) proteins, cyclic nucleotide-gated channels (CNGCs), and annexins, have been linked to Ca2C fluxes in plant cells.4 Because cytosolic Ca2C elevation is one of the earliest responses of plant cells to stress treatments,3,5 and Ca2C-binding proteins are required for several environmental stress responses,6 stress-activated Ca2C channels are candidates that may link stress stimuli to Ca2C-dependent downstream responses.7 Aequorin is a Ca2C-activated photoprotein isolated from the hydrozoan Aequorea victoria. The protein interacts with the green fluorescent protein (GFP) to produce green light by resonant energy transfer, while aequorin by itself generates blue light.8 Aequorin-based platform to research Ca2C signaling in Arabidopsis Aequorin is also a holoprotein composed of 2 distinct units, the apoprotein that is called apoaequorin, and the prosthetic group coelenterazine, the luciferin. Apoprotein can stably bind coelenterazine and oxygen is required for the regeneration to the active form of aequorin.9-10 In e1057366-1
the presence of Ca2C, aequorin undergoes a conformational change and through oxidation converts its prosthetic group, coelenterazine, into excited coelenteramide and CO2.11 As the excited coelenteramide relaxes to the ground state, blue light (wavelength of 465 nm) is emitted. Before coelenteramide is exchanged out, the entire protein is still fluorescent blue.12-13 There are currently 2 methods to noninvasively evaluate the Ca2C levels in plants. With high quantum yield, the fluorescence resonance energy transfer (FRET)-based probes are suitable to measure the Ca2C signatures in the cellular or subcellular resolutions.14 Yellow cameleon YC2.115 and the improved YC3.616 are currently used to monitor the Ca2C level in the cytoplasm. Although the aequorinbased probe gives a low quantum yield, it is more suitable for cell population or whole plant measurement of Ca2C levels.14 Using plants transformed with aequorin, progress has been made in understanding how plants respond to different stimuli. This includes abiotic stimuli, heat shock,17 drought,18-20 salinity,1819 cold-shock,5,19,21-23 mechanical perturbation,5,22-24 pH19,22 and ROS19,25, and biotic stimuli, salicylic acid,26 symbiosis signaling26 and microbe/pathogen-associated molecular patterns.27 Interestingly, aequorin was also reported to fuse to different targeting peptides or proteins to localize it to different subcellular localizations, indicating its adaptability to measure Ca2C concentration in vivo.28 Identification of important genes by screening Ca2C insensitive mutants Based on the aequorin-based system, 2 novel sensors have been discovered by a forward genetic screening of ethylmethane-sulfonate (EMS) mutagenized Arabidopsis seedlings. The first sensor was an ATP receptor. The authors identified 2 mutants lacking a cytoplasmic calcium response to ATP addition. The two allelic mutants were predicted to encode a lectin receptor kinaseI.9 (LecRK-I.9, At5g60300), located on the plasma membrane. The receptor binds extracellular ATP at the cell surface, with preferred affinity for ATP. It has a very different molecular structure from the known animal receptors. Given this unique
e1057366-2
structure, it was renamed P2K (K for kinase) for this previously unknown family of purinoreceptors. Further biological functions will be found for this interesting signal molecule in plants.29 The second sensor was also a plasma membrane protein. Arabidopsis mutants that exhibit low sensitivities to osmoticinduced Ca2C increases were identified. One mutant, reduced hyperosmolalityinduced Ca2C increase 1 (osca1), displayed impaired osmotic Ca2C signaling in guard and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 forms osmotic-gated Ca2C-permeable channels, may be an osmosensor. OSCA1 was identified as a novel gene encoding a protein of 772 amino acid residues (At4g04340). Though various abiotic and biotic stimuli trigger Ca2C increases by activating Ca2C channels in plants, OSCA1 was the first example of such a channel.20 Hou et al. (2014) reported the cloning of AtCSC1 (Calcium permeable Stressgated cation Channel 1) (At4g22120), an osmosensitive Ca2C permeable cation channel, which is a close homolog of OSCA1. Whether AtCSC1 functions as an osmosensitive channel in the plasma membrane or endomembranes in planta as well as its physiological functions remains to be determined.7 Taking a similar approach, interesting results were obtained from other groups. Pan et al. (2012) reported an easy and efficient method in a forward genetic screen for Ca2C signal-deficient mutants in Arabidopsis.30 Wild-type plants (Col-0) were transformed with 35S:apoaequorin construct pMAQ. The transformants expressing apoaequorin stably were then transformed with T-DNA vector pSKI105. Basta-resistant seedlings (T1 generation) were isolated and T2 generation seeds were collected for screening. Using this method, 121 mutants with disrupted NaCl- and H2O2-induced Ca2C signals were isolated. Furthermore, one gene was identified, Actin-Related Protein2 (Arp2) (At3g27000), as a regulator of Ca2C in response to salt stress. In this study, the authors showed that the Arp2/3 complex functions to regulate mitochondrial-dependent changes in Ca2C during plant growth in response to salt, indicating
Plant Signaling & Behavior
that changes in cytoskeletal organization function upstream of changes in Ca2C. In addition, Arp2/3 complex-mediated actin dynamics participate in regulating mitochondria movement but are not required for the association of mitochondria with micro-filaments.31 Aequorin-based luminescence imaging in rice roots Biotic or abiotic stress threatens the yield of rice. In order to increase the tolerance of rice to various environmental stresses, the genes involved in the stress perception and response need to be identified. However, compared with that of Arabidopsis, the mechanism of Ca2C signaling in rice is far behind. To date, only 2 aequorin transgenic rice articles have been reported.27,32 To investigate the Ca2C and protein phosphorylation changes induced by microbe/ pathogen-associated molecular patterns (MAMPs/PAMPs), Kurusu et al. (2011) established a transgenic rice cell line stably expressing apoaequorin and characterized the interrelationship between MAMP-induced changes in Ca2C, production of reactive oxygen species (ROS) and protein phosphorylation.27 Despite the progress achieved by rice culture cells, stable transgenic rice expressing apoaequorin is needed to investigate the changes of Ca2C in response to various environmental stimuli in rice. Salinity is one of the most common abiotic stresses encountered by rice, which is classified as a salt-sensitive crop in early stages of development, and limits its productivity.33-34 Salt overly sensitive (SOS) signaling is a well-defined pathway, which explains how plants respond to salt stress.35 Salt and reactive oxygen species (ROS) stresses are able to increase the concentration of Ca2C, which is caused by the flux of Ca2C.36 But the differences between these 2 processes are largely unknown. A recently published article introduced recombinant aequorin into rice and examined the change in Ca2C in response to salt and ROS stresses.32 In that paper, the apoaequorin gene was constitutively expressed under control of 35S promoter. The results revealed that the aequorin-based luminescence signal was only observed in roots, but RT-PCR
Volume 10 Issue 11
Funding
This work was supported by National Science Foundation of China under grant No. 31301000 and No. 31200913 to J.N. and Z.Y. respectively.
References
Figure 1. Transgenic rice harboring aequorin showed luminescence in roots.31 (A) Pseudocolor image of aequorin luminescence in the whole plant. Dotted luminescence signals (indicated by arrows) were observed in shoots after the addition of surfactant. The relationship between luminescence intensity and the pseudocolor images are scaled by pseudocolor bars. (B) Bright-field image of the same seedlings. WT, Wild-type rice seedlings. AQ, transgenic rice harboring aequorin.
showed that apoaequorin was expressed in all tissues. Further investigation found that it’s the leaf wax which prevents the permeating of coelenterazine as dotted signals in shoots were observed by adding surfactant (Silwet L-77) (Fig. 1). That means the transgenic rice is a good model to reflect the Ca2C level in rice roots. To further investigate the source of Ca2C in NaCl- and H2O2-induced Ca2C responses, different Ca2C inhibitors were used. Tests were performed using GdCl3-, LaCl3-, neomycin- and thapsigargintreated plants on the Ca2C increase in response to NaCl and H2O2 respectively.37-39 Results showed that these inhibitors differentially inhibited NaCland H2O2-induced Ca2C responses, indicating different sources of Ca2C induction by NaCl and H2O2. According to the time course of Ca2C changes in response to NaCl and H2O2 treatment in rice roots, together with the Ca2C inhibitors’ results, it was suggested that the salt sensors may be on the surface of the rice root and are closely coupled with Ca2C channels, while the effect of H2O2 on calcium signaling is less direct. SERF1 functions as a central hub in the ROS-dependent signaling during the initial response to salt stress in rice.40 Zhang et al. (2015)32 examined NaCland H2O2-induced expression levels of
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SERF1 and other initial response genes with or without Ca2C inhibitors. Considering the fact that a Ca2C spike is the first response to salt stress,18 and salt-induced ROS accumulation is regulated by Ca2C,41 together with the gene expression analysis, a Ca2C and H2O2 mediated molecular signaling model for the initial response to NaCl in rice roots was proposed.32 Prospect Lacking an efficient method to isolate mutants in Ca2C signal generation process may limit Ca2C signaling research in rice. Typical forward genetic screening is always useful to find genes involved in Ca2C signaling. Looking back at existing research in rice, rice calcium signal research has only just begun. Following the Arabidopsis mature research methods and techniques, especially the mutant screening system, we expect to find several important Ca2C related calcium sensors which have important agronomic traits in the near future. We are looking forward to great advances in rice calcium signaling research.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Plant Signaling & Behavior
1. Brownlee C. Plant signaling: calcium first and second. Curr Biol 2003; 13:pR923-4; PMID:14654020; http:// dx.doi.org/10.1016/j.cub.2003.11.016 2. Hepler PK. Calcium: a central regulator of plant growth and development. Plant Cell 2005; 17:2142-55; PMID:1182479; http://dx.doi.org/ 10.1105/tpc.105.032508 3. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem 2007; 76:387-417; PMID:4196875; http://doi : 10.1146/annurev.biochem.75.103004.142819 4. Swarbreck SM, Cola¸c o R, Davies JM. Plant calcium-permeable channels. Plant Physiol 2013; 163:514-22; PMID: 23860348; http://dx.doi.org/ 10.1104/pp.113.220855 5. Knight MR, Campbell AK, Smith SM, Trewavas AJ. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 1991; 352:524-6; PMID:1865907; http://dx. doi.org/10.1038/352524a0 6. Luan S, Kudla J, Rodriguez-Concepcion M, Yalovsky S, Gruissem W. Calmodulins and calcineurin B-like proteins: calcium sensors for specific signal response coupling in plants. Plant Cell 2002; 14 Suppl:S389-400; PMID:12045290; http://www.plantcell.org/cgi/doi/ 10.1105/tpc.001115 7. Hou CC, Tian W, Kleist T, He K, Garcia V, Bai FL, Hao YL, Luan S, Li L. DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes. Cell Res 2014; 24:632-5; PMID:24503647; http://dx.doi. org/10.1038/cr.2014.14 8. Shimomura O. A short story of aequorin. Biol Bull 1995; 189:1-5; http://dx.doi.org/10.2307/1542194 9. Shimomura O, Johnson FH. Regeneration of the photoprotein aequorin. Nature 1975; 256:236-8; PMID:239351; http://dx.doi.org/10.1038/256236a0 10. Shimomura O, Johnson FH. Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci U S A 1978; 75:2611-5; PMID:275832; http://dx.doi.org/10.1073/pnas.75.6.261111 11. Shimomura O, Johnson FH, Morise H. Mechanism of the luminescent intramolecular reaction of aequorin. Biochemistry 1974; 13:3278-86; PMID:4152180; http://dx.doi.org/10.1021/bi00713a016 12. Shimomura O, Johnson FH. Calcium binding, quantum yield, and emitting molecule in aequorin bioluminescence. Nature 1970; 227:1356-7; PMID:4393938; http://dx.doi.org/10.1038/2271356a0 13. Inouye S, Sasaki S. Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase. FEBS Lett 2006; 580:1977-82; PMID:15527769; http://dx.doi.org/10.1016/j.febslet.2006.02.065 14. Monshausen GB. Visualizing Ca2C signatures in plants. Curr Opin Plant Biol 2012; 15:677-82; PMID: 23044039; http://dx.doi.org/10.1016/j.pbi.2012.09.014 15. Miyawaki A, Griesbeck O, Heim R, Tsien RY. Dynamic and quantitative Ca2C measurements using improved cameleons. Proc Natl Acad Sci U S A 1999; 96: 2135-40; PMID:10051607; http://dx.doi.org/ 10.1073/pnas.96.5.2135 16. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A. Expanded dynamic range of fluorescent indicators for Ca2C by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 2004; 101:10554-9; PMID:15247428; http://dx. doi.org/10.1073/pnas.0400417101
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17. Gong M, van der Luit AH, Knight MR, Trewavas AJ. Heat-shock-induced changes in intracellular Ca2C level in tobacco seedlings in Relation to thermotolerance. Plant Physiol 1998; 116:429-37; http://dx.doi.org/ 10.1104/pp.116.1.429 18. Knight H, Trewavas AJ, Knight MR. Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J 1997; 12:1067-78; PMID:9418048; http:// dx.doi.org/10.1046/j.1365-313X.1997.12051067.x 19. Zhu X, Feng Y, Liang G, Liu N, Zhu JK. Aequorinbased luminescence imaging reveals stimulus- and tissue-specific Ca2C dynamics in Arabidopsis plants. Mol Plant 2013; 6:444-55; PMID:23371933; http://dx.doi. org/10.1093/mp/sst013 20. Yuan F, Yang H, Xue Y, Kong D, Ye R, Li C, Zhang J, Theprungsirikul L, Shrift T, Krichilsky B, et al. OSCA1 mediates osmotic-stress-evoked Ca2C increases vital for osmosensing in Arabidopsis. Nature 2014; 514:367-71; PMID: 25162526; http://dx.doi.org/ 10.1038/nature13593 21. Knight H, Trewavas AJ, Knight MR. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 1996; 8:489-503; PMID:8721751; http://dx.doi. org/10.2307/3870327 22. Russell AJ, Knight MR, Cove DJ, Knight CD, Trewavas AJ, Wang TL. The moss, Physcomitrella patens, transformed with apoaequorin cDNA responds to cold shock, mechanical perturbation and pH with transient increases in cytoplasmic calcium. Transgenic Res 1996; 5:167-70; PMID:8673143; http://dx.doi.org/10.1007/ BF01969705 23. Meyerhoff O, Muller K, Roelfsema MR, Latz A, Lacombe B, Hedrich R, Dietrich P, Becker D. AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold. Planta 2005; 222:418-27; PMID:15864638; http://dx.doi.org/10.1007/s00425005-1551-3 24. Haley A, Russell AJ, Wood N, Allan AC, Knight M, Campbell AK, Trewavas AJ. Effects of mechanical signaling on plant cell cytosolic calcium. Proc Natl Acad Sci U S A 1995; 92:4124-8; PMID:11536690; http:// dx.doi.org/10.1073/pnas.92.10.4124 25. Monshausen GB, Bibikova TN, Weisenseel MH, Gilroy S. Ca2C regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots. Plant Cell 2009; 21:2341-56; PMID:19654264; http://dx.doi.org/10.1105/ tpc.109.068395
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26. Kawano T, Sahashi N, Takahashi K, Uozumi N, Muto S. Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: the earliest events in salicylic acid signal transduction. Plant Cell Physiol 1998; 39:721-30; http://dx.doi.org/10.1093/oxfordjournals. pcp.a029426 27. Kurusu T, Hamada H, Sugiyama Y, Yagala T, Kadota Y, Furuichi T, Hayashi T, Umemura K, Komatsu S, Miyao A, et al. Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2C transients by protein phosphorylation. J Plant Res 2011; 124:415-24; PMID:21063744; http://dx.doi. org/10.1007/s10265-010-0388-4 28. Mehlmer N, Parvin N, Hurst CH, Knight MR, Teige M, Vothknecht UC. A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana. J Exp Bot 2012; 63: 1751-61; PMID: 22213817; http://dx.doi.org/ 10.1093/jxb/err406 29. Choi J, Tanaka K, Cao Y, Qi Y, Qiu J, Liang Y, Lee SY, Stacey G. Identification of a plant receptor for extracellular ATP. Science 2014; 343:290-4; PMID: 24436418; http://dx.doi.org/10.1126/science. 343.6168.290 30. Pan Z, Zhao Y, Zheng Y, Liu J, Jiang X, Guo Y. A high-throughput method for screening Arabidopsis mutants with disordered abiotic stress-induced calcium signal. J Genet Genomics 2012; 39:225-35; PMID:22624884; http://dx.doi.org/10.1016/j. jgg.2012.04.002 31. Zhao Y, Pan Z, Zhang Y, Qu X, Zhang Y, Yang Y, Jiang X, Huang S, Yuan M, Schumaker KS, et al. The actin-related protein2/3 complex regulates mitochondrial-associated calcium signaling during salt stress in Arabidopsis. Plant Cell 2013; 25:454459; PMID:24280386; http://dx.doi.org/10.1105/ tpc.113.117887 32. Zhang YY, Wang YF, Taylor JL, Jiang ZH, Zhang S, Mei FL, Wu YR, Wu P, Ni J. Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots. J Exp Bot 2015; 66:2535-45; PMID:25754405; http://dx.doi.org/10.1093/jxb/erv043 33. Lutts S, Kinet J, Bouharmont J. Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J Exp Bot 1995; 46:1843-52; http://dx.doi.org/10.1093/jxb/ 46.12.1843
Plant Signaling & Behavior
34. Todaka D, Nakashima K, Shinozaki K, YamaguchiShinozaki K. Toward understanding transcriptional regulatory networks in abiotic stress responses and tolerance in rice. Rice 2012; 5:6; PMCID:PMC3834508; http://dx.doi.org/10.1186/1939-8433-5-6 35. Zhu JK. Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physiol 2000; 124:941948; PMID:11080272; http://dx.doi.org/10.1104/ pp.124.3.941 36. Ji H, Pardo JM, Batelli G, Van Oosten MJ, Bressan RA, Li X. The Salt Overly Sensitive (SOS) pathway: established and emerging roles. Mol Plant 2013; 6:27586; PMID:23355543; http://dx.doi.org/10.1093/mp/ sst017 37. Tracy FE, Gilliham M, Dodd AN, Webb AA, Tester M. NaCl-induced changes in cytosolic free Ca2C in Arabidopsis thaliana are heterogeneous and modified by external ionic composition. Plant Cell Environ 2008; 31:1063-73; PMID:18419736; http://dx.doi.org/ 10.1111/j.1365-3040.2008.01817.x 38. Munnik T, Irvine RF, Musgrave A. Phospholipid signalling in plants. Biochimica Biophysica Acta 1998; 1389:222-72; PMID:9512651; http://dx.doi.org/ 10.1016/S0005-2760(97)00158-6 39. Treiman M, Caspersen C, Christensen SB. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2C-ATPases. Trends Pharmacol Sci 1998; 19:131-5; PMID:9612087; http://dx.doi. org/10.1016/S0165-6147(98)01184-5 40. Schmidt R, Mieulet D, Hubberten H-M, Obata T, Hoefgen R, Fernie AR, Fisahn J, San Segundo B, Guiderdoni E, Schippers JH. SALT-RESPONSIVE ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice. Plant Cell 2013; 25:2115-31; PMID:23800963; http:// dx.doi.org/10.1105/tpc.113.113068 41. Drerup MM, Schlucking K, Hashimoto K, Manishankar P, Steinhorst L, Kuchitsu K, Kudla J. The Calcineurin B-like calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26 regulate the Arabidopsis NADPH oxidase RBOHF. Mol Plant 2013; 6:559-69; PMID:23335733; http:// dx.doi.org/10.1093/mp/sst009 42. Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 2012; 30:174-8; PMID:22267009; http://dx. doi.org/10.1038/nbt.2095
Volume 10 Issue 11
Enlightenment on the aequorin-based platform for screening Arabidopsis stress sensory channels related to calcium signaling Zhiming Yu1, Jemma L Taylor2, Yue He1, and Jun Ni1,* 1
College of Life and Environmental Sciences; Hangzhou Normal University; Hangzhou, China; 2School of Life Sciences; Gibbet Hill Campus; University of Warwick;
Coventry, United Kingdom
ree calcium ions (Ca2C) are an important signal molecule in response to a large array of external stimuli encountered by plants. Using the aequorin-based Ca2C recording system, tremendous progress has been made in understanding the Ca2C responses to biotic or abiotic stresses in dicotyledonous Arabidopsis. However, due to the lack of a similar detection system, little information has been obtained from the monocotyledonous rice (Oryza sativa). Recombinant aequorin has been introduced into rice, and the Ca2C responses to NaCl and H2O2 in rice roots were characterized. Although rice calcium signal sensor research has just started, the transgenic rice expressing aequorin provides a good platform to study rice adapted to different environmental conditions.
F
Keywords: aequorin, Arabidopsis, calcium signaling, sensor, Oryza sativa *Correspondence to: Jun Ni; Email: [email protected]. cn Submitted: 04/27/2015 Revised: 05/23/2015 Accepted: 05/27/2015 http://dx.doi.org/10.1080/15592324.2015.1057366 www.tandfonline.com
Plants live in a constantly changing environment from which they cannot physically escape. Plants therefore need signaling and response mechanisms to adapt to new local conditions. The primary messages which the plant responds to comprise of extracellular substances, including some hydrophilic molecules, so they cannot cross the bilayer cell membrane to initiate changes within the cell directly.1 This functional limitation necessitates the cell to devise signal transduction mechanisms to transduce primary into secondary messengers, so that the extracellular signal may be propagated intracellularly.1,2 Ca2C is one of the most important secondary messenger molecules. The movement of Ca2C into and out of the cytoplasm functions as a signal for many cellular processes.1,2 Ca2C permeable ion channels can transduce Plant Signaling & Behavior
environmental stimuli into Ca2C-encoded messages which regulate downstream gene expression. Sensory channels are transducers of primary messages to secondary messages. Using these sensors, plants set up a communication bridge between environmental stress and cellular signal cascades. In animals, Ca2C-permeable transient receptor potential (TRP) channels act as sensors for temperature, osmotic potential, and other environmental conditions.3 Although stress-gated Ca2C-permeable channels like TRPs in animals are not found in land plants, several families of proteins, including glutamate receptor-like (GLR) proteins, cyclic nucleotide-gated channels (CNGCs), and annexins, have been linked to Ca2C fluxes in plant cells.4 Because cytosolic Ca2C elevation is one of the earliest responses of plant cells to stress treatments,3,5 and Ca2C-binding proteins are required for several environmental stress responses,6 stress-activated Ca2C channels are candidates that may link stress stimuli to Ca2C-dependent downstream responses.7 Aequorin is a Ca2C-activated photoprotein isolated from the hydrozoan Aequorea victoria. The protein interacts with the green fluorescent protein (GFP) to produce green light by resonant energy transfer, while aequorin by itself generates blue light.8 Aequorin-based platform to research Ca2C signaling in Arabidopsis Aequorin is also a holoprotein composed of 2 distinct units, the apoprotein that is called apoaequorin, and the prosthetic group coelenterazine, the luciferin. Apoprotein can stably bind coelenterazine and oxygen is required for the regeneration to the active form of aequorin.9-10 In e1057366-1
the presence of Ca2C, aequorin undergoes a conformational change and through oxidation converts its prosthetic group, coelenterazine, into excited coelenteramide and CO2.11 As the excited coelenteramide relaxes to the ground state, blue light (wavelength of 465 nm) is emitted. Before coelenteramide is exchanged out, the entire protein is still fluorescent blue.12-13 There are currently 2 methods to noninvasively evaluate the Ca2C levels in plants. With high quantum yield, the fluorescence resonance energy transfer (FRET)-based probes are suitable to measure the Ca2C signatures in the cellular or subcellular resolutions.14 Yellow cameleon YC2.115 and the improved YC3.616 are currently used to monitor the Ca2C level in the cytoplasm. Although the aequorinbased probe gives a low quantum yield, it is more suitable for cell population or whole plant measurement of Ca2C levels.14 Using plants transformed with aequorin, progress has been made in understanding how plants respond to different stimuli. This includes abiotic stimuli, heat shock,17 drought,18-20 salinity,1819 cold-shock,5,19,21-23 mechanical perturbation,5,22-24 pH19,22 and ROS19,25, and biotic stimuli, salicylic acid,26 symbiosis signaling26 and microbe/pathogen-associated molecular patterns.27 Interestingly, aequorin was also reported to fuse to different targeting peptides or proteins to localize it to different subcellular localizations, indicating its adaptability to measure Ca2C concentration in vivo.28 Identification of important genes by screening Ca2C insensitive mutants Based on the aequorin-based system, 2 novel sensors have been discovered by a forward genetic screening of ethylmethane-sulfonate (EMS) mutagenized Arabidopsis seedlings. The first sensor was an ATP receptor. The authors identified 2 mutants lacking a cytoplasmic calcium response to ATP addition. The two allelic mutants were predicted to encode a lectin receptor kinaseI.9 (LecRK-I.9, At5g60300), located on the plasma membrane. The receptor binds extracellular ATP at the cell surface, with preferred affinity for ATP. It has a very different molecular structure from the known animal receptors. Given this unique
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structure, it was renamed P2K (K for kinase) for this previously unknown family of purinoreceptors. Further biological functions will be found for this interesting signal molecule in plants.29 The second sensor was also a plasma membrane protein. Arabidopsis mutants that exhibit low sensitivities to osmoticinduced Ca2C increases were identified. One mutant, reduced hyperosmolalityinduced Ca2C increase 1 (osca1), displayed impaired osmotic Ca2C signaling in guard and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 forms osmotic-gated Ca2C-permeable channels, may be an osmosensor. OSCA1 was identified as a novel gene encoding a protein of 772 amino acid residues (At4g04340). Though various abiotic and biotic stimuli trigger Ca2C increases by activating Ca2C channels in plants, OSCA1 was the first example of such a channel.20 Hou et al. (2014) reported the cloning of AtCSC1 (Calcium permeable Stressgated cation Channel 1) (At4g22120), an osmosensitive Ca2C permeable cation channel, which is a close homolog of OSCA1. Whether AtCSC1 functions as an osmosensitive channel in the plasma membrane or endomembranes in planta as well as its physiological functions remains to be determined.7 Taking a similar approach, interesting results were obtained from other groups. Pan et al. (2012) reported an easy and efficient method in a forward genetic screen for Ca2C signal-deficient mutants in Arabidopsis.30 Wild-type plants (Col-0) were transformed with 35S:apoaequorin construct pMAQ. The transformants expressing apoaequorin stably were then transformed with T-DNA vector pSKI105. Basta-resistant seedlings (T1 generation) were isolated and T2 generation seeds were collected for screening. Using this method, 121 mutants with disrupted NaCl- and H2O2-induced Ca2C signals were isolated. Furthermore, one gene was identified, Actin-Related Protein2 (Arp2) (At3g27000), as a regulator of Ca2C in response to salt stress. In this study, the authors showed that the Arp2/3 complex functions to regulate mitochondrial-dependent changes in Ca2C during plant growth in response to salt, indicating
Plant Signaling & Behavior
that changes in cytoskeletal organization function upstream of changes in Ca2C. In addition, Arp2/3 complex-mediated actin dynamics participate in regulating mitochondria movement but are not required for the association of mitochondria with micro-filaments.31 Aequorin-based luminescence imaging in rice roots Biotic or abiotic stress threatens the yield of rice. In order to increase the tolerance of rice to various environmental stresses, the genes involved in the stress perception and response need to be identified. However, compared with that of Arabidopsis, the mechanism of Ca2C signaling in rice is far behind. To date, only 2 aequorin transgenic rice articles have been reported.27,32 To investigate the Ca2C and protein phosphorylation changes induced by microbe/ pathogen-associated molecular patterns (MAMPs/PAMPs), Kurusu et al. (2011) established a transgenic rice cell line stably expressing apoaequorin and characterized the interrelationship between MAMP-induced changes in Ca2C, production of reactive oxygen species (ROS) and protein phosphorylation.27 Despite the progress achieved by rice culture cells, stable transgenic rice expressing apoaequorin is needed to investigate the changes of Ca2C in response to various environmental stimuli in rice. Salinity is one of the most common abiotic stresses encountered by rice, which is classified as a salt-sensitive crop in early stages of development, and limits its productivity.33-34 Salt overly sensitive (SOS) signaling is a well-defined pathway, which explains how plants respond to salt stress.35 Salt and reactive oxygen species (ROS) stresses are able to increase the concentration of Ca2C, which is caused by the flux of Ca2C.36 But the differences between these 2 processes are largely unknown. A recently published article introduced recombinant aequorin into rice and examined the change in Ca2C in response to salt and ROS stresses.32 In that paper, the apoaequorin gene was constitutively expressed under control of 35S promoter. The results revealed that the aequorin-based luminescence signal was only observed in roots, but RT-PCR
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Funding
This work was supported by National Science Foundation of China under grant No. 31301000 and No. 31200913 to J.N. and Z.Y. respectively.
References
Figure 1. Transgenic rice harboring aequorin showed luminescence in roots.31 (A) Pseudocolor image of aequorin luminescence in the whole plant. Dotted luminescence signals (indicated by arrows) were observed in shoots after the addition of surfactant. The relationship between luminescence intensity and the pseudocolor images are scaled by pseudocolor bars. (B) Bright-field image of the same seedlings. WT, Wild-type rice seedlings. AQ, transgenic rice harboring aequorin.
showed that apoaequorin was expressed in all tissues. Further investigation found that it’s the leaf wax which prevents the permeating of coelenterazine as dotted signals in shoots were observed by adding surfactant (Silwet L-77) (Fig. 1). That means the transgenic rice is a good model to reflect the Ca2C level in rice roots. To further investigate the source of Ca2C in NaCl- and H2O2-induced Ca2C responses, different Ca2C inhibitors were used. Tests were performed using GdCl3-, LaCl3-, neomycin- and thapsigargintreated plants on the Ca2C increase in response to NaCl and H2O2 respectively.37-39 Results showed that these inhibitors differentially inhibited NaCland H2O2-induced Ca2C responses, indicating different sources of Ca2C induction by NaCl and H2O2. According to the time course of Ca2C changes in response to NaCl and H2O2 treatment in rice roots, together with the Ca2C inhibitors’ results, it was suggested that the salt sensors may be on the surface of the rice root and are closely coupled with Ca2C channels, while the effect of H2O2 on calcium signaling is less direct. SERF1 functions as a central hub in the ROS-dependent signaling during the initial response to salt stress in rice.40 Zhang et al. (2015)32 examined NaCland H2O2-induced expression levels of
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SERF1 and other initial response genes with or without Ca2C inhibitors. Considering the fact that a Ca2C spike is the first response to salt stress,18 and salt-induced ROS accumulation is regulated by Ca2C,41 together with the gene expression analysis, a Ca2C and H2O2 mediated molecular signaling model for the initial response to NaCl in rice roots was proposed.32 Prospect Lacking an efficient method to isolate mutants in Ca2C signal generation process may limit Ca2C signaling research in rice. Typical forward genetic screening is always useful to find genes involved in Ca2C signaling. Looking back at existing research in rice, rice calcium signal research has only just begun. Following the Arabidopsis mature research methods and techniques, especially the mutant screening system, we expect to find several important Ca2C related calcium sensors which have important agronomic traits in the near future. We are looking forward to great advances in rice calcium signaling research.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Plant Signaling & Behavior
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