Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+influx and modulates generation of reactive oxygen species in cultured rice cells
- Takamitsu Kurusu†1, 2,
- Daisuke Nishikawa†1,
- Yukari Yamazaki†1,
- Mariko Gotoh1,
- Masataka Nakano3, 4,
- Haruyasu Hamada1,
- Takuya Yamanaka1,
- Kazuko Iida5,
- Yuko Nakagawa3,
- Hikaru Saji6,
- Kazuo Shinozaki7,
- Hidetoshi Iida3 and
- Kazuyuki Kuchitsu1, 2Email author
© Kurusu et al; licensee BioMed Central Ltd. 2012
Received: 28 June 2011
Accepted: 23 January 2012
Published: 23 January 2012
Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.
We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.
We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.
Plants need to sense and respond to mechanical stresses, such as wind, touch, and changes in osmotic pressure [1–3]. Elevation of cytosolic free Ca2+ concentration ([Ca2+]cyt) is induced in response to various stimuli, such as chemical, physical, and mechanical stimuli [2, 4–7]. During this process, [Ca2+]cyt levels rise through the opening of Ca2+ channels located on the plasma membrane and endomembranes. Electrophysiological and bioinformatic studies have revealed the existence of plasma membrane Ca2+-permeable channels activated by mechanical stimuli, although the structural entity involved and their physiological functions remain largely unknown [8–12].
Molecular and electrophysiological studies have shown that Arabidopsis thaliana MSL9 and MSL10, homologs of the bacterial mechanosensitive channel MscS, are required for mechanosensitive channel activity in the plasma membrane of root cells, and are more permeable to Cl- than Ca2+ [13, 14]. We have recently identified two plasma membrane proteins as putative Ca2+-permeable mechanosensitive channels, MCA1 (At4g35920) and MCA2 (At2g17780), from Arabidopsis [15, 16], and showed that ectopic overexpression of MCA1 increases Ca2+ uptake in roots, and also enhances [Ca2+]cyt elevation upon hypo-osmotic shock. However, the direct effects of MCA proteins on osmotic-induced Ca2+ influx through the plasma membrane and the osmotic signaling pathways are little understood.
Upon recognition of osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS) [17–20]. These downstream events are often prevented when Ca2+ influx is compromised by either Ca2+ chelators, such as ethylene glycol-bis-(2-aminoethylether)-N, N, N', N'-tetra acetic acid (EGTA), or Ca2+-channel blockers, such as La3+ . In tobacco cells, hypo-osmotic shock-induced ROS generation reportedly requires activation of mechanosensitive Ca2+ channels . These results suggest that Ca2+ influx mediated by mechanosensitive Ca2+ channels is involved in the induction of osmotic signals including ROS generation. However, in osmotic responses, molecular bases and regulation mechanisms remain poorly elucidated.
In the present study, we have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Studies of these lines with altered levels of this putative mechanosensitive Ca2+ channel indicated that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells.
Identification of OsMCA1 and its expression patterns
Full-length cDNA of OsMCA1 was obtained by a rapid amplification of cDNA ends (RACE)-PCR method. It encodes a polypeptide of 418 amino acid residues with a calculated molecular mass of 47,417 (GenBank Accession No. AB601973). The predicted protein showed 66.7% and 57.6% amino acid sequence identity compared with Arabidopsis MCA1 and MCA2, respectively; the TopPred program http://www.sbc.su.se/~erikw/toppred2/ suggests that OsMCA1 has two potential transmembrane segments (S1 and S2) (Additional file 1), while other transmembrane segment prediction programs suggest different numbers of putative transmembrane segments (data not shown). The PLAC8 motif was found by TMpred prediction http://www.ch.embnet.org/software/TMPRED_form.html in the C-terminal region (Additional file 1). A database search of the whole genome (Rice BLAST; http://riceblast.dna.affrc.go.jp/) indicated that rice has no other homolog of OsMCA1.
Intracellular localization of the OsMCA1 protein
Effects of OsMCA1 overexpression on Ca2+uptake in cultured rice cells
Effect of OsMCA1 suppression on growth and development in planta
The OsMCA1-suppressed lines showed slower growth in adult plants (Figure 4B, C). Though germination rates (data not shown) and seedling growth of suppression lines were comparable to controls in the Murashige and Skoog medium (MS medium) (Additional file 3), growth of suppression lines was remarkably retarded after transplantation into soil in a greenhouse, suggesting that OsMCA1 suppression leads to hypersensitivity to environmental stresses. This phenotype was exhibited in all 5 independent T2 transgenic RNAi lines; severity of the phenotypes correlated well with expression levels of OsMCA1 transcripts (Figure 4A, C). Furthermore, unlike Arabidopsis mca mutants, rachises of the OsMCA1-suppressed lines were significantly shorter than those of controls (Figure 4D, E), suggesting that OsMCA1 plays a different role from Arabidopsis MCAs in some developmental stages.
Effects of OsMCA1-suppression on cell growth and Ca2+sensitivity in cultured rice cells
Involvement of OsMCA1 in mechanical stress-induced [Ca2+]cytchanges
We also tried to examine the effect of overexpression of OsMCA1 on mechanical stress-triggered Ca2+ influx. However, we observed a strong reduction in the total aequorin luminescence in all transgenic cell lines overexpressing OsMCA1 (data not shown). Thus it was impossible to measure [Ca2+]cyt using OsMCA1-overexpressing lines. Real-time RT-PCR analysis revealed that the expression level of aequorin gene in the OsMCA1-overexpressing lines was comparable to the control (data not shown). Thus constitutive overexpression of OsMCA1 does not affect the expression but may affect the stability of aequorin protein or inhibit the aequorin chemiluminescence.
Effects of OsMCA1-overexpression on sensitivity to hypo-osmotic shock and generation of reactive oxygen species
Hypo-osmotic shock has been shown to trigger ROS generation following [Ca2+]cyt increase in cultured tobacco cells [18, 22]. As OsMCA1 has been suggested to affect regulation of hypo-osmotic shock-induced Ca2+ influx (Figure 6E), we investigated possible OsMCA1 involvement in hypo-osmotic shock-induced ROS generation in cultured rice cells, using two distinctive methods sensitive for superoxide anion radical (•O2-) and hydrogen peroxide (H2O2).
It has been suggested that Ca2+ plays a crucial role in mechanical sensing [7, 25]. However, little is known of the molecular mechanisms responsible for Ca2+ mobilization. Functional characterization of the OsMCA1-RNAi lines as well as overexpressors suggests that OsMCA1 is involved in hypo-osmotic shock-induced Ca2+ influx and ROS generation.
Possible functions of OsMCA1 in the regulation of growth and development of rice
OsMCA1-suppressed plants displayed stunted growth and shortened rachises (Figure 4D, E). These phenotypes are frequently observed under drought-stress conditions . The suppression of OsMCA1 might have affected adaptation to drought. Drought stress is known to lead to osmotic stress at a cellular level. Since hypo-osmotic shock-induced [Ca2+]cyt changes were impaired in the OsMCA1-suppressed lines, these lines may have defects in osmotic sensing/responses and ability to adapt to drought stress. Future studies to characterize physiological reactions to drought and mechanical signaling in OsMCA1-suppressed plants would further elucidate the in vivo roles of OsMCA1 in intact plants.
In Arabidopsis, the mca1 mca2 double mutant shows a growth defect in soil, and reduced accumulation of Ca2+ as well as enhanced sensitivity to Mg2+ . The balance of Ca2+ and Mg2+ in soil is an important factor for normal plant growth . Since the growth of the OsMCA1-suppressed lines was significantly restricted compared with the control under Ca2+-limitation (Figure 5), growth retardation in the OsMCA1-suppressed plants may be attributed to reduced Ca2+ uptake, resulting in a low Ca2+-Mg2+ ratio.
Possible involvement of OsMCA1 in osmotic signaling in cultured rice cells
The GFP-OsMCA1 fusion protein localized specifically to the plasma membrane (Figure 2), suggesting that OsMCA1 is a plasma membrane protein. In cultured rice cells, both hypo-osmotic shock- and TNP-induced [Ca2+]cyt transients, which were inhibited by La3+ and Gd3+, were impaired in OsMCA1-suppressed lines (Figures 6 and 7). The temporal pattern of the MAMP-induced [Ca2+]cyt transient was similar (data not shown), but was unaffected by OsMCA1 suppression (Figure 6D). These results suggest that OsMCA1 affects regulation of Ca2+ influx across the plasma membrane in response to mechanical stimulation in cultured rice cells.
Hypo-osmotic shock triggers ROS generation following a [Ca2+]cyt increase [18, 20, 22]. Extracellular Ca2+ is required for both Ca2+ influx and NADPH oxidase-mediated ROS generation induced by hypo-osmotic shock (Figures 6C and 8B, Additional file 4), suggesting that ROS generation requires Ca2+ influx across the plasma membrane. Overexpression of OsMCA1 enhances ROS generation (Figure 8D, Additional file 6). Binding Ca2+ to the EF-hand regions of cytosolic regulatory domains of plant NADPH oxidases directly activates them [28–30]. A functional NADPH oxidase AtrbohC/RHD2 reportedly affects mechanical stress-induced ROS generation in a Ca2+-dependent manner . Overproduction of the plasma membrane Ca2+-permeable channels may induce the mobilization of excess Ca2+ in response to mechanical stimuli, which may cause enhanced activation of NADPH oxidases.
In OsMCA1-suppressed lines challenged with hypo-osmotic shock, Ca2+ influx was partially impaired (Figure 6E), but no significant influence of the impairment on subsequent ROS generation was detected under our assay conditions (Figure 8A). Similar effects of overexpression and a loss-of-function mutation were also observed with another putative Ca2+-permeable channel, OsTPC1 . A certain level of Ca2+ increment may be sufficient for NADPH oxidase-mediated ROS generation. Alternatively, other Ca2+-permeable channels activated by hypo-osmotic shock may redundantly play a role in bypassing OsMCA1. It has been suggested that Arabidopsis MSL9 and MSL10, homologs of the bacterial mechanosensitive channel MscS, are required for mechanosensitive channel activity in root cell plasma membranes, and are able to translocate cations including Ca2+ [13, 14]. Rice MSL homologs may therefore be candidates for such Ca2+-permeable channels.
The present study indicates that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and NADPH oxidase-mediated ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.
Plant materials and cell culture
Surface-sterilized seeds of rice, Oryza sativa L. cv. Nipponbare, were germinated on MS medium  containing 0.8% agar and grown for 10 days in a growth chamber under long day conditions (16 h light/8 h darkness, 28°C). Seedlings were transplanted into soil and grown in a greenhouse (16 h light/8 h darkness, 28°C and 60% humidity). Calli were suspension-cultured at 25°C in a liquid L medium  containing 2,4-D (0.5 mg L-1) in the dark and subcultured in fresh medium every week. Cells were filtered through a 20-mesh screen every 2 weeks to make fine aggregates. Cells at 5 days after subculture were used for experiments with osmotic stress and defense responses. N-acetylchito-oligosaccharides were kindly provided by Prof. Naoto Shibuya (Meiji University).
Isolation of OsMCA1 cDNA
The estimated coding region of OsMCA1 was amplified by PCR using two primers: OsMCA1 forward, 5'-GAAGAAGAAGAAGAAGAAGAAGCCGAGTAG-3'; OsMCA1 reverse, 5'-TATTTATGCTTACCCTGCATTGTTTGTGTT-3'. Total RNA was isolated from rice leaves using Trizol (Invitrogen, Carlsbad, CA, USA) in accordance with manufacturer's protocol and quantified spectrophotometrically. First-strand cDNA was synthesized from 3 μg of total RNA with the oligo-dT primer and reverse transcriptase (Invitrogen). To obtain full-length cDNAs for OsMCA1 and to define the open reading frame, 3'-RACE PCR and 5'-RACE PCR were performed with a 3'-full RACE core kit (Takara, Ohtsu, Japan) and a 5'-RACE system (Invitrogen) in accordance with manufacturers' protocols.
RNA isolation and RT-PCR analyses
Total RNA was isolated using Trizol reagent in accordance with manufacturer's protocol and quantified using a spectrophotometer. First-strand cDNA was synthesized from 3 μg total RNA with an oligo-dT primer and reverse transcriptase. PCR amplification was performed with an initial denaturation at 95°C for 3 min followed by indicated numbers of cycles of incubations at 94°C for 30 s, 55°C for 90 s, and 72°C for 1 min by using specific primers for OsMCA1. Actin was used as a quantitative control . Aliquots of individual PCR products were resolved by agarose gel electrophoresis and visualized using ethidium bromide staining and exposure to UV light.
Real-time RT-PCR quantification
Real-time RT-PCR assays were performed as described by Kurusu et al. (2010) . First-strand cDNA was synthesized from 3 μg of total RNA using an oligo-dT primer and reverse transcriptase. Real-time PCR was performed using an ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) with SYBR Green real-time PCR Master Mix (Toyobo, Osaka, Japan) and OsMCA1 specific primers OsMCA1-RealF, 5'-TGGTCTCAAGCAGAGGATCATACA-3'; OsMCA1-RealR, 5'-CTCTGAACAGCAACCAAGCAAA-3'. Relative mRNA levels were calculated using the standard curve method and normalized to the corresponding OsActin1 gene levels. Standard samples of known template amounts were used to quantify PCR products.
Spatial pattern of OsMCA1 expression using OsMCA1p::GUS-expressing plants
A DNA fragment of the OsMCA1 promoter region was prepared using PCR by synthesizing the 5'-non-coding region spanning -1.5 to 0 kb from the OsMCA1 initiation codon, using rice (Nipponbare) genomic DNA as a template and the following primers: OsMCA1pF, 5'-CACCAACAACCCCTAACATGCCTAA-3'; OsMCA1pR, 5'-TGCCGTCGTCTACTCGGCTTCTTCT-3' (the CACC sequence used with the Gateway system), subcloned into a pENTR/D-TOPO cloning vector, and then cloned into a Ti-based promoterless GUS expression vector, pHGWFS7  using the LR clonase reaction; Agrobacterium-mediated transformation of rice calli was performed. Transformed calli were screened by hygromycin selection (50 μg mL-1); transgenic plants were then regenerated.
The T2 transgenic plants were grown at 28°C under a 16 h light/8 h dark cycle for experiments. Histochemical localization of GUS activity in situ was performed as follows. Samples were fixed for 1 h with 90% acetone in Eppendorf tubes placed on ice and washed four times with 100 mM sodium phosphate buffer, pH 7.0. Samples were then incubated for 24 h at 37°C in X-Gluc buffer (0.5 mg/mL 5-bromo-4-chloro-3-indolyl glucuronidase (Nacalai Tesque, Osaka, Japan), 50 mM sodium phosphate buffer, pH 7.0, 5% methanol), and then cleaned and fixed by rinsing for 1 h each with 50%, 70%, 90%, and 100% (v/v) ethanol successively. Fixed samples were stored in 100% ethanol before being photographed.
Generation of OsMCA1-overexpressing and suppressed lines
To generate RNA-silencing-triggered inverted repeat constructs, a region corresponding to 400 bp of the 3'-UTR of OsMCA1 was amplified using RNAiFW, 5'-CACC CTCTTATCCAAACTTGCCAT-3' and RNAiRV, 5'- AATGTTCCACAGGGGAAAAAGAATGTTCTC-3' as specific primers, subcloned into a pENTR/D-TOPO cloning vector, and cloned into a Ti-based RNAi vector, pANDA  using the LR clonase reaction. The construct was introduced into rice calli using Agrobacterium-mediated transformation, according to the method of Tanaka et al. 2001 . Transformed calli were screened by hygromycin selection (50 μg mL-1); transgenic plants were then regenerated. Transgenic cell lines derived from T2 plants were used for various analyses.
To overexpress OsMCA1 and GUS cDNAs, sequences were cloned into a Ti-based vector pPZP2H-lac  downstream of the maize Ubiquitin promoter, and Agrobacterium-mediated transformation of rice calli was performed. Transformed calli were screened by hygromycin selection (50 μg mL-1), followed by the regeneration of transgenic plants.
To express cytoplasm-targeted apoaequorin cDNA  in OsMCA1-suppressed plants, sequences were cloned into a Ti-based vector pSMAB704  downstream of a CaMV 35S promoter, and Agrobacterium-mediated transformation of rice calli was performed. Transformed calli were screened using bialaphos (Meiji Seika, Tokyo, Japan) selection (5 μg mL-1), followed by the regeneration of transgenic plants.
Subcellular localization of OsMCA1 in tobacco BY-2 cells
To generate transgenic BY-2 cells expressing GFP-OsMCA1, the coding region was amplified using OsMCA1(GFP)F, 5'-CACCATGGCGTCGTGGGAGAACCT-3' and OsMCA1(GFP)R, 5'-TTAGTGTTCCATGTACTGAA-3' as specific primers, subcloned into a pENTR/D-TOPO cloning vector, and then cloned into a pH7WGF2 vector encoding a N-terminal EGFP fusion  using the LR clonase reaction.
Transformation of BY-2 cells was carried out in accordance with An (1985)  with minor modifications as follows: 4 mL of 3-day-old exponentially growing culture was transferred to 90-mm Petri dishes and incubated at 28°C with 100 μL of fresh overnight-culture of Agrobacterium tumefaciens pGV2260 containing the binary vector pH7WGF2. After a 48-h co-cultivation, the tobacco cells were washed and plated on to LS agar medium containing hygromycin (50 μg mL-1) and carbenicillin (250 μg mL-1). Every 3-4 weeks, transformants were selected and transferred onto fresh medium for continued selection.
The fluorescent styryl membrane probe FM4-64 (Molecular Probes, Carlsbad, CA, USA) was kept as a 17 mM stock solution in sterile water, and used at a final concentration of 4.25 μM to label the vacuolar membrane (tonoplast). Five-day-old BY-2 cells were treated with FM4-64 for 3 h and washed twice with culture medium.
Measurement of cytosolic Ca2+concentration
Measurements of Ca2+ mobilization were made in accordance with the method described by Kurusu et al. (2011) . Briefly, apoaequorin-expressing rice cells (5 day after subculture) were incubated with 1 μM coelenterazine for at least 12 h at 25°C. Cell suspension (250 μL) was transferred to 1.1-cm-diameter culture tubes, and set in a luminometer (Lumicounter 2500, Microtech Nition, Chiba, Japan). In the luminometer, culture tubes rotated 17 revolutions every 3 s clockwise and counterclockwise in turn, agitating the cells. After a 15-min incubation to stabilize the cells, Ca2+-dependent aequorin luminescence was measured and expressed as relative luminescence units (rlu).
Ca2+uptake in cultured cells
Rice cells 5 days after subculture were used to measure Ca2+ uptake. The rice cells were incubated in Ca2+-free medium for at least 3 h at 25°C. The cell suspension (80 mg mL-1) was transferred to medium containing 0.1 mM CaCl2 and incubated for 1 h. Ca2+ uptake was initiated by adding 45CaCl2 solution to a final concentration of 33 kBq/g. Cells were then agitated at 25°C; 1 mL of cells was collected at 0, 15 and 45 min after the addition of 45CaCl2. Cells were filtered using Whatman filters (GF/C) presoaked with 5 mM CaCl2 and washed 5 times with an ice-cold solution of 5 mM CaCl2, and 2 mM LaCl3 to remove 45Ca2+ from cell walls. Radioactivity retained on each filter was counted as described previously .
Measurement of ROS
Rice cells (cv. Nipponbare) 5 days after subculture were used for measurement of •O2- and H2O2 in the extracellular medium. •O2--dependent chemiluminescence was monitored in growth medium supplemented with 20 μM methoxylated cypridina luciferin analog (MCLA (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-α]pyrazin-3-one); Invitrogen) using a luminometer (Lumicounter 2500) under the same conditions as for measuring [Ca2+]cyt .
To monitor H2O2 produced in extracellular medium, cells (80 mg mL-1) were washed and resuspended in 5 mM MES buffer (pH 7.0) containing 0.5 mM CaCl2, 0.5 mM K2SO4 and with or without 175 mM mannitol (Kurusu et al. 2005). A 25-μL aliquot of medium was mixed in a 96-well microtiter plate with 150 μl 50 mM Tris-HCl (pH 8.0) and 25 μL 0.462 mM luminol in 50 mM Tris-HCl, pH 8.0. Potassium ferricyanide (25 μL, 11.76 mM) was added, and H2O2-dependent chemiluminescence was recorded for 15 s using a luminometer (MicroLumat Plus LB96V, Berthold Technologies, Bad Wildbad, Germany).
Statistical significance was determined using an unpaired Student's t test; P < 0.05 indicated significance.
YN Present address: Laboratory of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8510, Japan.
1,2-bis-(2-aminophenoxy)ethane-N, N, N', N'-tetra acetic acid
cytosolic free Ca2+ concentration
differential interference contrast
ethylene glycol-bis-(2-aminoethylether)-N, N, N', N'-tetra acetic acid
green fluorescent protein
microbe-associated molecular pattern
- • O2-:
superoxide anion radical
- MS medium:
Murashige and Skoog medium
rapid amplification of cDNA ends
relative luminescence units
- RNAi :
reactive oxygen species
We would like to thank Mr. Yasuhiro Sakurai for helpful technical assistance, Drs. Hiroaki Shimada and Tadamasa Sasaki for helpful technical suggestions, Drs. Daisuke Miki and Ko Shimamoto for the RNAi plasmid (pANDA vector), and Dr. Naoto Shibuya for the gift of N-acethylchitoheptaose.
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science & Technology for Scientific Research on Innovative Areas (21200067) to TK, for Exploratory Research (21658118) to KK, for Young Scientists (B) (21780041) to TK, for Scientific Research on Priority Area (21026009) to HI, for Scientific Research B (19370023) to KK and (21370017) to HI, and by grants from Japan Science and Technology Agency, for Adaptable and Seamless Technology Transfer Program through target-driven R&D (AS221Z03504E) to TK and for CREST to HI and KK.
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