284 44(3) 2009 Ca 2+ (Knight et al., 1996) CBF1(C-repeat-binding factor 1), RNA M-MLV 1 c DNA DNA A petala2/, SYBR Premix Ex Taq (AP 2/EREBP)(Stocking

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1 Chinese Bulletin of Botany 2009, 44 (3): , doi: /j.issn CBF1 Ca 2+ *,,,, , Ca 2+ (Ar abidopsis thaliana) CBF1, CBF1 Ca 2+, CBF1 Ca 2+, 2+, Ca 2+ EGT A LaCl 3, 2+ Ca CB F1 Ca 2+, Ca 2+ CBF 1 CBF 1 Ca 2+,,, CBF1,,, (2009). CBF1 Ca , , Pickard, 1993) Ca 2+ Ca 2+, Ca 2+ night, et al., 1996), Ca 2+, ( c o l d acclimation)(thomashow, 1999), Ca 2+, Ca 2+ (Plieth, 2001) Prasher, (1985) (Aequorea victoria) (apoaequor in) ABA ( Escherichia coli), ( Chinnusamy et al., 2006; Maeda et al., 2006; Suzuki and Mittler, 2006),, 3 Ca , Ca 2+ 3, Ca 2+, EF ; 400 kda Ca 2+ ([ (coelenterazine), Ca 2+ ] cyt ), 3 Ca 2+ [ Ca 2+ ] cyt,,, (Rudd and, coelen teramide Franklin-Tong, 2001) Knight (1996 ) CO 2 1, 465 nm Ca 2+ (Charbonneau et al., 1985) Ca 2+, C a 2 + ( Ding and,, : ; : : ( No ) * E -mail: songcp@henu.edu.cn

2 284 44(3) 2009 Ca 2+ (Knight et al., 1996) CBF1(C-repeat-binding factor 1), RNA M-MLV 1 c DNA DNA A petala2/, SYBR Premix Ex Taq (AP 2/EREBP)(Stockinger et al., 1997), real-time PCR,, DNA CRT/DRE,, ; 95 C 10, 57 C 20, 72 C 15, 40 ; (responsive to dehydration, RD) Melt 72 C 99 C, 1 45, (cold regulated, COR) (low-temperature induced, LTI) (Zhu, 2001) J aglo-ottosen (1998) CBF1, PCR : CBF1 Sense, 5'-GCATGTCTCAACTT- COR CGCTGA-3', CBF1 Anti-sense, 5'-ATCGTCTCCTCC-, GGCAAGTCA-3', Ubiquitin Anti-sense, 5'-CACAAAC-, (Zhu et al., GAGGGCTGGAACA-3' 2007), C a 2+ ;, 1.3 (Ca 2+ signature), (M iao et al., 2006) C BF1 pmaq2 HVA1-2.2 GV3101, C a 2+ ( Miao et al., 2006) 4, C a 2+,, F 1 Ca 2+,, PCR ( Arabidopsis thaliana L.) CBF1 MF Thomashow 7 8, W assilewskija (Ws)-2, 2. 5 mmol. L 1 pmaq2 HVA H Knight, GV C a 2+ SYBR Premix Ex Taq T akara, rizol T Invitrogen, Promega, EGTA LaCl Real-time PCR M-MLV 100 ml ( ) Sigma, , Trizol 3 Real-time PCR : 95 C 60 (early responsive to dehydration, ERD) 1 C 5 3 Realtime PCR MX3005P(Stratagene) Ubiquitin Real-time ATGTCCAG-3'; Ubiquitin Sense, 5'-ATTACCCGATG- PCR Sense, 5'-ATGACCAGCGAACAATACTCAGT-3'; Antisense, 5'-TTAGGGGACAGCTCCACCGTAGA-3', 1 2, (20/ 20n, Turner Biosystems), ) 20, 0.6 ml (0 C) MS 14 4 C, 60, 0.6 ml

3 : CBF1 Ca mol. L 1 CaCl 2 20%, Ca 2+ : pca ( log k) k (ra te constant), ( Knight et al., 1996) 1.6, La Cl 3 EGTA (ethylene glycoltetraacetic acid), 10 mmol. L 1 20 mmol. L 1, 1, 1. 5 C a 2+ Figure 1 Analysis of the transcript levels of CBF1 gene Ca CBF1 CB F1, rea l- time PCR CBF1,, CBF1 ; 1, ;, 1 CBF, 6, CBF 1 CO R, Figure 2 PCR analysis of aequorin gene in transgenic CBF1 Ca 2+ Arabidopsis lines M: Marker; 1: Wild-type; 2 4: Transgenic pmaq2 lines; 5 7:, pmaq2 Transgenic HVA1-2.2 lines HVA1-2.2 CBF1, ( 2) 2, CBF1, Ca 2+, CBF1, Ca 2+ (1.69±0.063) mmol. L 1, 1 CBF1 real-time PCR under cold stress by using real-time PCR PCR M: ; 1: ; 2 4: pmaq2 ; 5 7: HVA1-2.2 (1.43±0.039) mmol. L 1,, CBF1 2.3 CBF1 C a 2+ C a 2+, (0 C) CBF1 Ca 2+ Ca 2+, 3A, HVA1-2.2,

4 286 44(3) E GTA LaCl 3 CBF1 C a 2 +, ( Knight et al., 1996) Ca 2+ (Knight et al., 1996), 3B CB 2+ F1 Ca, Ca 2+, CBF1, Ca 2 + ( 1.3 2±0.0 36) Ca 2+ mmol. L 1, ( 1.29±0.068) mmol. L 1, Ca 2+, CB F1 Ca 2+ Ca 2+ 2+,, Ca Ca 2+ CBF1 Ca 2+, Ca 2+ EGTA, Ca 2+, Ca 2+, Ca mmol. L 1 EGTA, Ca 2+, 4A 4B, EGTA, CBF1 Ca 2 + ; Ca 2+ (1. 43±0.039) mmol. L 1 (0.898±0.079) mmol. L 1, CBF1 (1.69± 0.063) mmol. L 1 (1.01±0.039) mmol. L 1 ( 4C) LaCl 3 Ca 2+, Ca mmol. L 1 LaCl 3, EGTA ( E), CBF1 C a 2+ (0.761± 0.094) mmol. L 1 (0.81±0.086) mmol. L 1 3 CBF1 ( CBF1-OX) ( 4F) EGTA LaCl 3 Ca 2+ (A) Ca 2+ ; (B) Ca 2+ Ca 2+, CBF 1 Figure 3 Kinetic changes of the cold-induced [Ca 2+ ] in CBF1 overexpression (CBF1-OX) mutant plants (A) The concentration of cytosolic free calcium ([Ca 2+ ] cyt ) enhancement; (B) The concentration of calcium adjacent to the vacuolar membrane ([Ca 2+ ] md ) enhancement. Mean values ± SE are given in the text Ca 2+ ( 4C, F) 2.5, 2 (1), (Jaglo-Ottosen et al., 1998; Chinnusamy et al., 2006; 2+ Dong et al., 2006); (2) Ca 2+ ( Knight et al., 1998)

5 : CBF1 Ca EGTA(ethylene glycol tetraacetic acid) LaCl 3 CBF1 ( CBF1-OX)(B), (E) (W T)(A), ( D ) [ Ca 2+ ] cyt (A) (B) (D) ( Figure 4 E); (C) ( F) EGTA LaCl 3 Ca 2+ Effects of EGTA (ethylene glycol tetraacetic acid) and LaCl 3 on cold-shock induced [Ca 2+ ] cyt transients in CBF1 overexpression mutant (CBF1-OX) (B), (E) and wild-type Arabidopsis thaliana (WT) (A), (D) A representative curve is shown in (A), (B) and (D), (E); (C) is the average [Ca 2+ ] cyt achieved per seedling in (A) and (B); (F) is the average [Ca 2+ ] cyt achieved per seedling in (D) and (E). Mean values ± SE are given in the text, Ca 2+, Ca 2+ Ca 2+, Ca 2+ EG TA Ca 2+ La Cl 3 (Knight et al., 1996) Ca 2+, CB F1 CBF1 Ca 2+, Ca 2+ (Jaglo-Ottosen et al., 1998) ( 4C, F), CBF1 Ca 2+ Ca 2+, CBF1 Ca 2+, Ca 2+ Ca 2+, CBF1, Ca 2+, (Thomashow, 1999; Uemura et Ca 2+ ( 3), CBF 1 al., 2006), Ca 2+, Ca 2+,,

6 288 44(3) 2009 (Plieth, alters drought calcium signaling pathways in Arabidopsis. Plant 2001) Ca 2+ J 16, Knight H, Trewavas AJ, Knight MR (1996). Cold calcium signaling in Arabidopsis involves two cellular pools and a change ( Hirschi, 1999), CBF1, in calcium signature after acclimation. Plant Cell 8, 489 Ca 2+ CBF1 Ca 2+ play a crucial role in low-temperature adaptation and phloem, loading in Arabidopsis. Plant Cell 18, Ca 2+, Miao Y, Lv D, Wang P, Wang XC, Chen J, Miao C, Song CP Ca 2+, (2006). An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 18, H Knight Plieth C (2001). Plant calcium signaling and monitoring: pros and pmaq2 HVA1-2.2 MF cons and recent experimental approaches. Protoplasma 218, Thomashow CBF1 Prasher DC, McCann RO, Cormier MJ (1985). Cloning and MR Knight expression of the cdna cloning for aequorin, a bioluminescence calcium-binding protein. Biochem Biophys Res Commun Charbonneau H, Walsh KA, McCann RO, Prendergast FG, Cormier MJ, Vanaman TC (1985). Amino acid sequence of the calcium-dependent photoprotein aequorin. Biochemistry 24, Chinnusamy V, Zhu JH, Zhu JK (2006). Gene regulation during cold acclimation in plants. Physiol Plant 126, Ding JP, Pickard BG (1993). Modulation of mechanosensitive calcium-selective cation channels by temperature. Plant J 3, Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK (2006). The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci USA 103, Hirschi KD (1999). Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity. Plant Cell 11, Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998). Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, Knight H, Brandt S, Knight MR (1998). A history of stress 503. Maeda H, Song W, Sage TL, DellaPenna D (2006). Tocopherols 126, Rudd JJ, Franklin-Tong VE (2001). Unravelling response-specificity in Ca 2+ signaling pathways in plant cells. New Phytol 151, Stockinger EJ, Gilmour SJ, Thomashow MF (1997). Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the CRT/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94, Suzuki N, Mittler R (2006). Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction. Physiol Plant 126, Thomashow MF (1999). Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50, Uemura M, Tominaga Y, Nakagawara Y, Shigematsu S, Minami A, Kawamura Y (2006). Responses of the plasma membrane to low temperatures. Physiol Plant 126, Zhu J, Dong CH, Zhu JK (2007). Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr Opin Plant Biol 10, Zhu JK (2001). Cell signaling under salt, water and cold stress. Curr Opin Plant Biol 4,

7 : CBF1 Ca Intracellular Ca 2+ Changes During Cold Stress in CBF1- overexpress Arabidopsis Guozeng Zhang, Ling Bai, Chunpeng Song * The Laboratory of Plant Stress Biology, Department of Biology, Henan University, Kaifeng , China Abstract Plant growth is greatly affected by low temperature. Cold shock elicits an immediate increase in free Ca 2+ concentration in plant cells. Here, we focused on the expression of CBF1 under low temperature treatment and the cold-triggered Ca 2+ responses in CBF1-overexpressing Arabidopsis. CBF1 was induced by low temperature. To address the relation between CBF1 and Ca 2+, we measured cold-triggered Ca 2+ responses in CBF1-overexpressing Arabidopsis. CBF1-overexpressing plants exhibited a higher mean peak Ca 2+ concentration than did wild-type plants, but the Ca 2+ signature adjacent to the vacuolar membrane increased to similar levels in both plants. In Arabidopsis seedlings pretreated with EGTA or LaCl 3, the cold shock-induced increase of Ca 2+ concentration was inhibited. The cytoplasmic Ca 2+ concentration in CBF1-overexpressing and wild-type plants decreased to the same level. Thus, the results suggest that Ca 2+ is involved in the process of the CBF1 response to low-temperature signal transduction, and the CBF1-overexpress may enhance freezing tolerance of plants by increasing the cytoplasmic free Ca 2+ concentration. Key words aequorin, Arabidopsis thaliana, calcium, CBF1, cold stress Zhang GZ, Bai L, Song CP (2009). Intracellular Ca 2+ changes during cold stress in CBF1-overexpress Arabidopsis. Chin Bull Bot 44, * Author for correspondence. songcp@henu.edu.cn ( :

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