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CbADH1 improves plant cold tolerance

CbADH1 improves plant cold tolerance PLANT SIGNALING & BEHAVIOR 2019, VOL. 14, NO. 7, e1612680 (5 pages) https://doi.org/10.1080/15592324.2019.1612680 RESEARCH PAPER a a b Yuan Song , Lijun Liu , and Xiang Ma Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China; Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, China ABSTRACT ARTICLE HISTORY Received 7 March 2019 ADH1 (alcohol dehydrogenase 1) was involved in plant growth and development and responded to Revised 19 April 2019 various stresses. We published a cold-induced alcohol dehydrogenase 1 gene from C. bungeana, Accepted 23 April 2019 CbADH1, which exists 43 unique amino acids. Here, we confirmed that overexpression of CbADH1 in Arabidopsis and tobacco significantly improved cold shock tolerance through electrolyte leakage, semi- KEYWORDS lethal temperature, phenotypic and survival analysis. These results indicate that CbADH1 is the candi- ADH1 (alcohol date gene to improve the ability of plant freezing resistance, and it has great application value. dehydrogenase 1); cold tolerance; Chorispora bungeana Introduction Results Chorispora bungeana grows in the alpine regions and is Screening of homozygous lines overexpressing CbADH1 a perennial alpine subnival plant species experiences frequent In order to further study the function of CbADH1, CbADH1 temperature fluctuations during the growth season, which fused with pEarlyGate101-YFP vector was introduced into wild- has a strong freeze tolerance. We found an alcohol dehydro- type Arabidopsis thaliana (Col.) by dipping flower, and the homo- genase-like protein that was specifically expressed in cold zygous lines expressing CbADH1 were obtained. Quantitative stress according to the results of two-dimensional electro- PCR was used to identify the changes of CbADH1 gene expres- phoresis of proteins. We cloned and characterized CbADH1 sion in homozygotes. As shown in Figure 1, the gene expression from C. bungeana. Multiple alignment result showed that the was increased in the four homozygous lines we selected. Among N-terminal domain of CbADH1 existed 43 unique amino them, the gene expression in 1–6and 9–4 lines increased four-fold acids, and the subcellular localization was affected, suggesting and eight-fold, respectively, and these two lines were used in different biological functions. subsequent antifreeze tests (Figure 1). The classic alcohol dehydrogenase (ADH, alcohol: NAD The seedlings of wild-type and transgenic plants of 3-week oxidoreductase, EC1.1.1.1) is a Zn-binding enzyme that acts size were cultured at 4 °C for 7 d and the control lines were as a dimer and relies on a NAD (P) co-factor to interconvert cultured in medium grew normally for 7 d, and the freezing ethanol and acetaldehyde (as well as other short linear alco- resistance experiment was carried out at the same time. hol/aldehyde pairs). ADH activity is subject to various 4 5,6 stresses, including biotic and abiotic stress. However, the function mechanism is not clear. ADH is related to the fer- CbADH1 improves the cold tolerance of plant mentative metabolism reduces acetaldehyde to ethanol, regen- independent of cold acclimation erates NAD+ and 2ATP, and protects cells from cytoplasmic As shown in Figure 2, transgenic plants are more freeze-resistant acidosis. Ethanol enhances ATPase synthesis in different than wild-type plants. The half lethal temperature (LT50) of non- 8,9 species. ATPase-catalyzed proton pumping may contribute cold acclimated (Figure 2(a)), WT lines were −4.05 °C, while the to chilling tolerance in plants. ADH1 plays a key role in LT50 of transgenic lines was −5.97 °C. Under cold acclimation maintaining the stability of the membrane structure for the (Figure 2(b)), the LT50 of transgenic lines also decreased by about enhancement of cold resistance in plants. one degree (−7.01 °C) compared with −6°Cofwildtypelines. We also discussed Metabolite Profiling of adh1 Mutant This result has been mentioned in our previous study. These Response to Cold Stress in Arabidopsis, the results showed that results suggest that CbADH1 can enhance the cold tolerance of soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine) plants, which is independent or partly dependent on cold changed accordingly. acclimation. In this study, we proved CbADH1 significantly improved In order to find the functional difference of ADH1 gene of cold shock response through physiological and biochemical C. bungeana (CbADH1) and of A. thaliana (AtADH1) under indicators, indicating CbADH1 has strong antifreeze function cold stress, CbADH1 (including 43 unique amino acids), and great application prospect. CbADH1 and AtADH1 were overexpressed in tobacco CONTACT Yuan Song songyuan@lzu.edu.cn Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China © 2019 Taylor & Francis Group, LLC e1612680-2 Y. SONG ET AL. overexpression of CbADH1 and CbADH1 significantly enhanced the survival rate of Arabidopsis and tobacco under sudden cold stress (Figures 3 and 4). The results showed that CbADH1 could enhance the ability of sudden cooling toler- ance in plants. Discussion According to our previous work, we cloned the CbADH1 gene of C. bungeana. The results of multiple sequence align- ment showed that it contained three conserved domains of ADH1 family. Evolutionary analysis also showed high homol- ogy with the ADH1 gene of other species. Here we found CbADH1 can powerfully improve the freezing resistance, providing a candidate gene for genetic engineering. The structure of ADH1 and the binding site to substrate are Figure 1. Relative expression level of CbADH1 in homozygous seedlings. Data presented are the mean values of triplicate independent experiments ± SD, and very conservative. However, the 5‘ terminal deleted a specific asterisks indicate the significant differences in the expression level compared to 2 sequence encoding 43 amino acids, and we marked a triangle WT. 2 here. It could form different complexes because of the exis- (*p < .05, **p < .01, t-test, Tukey) . △ tence of 5‘ terminal specialsequencesothattheyshowdifferent activity in transgenic plants. The 22nd serine is the unique phos- and Arabidopsis thaliana, respectively. After plants were cul- phorylation site of cAMP and cGMP-dependent protein kinase in tured for four weeks in room temperature, the freezing resis- CbADH1, which may affect its localization. And the specific tance of wild and transgenic homozygous lines was analyzed functionsneededtobefurther studied. At the sametime, the5‘ by sudden cold treatments. The transgenic lines of flanking sequence of 4450 bp was cloned. The upstream 2000 bp Arabidopsis thaliana were treated with sudden cold treatment sequence of ATG was analyzed by PLACE website. It was found at −10 °C for 3 h and the transgenic tobacco at −4 °C for 3 that there are many cis-acting elements related to plant growth h. The survival rate of plants was counted after recovering at and development and abiotic stress response. 23 °C for 7 d. The results showed that both AtADH1 and The expression pattern analysis showed that CbADH1 was CbADH1 could enhance the freezing resistance of Arabidopsis highly expressed in young leaves and in flowers as well as in thaliana and tobacco plants. Compared with AtADH1, the cotyledons and hypocotyls after 12 d of germination. This Figure 2. Effect of freezing temperature on electrolyte leakage from the transgene lines and WT plants under (a) non-acclimation condition (NA) and (b) cold acclimation (CA) at 4 °C for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences compared toWT. (*p < .05, **p < .01, t-test, Tukey). PLANT SIGNALING & BEHAVIOR e1612680-3 Figure 3. CbADH1 conferred enhanced cold-shock resistance in Arabidopsis. (a) The photograph showing 28-d-old of transgenic Arabidopsis and WT plants in the growth chamber. (b) Recovery photograph of transgene Arabidopsis and WT in cold shock treatment at −10 °C for 3 h and recovery for 7 d. (c) The survival rate of different transgene plants after recovery for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences. (*p < .05, **p < .01, t-test, Tukey). expression pattern is similar to that of Arabidopsis thaliana. In CbADH1 could be used as a candidate gene for improving cold addition, GUS activity occurs in pollen grains, suggesting that tolerance of plants. However, under cold acclimation, the semi- CbADH1 may be involved in pollinator attraction, seed propaga- lethal temperature of CbADH1 transgenic lines was only scanty tion and defense against animal predation in alpine margin than that of the wild type (Figure 2(b)), suggesting that environments. cold acclimation had little effect on the role of CbADH1 in cold The transcriptional level of CbADH1 increased in different stress. The transcription data showed that there was no homolog abiotic stress, which may be related to the stress-responsive of CBFs in Chorispora bungeana, suggesting that cold acclimation binding elements in its promoter region. Previous studies showed hadnoorveryweakeffect on C. bungeana.Theresultsshowed that both biological and abiotic stress induced the expression of that CbADH1 significantly enhanced the tolerance of transgenic ADH1, but most of the studies focused on hypoxia stress, such as plants under sudden cold stress (Figures 3 and 4). It provides the study of water flooding stress in rice, metabolic pathway of theoretical support for the application of genes in Chorispora adh1 mutant in Chlamydomonas reinhardtii under hypoxia, bungeana as the candidate genes for freezing resistance in crop cold tolerance in diploid strawberry, suggesting that ethanol genetic improvement. dehydrogenase could be used as a molecular marker for the studyofcoldtolerance.The ADHenzymeactivity in Materials and methods C. bungeana increased under cold stress, which was consistent Experiment on freezing resistance of Arabidopsis with the results of previous studies. It was found that the transgenic lines expression of ADH1 and enzyme activity increased under cold stress compared with the control group. The transgenic lines of Arabidopsis thaliana were infected with The semi-lethal temperature was lower in CbADH1 transgenic the constructed subcellular localizing vector pEarlyGate101 into lines than the wild type without cold acclimation (Figure 2(a)), so the wild-type plants of Arabidopsis thaliana in antifreeze e1612680-4 Y. SONG ET AL. Figure 4. CbADH1 conferred enhanced cold-shock resistance in N. benthamiana. (a) The photograph showing 28-d-old of transgenic tobacco and WT plants in the growth chamber. (b) Recovery photograph of transgene tobacco and WT in cold shock treatment at −4 °C for 3 h and recovery for 7 d. (c) The survival rate of different transgene plants after recovery for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences. (*p < .05, **p < .01, t-test, Tukey). experiment. The homozygous lines were screened by a herbicidal activated in 50 ml of LB (Kan + Gen + Rif), 28 °C for 8 min, and agent for antifreeze test. All seeds were sown on half-strength re-suspended 1/2 MS (add 2% sucrose, 50 uM acetosyringone), Murashige and Skoog (MS) germination medium, supplemen- and the OD600 is 0.8. Then, the leaves of tobacco seedlings were ted with 1% sucrose and 0.8% agar at pH 5.7. cultured for four weeks, and the perforator was used to form The T-DNA mutant adh1–2 with BASTA resistance, and the a small leaf disc. The leaves were immersed in the bacterial open reading frames of CbADH1 and AtADH1 were fused to solution and shook at 28 °C avoiding light (70–80 rpm) 30 min. pMDC83 by Gateway system. GV3101 was introduced into The leaves were sandwiched on the ultra-clean worktable, and the Arabidopsis thaliana wild type and mutant lines. The homozy- aseptic filter paper was used to dry the bacterial solution on the gous strains were screened on MS medium containing 50 mg/L surface of the leaves and moved to the 1/2 MS containing 1 mg/L hygromycin. Over-expressed CbADH1 and AtADH1 transgenic 6-BA for 2 d. Then transferred to the screening medium for 1 homozygous lines were cultured for 4 weeks and then treated month (1/2 MS 2% sucrose 0.6% Agar 2 mg/L 6-BA 500 mg/L car with cold stress. The treatment condition was 0 °C for 1.5 h, then 50 mg/L hyg 0.5 mg/L NAA), and car (carboxybenzyl) was to decreased by 1 °C per hour until −10 °C for 3 h, then restored to inhibit bacteria. Finally, the plantlets were transferred to the 4 °C for 12 h. The plants were recovered in room temperature for medium (1/2 MS with 2% sucrose 0.5% Agar 500 mg/L car 7 d. The phenotype was observed and photographed, and the 50 mg/L hyg) for 20 d. Rooting was induced on rooting medium survival rate was counted. (1/2 MS with 2% sucrose 0.5% Agar 500 mg/L car 50 mg/L hyg 0.1 mg/L NAA), and then cultured in soil. The seeds of T1 generation were obtained. Then, the homozygous strain was Establishment of transgenic tobacco system and freezing screened on the MS medium containing hygromycin. resistance experiment Wild-type and over-expressed CbADH1 and AtADH1 trans- genic homozygous lines were cultured for 4 weeks and then Tobacco transformation experiment was carried out by leaf disk treated with cold treatment. The treatment conditions were 4 °C method. CDs regions of CbADH1 and AtADH1 were fused to for 0.5 h, then from 0 °C to −4°Cfor 3hdecreasedby1°C every pMDC83 and introduced into tobacco. First, agrobacterium is PLANT SIGNALING & BEHAVIOR e1612680-5 Chorispora bungeana in cold stress. Gene. 2017;636:1–16. half hour. After 4 °C 12 h, the recovery in room temperature for 7 doi:10.1016/j.gene.2017.09.015. d. The phenotype was observed and photographed, and the sur- 3. Strommer J. The plant ADH gene family. Plant J. 2011;66 vival rate was counted. (1):128–142. doi:10.1111/j.1365-313X.2010.04458.x. 4. Dolferus R, Jacobs M, Peacock WJ, Dennis ES. Differential interactions of promoter elements in stress responses of the Electrolyte leakage tests and lethal temperature Arabidopsis Adh gene. Plant Physiol. 1994;105:1075–1087. calculation 5. Pathuri IP, Reitberger IE, Huckelhoven R, Proels RK. Alcohol dehydrogenase 1 of barley modulates susceptibility to the parasitic Electrolyte leakage tests were performed as previously described fungus Blumeria graminis f.sp. hordei. J Exp Bot. 2011;62 45. The third to fifth leaves from 18-d-old plants were added to (10):3449–3457. doi:10.1093/jxb/err017. capped test tubes (15 ml) and placed in a bath-type programmable 6. Komatsu S, Deschamps T, Hiraga S, Kato M, Chiba M, freezer (XT5201-D31-R40C, XuTemp, China). The petiole was Hashiguchi A, Tougou M, Shimamura SYasue H. Characterization of a novel flooding stress-responsive alcohol placed facing downward into the bottom of the tube (without dehydrogenase expressed in soybean roots. Plant Mol Biol. damaging the leaves). Water was added to the bottom of the 2011;77(3):309–322. doi:10.1007/s11103-011-9812-y. tube until the petiole was fully submerged. Samples were kept on 7. Drew MC. Oxygen deficiency and root metabolism: injury and ice for 30 min. The plants were exposed to freezing temperatures acclimation under hypoxia and anoxia. Annu Rev Plant Physiol ranging from 0 to −20 °C (0, −4, −6, −8, −10, −12, −14, −16, −18, Plant Mol Biol. 1997;48:223–250. doi:10.1146/annurev. arplant.48.1.223. and −20 °C). Tubes were placed on ice after removal from the bath 8. Lalitha T, Ramakrishnan CV, Telang SD. Interaction of alcohol and until removal of all tubes was completed. Leaves were then and protein deficiency on adult rat brain lipids. Indian J Biochem immersed in 10 ml distilled water and placed on a shaker for 2 h at Biophys. 1987;24:238–240. 4 °C. Electrolyte leakage was determined as the ratio of before and 9. Lalitha T, Ramakrishnan CV, Telang SD. Interaction of alcohol after sample boiling via a conductivity meter (DDSJ-308, Leici, and protein deficiency on rat brain synaptosomal (Na+-K+)- ATPase. Neurochem Res. 1988;13:963–966. China). The freezing temperature that caused a 50% electrolyte 10. Low R, Rockel B, Kirsch M, Ratajczak R, Hortensteiner S, leakage (TEL50) was calculated from plotted data of relative elec- Martinoia E, Luttge URausch T. Early salt stress effects on the trolyte leakage as the log EC50 value of sigmoidal curve fitting to differential expression of vacuolar H(+)-ATPase genes in roots the leakage values using SPSS PASW Statistics 18.0 (IBM SPSS and leaves of Mesembryanthemum crystallinum. Plant Physiol. software, USA). 1996;110:259–265. 11. Song Y, Liu L, Wei Y, Li G, Yue XAn L. Metabolite profiling of ADH1 mutant response to cold stress in Arabidopsis. Front Plant Acknowledgments Sci. 2016;7:2072. 12. Higo K, Ugawa Y, Iwamoto MKorenaga T. Plant cis-acting reg- This work was supported by the Key Laboratory of Superior Forage ulatory DNA elements (PLACE) database: 1999. Nucleic Acids Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the Res. 1999;27:297–300. National Natural Science Foundation of China (No. 31872682); the 13. Takahashi H, Saika H, Matsumura H, Nagamura Y, Tsutsumi N. Fundamental Research Funds for the Central Universities (lzujbky-2018- Nishizawa NK, Nakazono M Cell division and cell elongation in 110 and lzujbky-2018-kb05). Also thank Core Facility of School of Life the coleoptile of rice alcohol dehydrogenase 1-deficient mutant Science, Lanzhou Unviersity for technical support. are reduced under complete submergence. Ann Bot. 2011;108 (2):253–261. doi:10.1093/aob/mcr137. 14. Magneschi L, Catalanotti C, Subramanian V, Dubini A, Yang W, Disclosure of Potential Conflicts of Interest Mus F, Posewitz MC, Seibert MPerata P, Grossman AR. A mutant No potential conflicts of interest were disclosed. in the ADH1 gene of Chlamydomonas reinhardtii elicits meta- bolic restructuring during anaerobiosis. Plant Physiol. 2012;158 (3):1293–1305. doi:10.1104/pp.111.191569. Funding 15. Davik J, Koehler G, From B, Torp T, Rohloff J, Eidem P, Wilson RC,SonstebyA,Randall SK,AlsheikhM.Dehydrin This work was supported by the Key Laboratory of Superior Forage alcohol dehydrogenase, and central metabolite levels are asso- Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the ciated with cold tolerance in diploid strawberry (Fragaria National Natural Science Foundation of China (No. 31872682); the spp.). Planta. 2013;237(1):265–277. doi:10.1007/s00425-012- Fundamental Research Funds for the Central Universities (lzujbky- 1771-2. 2018-110 and lzujbky-2018-kb05). 16. Peters J, SFrenkel C. Relationship between alcohol dehydrogenase activity and low-temperature in two maize genotypes, Silverado F1 and Adh1-Adh2- doubly null. Plant Physiol Biochem. 2004;42 ORCID (10):841–846. doi:10.1016/j.plaphy.2004.10.004. 17. Zhao Z,TanL,DangC,Zhang H,Wu QAnL.Deep- Xiang Ma http://orcid.org/0000-0002-7014-3774 sequencing transcriptome analysis of chilling tolerance mechanisms of a subnival alpine plant, Chorispora bungeana.BMC PlantBiol. 2012;12:222. doi:10.1186/1471- References 2229-12-222. 18. Murashige TSkoog F. A revised medium for rapid growth and 1. Song Y, Liu L, Feng Y, Wei Y, Yue X, He W, Zhang HAn L. Chilling- bioassay with tobacco tissue cultures. Plant Physiol. and freezing- induced alterations in cytosine methylation and its 1962;15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x. association with the cold tolerance of an alpine subnival plant, 19. Horsch RB. A simple and general method for transferring genes Chorispora bungeana.PLoS One. 2015;10:e0135485. into plants. Science. 1985;227(4691):1229–1231. doi:10.1126/ 2. Liu L, Song Y, Xu J, Li D, Li GAn L. Differential expression by science.227.4691.1229. chromatin modifications of alcohol dehydrogenase 1 of http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Signaling & Behavior Taylor & Francis

CbADH1 improves plant cold tolerance

Plant Signaling & Behavior , Volume 14 (7): 1 – Jul 3, 2019

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PLANT SIGNALING & BEHAVIOR 2019, VOL. 14, NO. 7, e1612680 (5 pages) https://doi.org/10.1080/15592324.2019.1612680 RESEARCH PAPER a a b Yuan Song , Lijun Liu , and Xiang Ma Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China; Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, China ABSTRACT ARTICLE HISTORY Received 7 March 2019 ADH1 (alcohol dehydrogenase 1) was involved in plant growth and development and responded to Revised 19 April 2019 various stresses. We published a cold-induced alcohol dehydrogenase 1 gene from C. bungeana, Accepted 23 April 2019 CbADH1, which exists 43 unique amino acids. Here, we confirmed that overexpression of CbADH1 in Arabidopsis and tobacco significantly improved cold shock tolerance through electrolyte leakage, semi- KEYWORDS lethal temperature, phenotypic and survival analysis. These results indicate that CbADH1 is the candi- ADH1 (alcohol date gene to improve the ability of plant freezing resistance, and it has great application value. dehydrogenase 1); cold tolerance; Chorispora bungeana Introduction Results Chorispora bungeana grows in the alpine regions and is Screening of homozygous lines overexpressing CbADH1 a perennial alpine subnival plant species experiences frequent In order to further study the function of CbADH1, CbADH1 temperature fluctuations during the growth season, which fused with pEarlyGate101-YFP vector was introduced into wild- has a strong freeze tolerance. We found an alcohol dehydro- type Arabidopsis thaliana (Col.) by dipping flower, and the homo- genase-like protein that was specifically expressed in cold zygous lines expressing CbADH1 were obtained. Quantitative stress according to the results of two-dimensional electro- PCR was used to identify the changes of CbADH1 gene expres- phoresis of proteins. We cloned and characterized CbADH1 sion in homozygotes. As shown in Figure 1, the gene expression from C. bungeana. Multiple alignment result showed that the was increased in the four homozygous lines we selected. Among N-terminal domain of CbADH1 existed 43 unique amino them, the gene expression in 1–6and 9–4 lines increased four-fold acids, and the subcellular localization was affected, suggesting and eight-fold, respectively, and these two lines were used in different biological functions. subsequent antifreeze tests (Figure 1). The classic alcohol dehydrogenase (ADH, alcohol: NAD The seedlings of wild-type and transgenic plants of 3-week oxidoreductase, EC1.1.1.1) is a Zn-binding enzyme that acts size were cultured at 4 °C for 7 d and the control lines were as a dimer and relies on a NAD (P) co-factor to interconvert cultured in medium grew normally for 7 d, and the freezing ethanol and acetaldehyde (as well as other short linear alco- resistance experiment was carried out at the same time. hol/aldehyde pairs). ADH activity is subject to various 4 5,6 stresses, including biotic and abiotic stress. However, the function mechanism is not clear. ADH is related to the fer- CbADH1 improves the cold tolerance of plant mentative metabolism reduces acetaldehyde to ethanol, regen- independent of cold acclimation erates NAD+ and 2ATP, and protects cells from cytoplasmic As shown in Figure 2, transgenic plants are more freeze-resistant acidosis. Ethanol enhances ATPase synthesis in different than wild-type plants. The half lethal temperature (LT50) of non- 8,9 species. ATPase-catalyzed proton pumping may contribute cold acclimated (Figure 2(a)), WT lines were −4.05 °C, while the to chilling tolerance in plants. ADH1 plays a key role in LT50 of transgenic lines was −5.97 °C. Under cold acclimation maintaining the stability of the membrane structure for the (Figure 2(b)), the LT50 of transgenic lines also decreased by about enhancement of cold resistance in plants. one degree (−7.01 °C) compared with −6°Cofwildtypelines. We also discussed Metabolite Profiling of adh1 Mutant This result has been mentioned in our previous study. These Response to Cold Stress in Arabidopsis, the results showed that results suggest that CbADH1 can enhance the cold tolerance of soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine) plants, which is independent or partly dependent on cold changed accordingly. acclimation. In this study, we proved CbADH1 significantly improved In order to find the functional difference of ADH1 gene of cold shock response through physiological and biochemical C. bungeana (CbADH1) and of A. thaliana (AtADH1) under indicators, indicating CbADH1 has strong antifreeze function cold stress, CbADH1 (including 43 unique amino acids), and great application prospect. CbADH1 and AtADH1 were overexpressed in tobacco CONTACT Yuan Song songyuan@lzu.edu.cn Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China © 2019 Taylor & Francis Group, LLC e1612680-2 Y. SONG ET AL. overexpression of CbADH1 and CbADH1 significantly enhanced the survival rate of Arabidopsis and tobacco under sudden cold stress (Figures 3 and 4). The results showed that CbADH1 could enhance the ability of sudden cooling toler- ance in plants. Discussion According to our previous work, we cloned the CbADH1 gene of C. bungeana. The results of multiple sequence align- ment showed that it contained three conserved domains of ADH1 family. Evolutionary analysis also showed high homol- ogy with the ADH1 gene of other species. Here we found CbADH1 can powerfully improve the freezing resistance, providing a candidate gene for genetic engineering. The structure of ADH1 and the binding site to substrate are Figure 1. Relative expression level of CbADH1 in homozygous seedlings. Data presented are the mean values of triplicate independent experiments ± SD, and very conservative. However, the 5‘ terminal deleted a specific asterisks indicate the significant differences in the expression level compared to 2 sequence encoding 43 amino acids, and we marked a triangle WT. 2 here. It could form different complexes because of the exis- (*p < .05, **p < .01, t-test, Tukey) . △ tence of 5‘ terminal specialsequencesothattheyshowdifferent activity in transgenic plants. The 22nd serine is the unique phos- and Arabidopsis thaliana, respectively. After plants were cul- phorylation site of cAMP and cGMP-dependent protein kinase in tured for four weeks in room temperature, the freezing resis- CbADH1, which may affect its localization. And the specific tance of wild and transgenic homozygous lines was analyzed functionsneededtobefurther studied. At the sametime, the5‘ by sudden cold treatments. The transgenic lines of flanking sequence of 4450 bp was cloned. The upstream 2000 bp Arabidopsis thaliana were treated with sudden cold treatment sequence of ATG was analyzed by PLACE website. It was found at −10 °C for 3 h and the transgenic tobacco at −4 °C for 3 that there are many cis-acting elements related to plant growth h. The survival rate of plants was counted after recovering at and development and abiotic stress response. 23 °C for 7 d. The results showed that both AtADH1 and The expression pattern analysis showed that CbADH1 was CbADH1 could enhance the freezing resistance of Arabidopsis highly expressed in young leaves and in flowers as well as in thaliana and tobacco plants. Compared with AtADH1, the cotyledons and hypocotyls after 12 d of germination. This Figure 2. Effect of freezing temperature on electrolyte leakage from the transgene lines and WT plants under (a) non-acclimation condition (NA) and (b) cold acclimation (CA) at 4 °C for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences compared toWT. (*p < .05, **p < .01, t-test, Tukey). PLANT SIGNALING & BEHAVIOR e1612680-3 Figure 3. CbADH1 conferred enhanced cold-shock resistance in Arabidopsis. (a) The photograph showing 28-d-old of transgenic Arabidopsis and WT plants in the growth chamber. (b) Recovery photograph of transgene Arabidopsis and WT in cold shock treatment at −10 °C for 3 h and recovery for 7 d. (c) The survival rate of different transgene plants after recovery for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences. (*p < .05, **p < .01, t-test, Tukey). expression pattern is similar to that of Arabidopsis thaliana. In CbADH1 could be used as a candidate gene for improving cold addition, GUS activity occurs in pollen grains, suggesting that tolerance of plants. However, under cold acclimation, the semi- CbADH1 may be involved in pollinator attraction, seed propaga- lethal temperature of CbADH1 transgenic lines was only scanty tion and defense against animal predation in alpine margin than that of the wild type (Figure 2(b)), suggesting that environments. cold acclimation had little effect on the role of CbADH1 in cold The transcriptional level of CbADH1 increased in different stress. The transcription data showed that there was no homolog abiotic stress, which may be related to the stress-responsive of CBFs in Chorispora bungeana, suggesting that cold acclimation binding elements in its promoter region. Previous studies showed hadnoorveryweakeffect on C. bungeana.Theresultsshowed that both biological and abiotic stress induced the expression of that CbADH1 significantly enhanced the tolerance of transgenic ADH1, but most of the studies focused on hypoxia stress, such as plants under sudden cold stress (Figures 3 and 4). It provides the study of water flooding stress in rice, metabolic pathway of theoretical support for the application of genes in Chorispora adh1 mutant in Chlamydomonas reinhardtii under hypoxia, bungeana as the candidate genes for freezing resistance in crop cold tolerance in diploid strawberry, suggesting that ethanol genetic improvement. dehydrogenase could be used as a molecular marker for the studyofcoldtolerance.The ADHenzymeactivity in Materials and methods C. bungeana increased under cold stress, which was consistent Experiment on freezing resistance of Arabidopsis with the results of previous studies. It was found that the transgenic lines expression of ADH1 and enzyme activity increased under cold stress compared with the control group. The transgenic lines of Arabidopsis thaliana were infected with The semi-lethal temperature was lower in CbADH1 transgenic the constructed subcellular localizing vector pEarlyGate101 into lines than the wild type without cold acclimation (Figure 2(a)), so the wild-type plants of Arabidopsis thaliana in antifreeze e1612680-4 Y. SONG ET AL. Figure 4. CbADH1 conferred enhanced cold-shock resistance in N. benthamiana. (a) The photograph showing 28-d-old of transgenic tobacco and WT plants in the growth chamber. (b) Recovery photograph of transgene tobacco and WT in cold shock treatment at −4 °C for 3 h and recovery for 7 d. (c) The survival rate of different transgene plants after recovery for 7 d. Data are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences. (*p < .05, **p < .01, t-test, Tukey). experiment. The homozygous lines were screened by a herbicidal activated in 50 ml of LB (Kan + Gen + Rif), 28 °C for 8 min, and agent for antifreeze test. All seeds were sown on half-strength re-suspended 1/2 MS (add 2% sucrose, 50 uM acetosyringone), Murashige and Skoog (MS) germination medium, supplemen- and the OD600 is 0.8. Then, the leaves of tobacco seedlings were ted with 1% sucrose and 0.8% agar at pH 5.7. cultured for four weeks, and the perforator was used to form The T-DNA mutant adh1–2 with BASTA resistance, and the a small leaf disc. The leaves were immersed in the bacterial open reading frames of CbADH1 and AtADH1 were fused to solution and shook at 28 °C avoiding light (70–80 rpm) 30 min. pMDC83 by Gateway system. GV3101 was introduced into The leaves were sandwiched on the ultra-clean worktable, and the Arabidopsis thaliana wild type and mutant lines. The homozy- aseptic filter paper was used to dry the bacterial solution on the gous strains were screened on MS medium containing 50 mg/L surface of the leaves and moved to the 1/2 MS containing 1 mg/L hygromycin. Over-expressed CbADH1 and AtADH1 transgenic 6-BA for 2 d. Then transferred to the screening medium for 1 homozygous lines were cultured for 4 weeks and then treated month (1/2 MS 2% sucrose 0.6% Agar 2 mg/L 6-BA 500 mg/L car with cold stress. The treatment condition was 0 °C for 1.5 h, then 50 mg/L hyg 0.5 mg/L NAA), and car (carboxybenzyl) was to decreased by 1 °C per hour until −10 °C for 3 h, then restored to inhibit bacteria. Finally, the plantlets were transferred to the 4 °C for 12 h. The plants were recovered in room temperature for medium (1/2 MS with 2% sucrose 0.5% Agar 500 mg/L car 7 d. The phenotype was observed and photographed, and the 50 mg/L hyg) for 20 d. Rooting was induced on rooting medium survival rate was counted. (1/2 MS with 2% sucrose 0.5% Agar 500 mg/L car 50 mg/L hyg 0.1 mg/L NAA), and then cultured in soil. The seeds of T1 generation were obtained. Then, the homozygous strain was Establishment of transgenic tobacco system and freezing screened on the MS medium containing hygromycin. resistance experiment Wild-type and over-expressed CbADH1 and AtADH1 trans- genic homozygous lines were cultured for 4 weeks and then Tobacco transformation experiment was carried out by leaf disk treated with cold treatment. The treatment conditions were 4 °C method. CDs regions of CbADH1 and AtADH1 were fused to for 0.5 h, then from 0 °C to −4°Cfor 3hdecreasedby1°C every pMDC83 and introduced into tobacco. First, agrobacterium is PLANT SIGNALING & BEHAVIOR e1612680-5 Chorispora bungeana in cold stress. Gene. 2017;636:1–16. half hour. After 4 °C 12 h, the recovery in room temperature for 7 doi:10.1016/j.gene.2017.09.015. d. The phenotype was observed and photographed, and the sur- 3. Strommer J. The plant ADH gene family. Plant J. 2011;66 vival rate was counted. (1):128–142. doi:10.1111/j.1365-313X.2010.04458.x. 4. Dolferus R, Jacobs M, Peacock WJ, Dennis ES. Differential interactions of promoter elements in stress responses of the Electrolyte leakage tests and lethal temperature Arabidopsis Adh gene. Plant Physiol. 1994;105:1075–1087. calculation 5. Pathuri IP, Reitberger IE, Huckelhoven R, Proels RK. Alcohol dehydrogenase 1 of barley modulates susceptibility to the parasitic Electrolyte leakage tests were performed as previously described fungus Blumeria graminis f.sp. hordei. J Exp Bot. 2011;62 45. The third to fifth leaves from 18-d-old plants were added to (10):3449–3457. doi:10.1093/jxb/err017. capped test tubes (15 ml) and placed in a bath-type programmable 6. Komatsu S, Deschamps T, Hiraga S, Kato M, Chiba M, freezer (XT5201-D31-R40C, XuTemp, China). The petiole was Hashiguchi A, Tougou M, Shimamura SYasue H. Characterization of a novel flooding stress-responsive alcohol placed facing downward into the bottom of the tube (without dehydrogenase expressed in soybean roots. Plant Mol Biol. damaging the leaves). Water was added to the bottom of the 2011;77(3):309–322. doi:10.1007/s11103-011-9812-y. tube until the petiole was fully submerged. Samples were kept on 7. Drew MC. Oxygen deficiency and root metabolism: injury and ice for 30 min. The plants were exposed to freezing temperatures acclimation under hypoxia and anoxia. Annu Rev Plant Physiol ranging from 0 to −20 °C (0, −4, −6, −8, −10, −12, −14, −16, −18, Plant Mol Biol. 1997;48:223–250. doi:10.1146/annurev. arplant.48.1.223. and −20 °C). Tubes were placed on ice after removal from the bath 8. Lalitha T, Ramakrishnan CV, Telang SD. Interaction of alcohol and until removal of all tubes was completed. Leaves were then and protein deficiency on adult rat brain lipids. Indian J Biochem immersed in 10 ml distilled water and placed on a shaker for 2 h at Biophys. 1987;24:238–240. 4 °C. Electrolyte leakage was determined as the ratio of before and 9. Lalitha T, Ramakrishnan CV, Telang SD. Interaction of alcohol after sample boiling via a conductivity meter (DDSJ-308, Leici, and protein deficiency on rat brain synaptosomal (Na+-K+)- ATPase. Neurochem Res. 1988;13:963–966. China). The freezing temperature that caused a 50% electrolyte 10. Low R, Rockel B, Kirsch M, Ratajczak R, Hortensteiner S, leakage (TEL50) was calculated from plotted data of relative elec- Martinoia E, Luttge URausch T. Early salt stress effects on the trolyte leakage as the log EC50 value of sigmoidal curve fitting to differential expression of vacuolar H(+)-ATPase genes in roots the leakage values using SPSS PASW Statistics 18.0 (IBM SPSS and leaves of Mesembryanthemum crystallinum. Plant Physiol. software, USA). 1996;110:259–265. 11. Song Y, Liu L, Wei Y, Li G, Yue XAn L. Metabolite profiling of ADH1 mutant response to cold stress in Arabidopsis. Front Plant Acknowledgments Sci. 2016;7:2072. 12. Higo K, Ugawa Y, Iwamoto MKorenaga T. Plant cis-acting reg- This work was supported by the Key Laboratory of Superior Forage ulatory DNA elements (PLACE) database: 1999. Nucleic Acids Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the Res. 1999;27:297–300. National Natural Science Foundation of China (No. 31872682); the 13. Takahashi H, Saika H, Matsumura H, Nagamura Y, Tsutsumi N. Fundamental Research Funds for the Central Universities (lzujbky-2018- Nishizawa NK, Nakazono M Cell division and cell elongation in 110 and lzujbky-2018-kb05). Also thank Core Facility of School of Life the coleoptile of rice alcohol dehydrogenase 1-deficient mutant Science, Lanzhou Unviersity for technical support. are reduced under complete submergence. Ann Bot. 2011;108 (2):253–261. doi:10.1093/aob/mcr137. 14. Magneschi L, Catalanotti C, Subramanian V, Dubini A, Yang W, Disclosure of Potential Conflicts of Interest Mus F, Posewitz MC, Seibert MPerata P, Grossman AR. A mutant No potential conflicts of interest were disclosed. in the ADH1 gene of Chlamydomonas reinhardtii elicits meta- bolic restructuring during anaerobiosis. Plant Physiol. 2012;158 (3):1293–1305. doi:10.1104/pp.111.191569. Funding 15. Davik J, Koehler G, From B, Torp T, Rohloff J, Eidem P, Wilson RC,SonstebyA,Randall SK,AlsheikhM.Dehydrin This work was supported by the Key Laboratory of Superior Forage alcohol dehydrogenase, and central metabolite levels are asso- Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the ciated with cold tolerance in diploid strawberry (Fragaria National Natural Science Foundation of China (No. 31872682); the spp.). Planta. 2013;237(1):265–277. doi:10.1007/s00425-012- Fundamental Research Funds for the Central Universities (lzujbky- 1771-2. 2018-110 and lzujbky-2018-kb05). 16. 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A simple and general method for transferring genes Chorispora bungeana.PLoS One. 2015;10:e0135485. into plants. Science. 1985;227(4691):1229–1231. doi:10.1126/ 2. Liu L, Song Y, Xu J, Li D, Li GAn L. Differential expression by science.227.4691.1229. chromatin modifications of alcohol dehydrogenase 1 of

Journal

Plant Signaling & BehaviorTaylor & Francis

Published: Jul 3, 2019

Keywords: ADH1 (alcohol dehydrogenase 1); cold tolerance; Chorispora bungeana

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