CbADH1 improves plant cold tolerance

ABSTRACT

ADH1 (alcohol dehydrogenase 1) was involved in plant growth and development and responded to various stresses. We published a cold-induced alcohol dehydrogenase 1 gene from C. bungeana, 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-lethal temperature, phenotypic and survival analysis. These results indicate that CbADH1 is the candidate gene to improve the ability of plant freezing resistance, and it has great application value.

KEYWORDS:

• ADH1 (alcohol dehydrogenase 1)
• cold tolerance
• Chorispora bungeana

Introduction

Chorispora bungeana grows in the alpine regions and is a perennial alpine subnival plant species experiences frequent temperature fluctuations during the growth season,¹ which has a strong freeze tolerance. We found an alcohol dehydrogenase-like protein that was specifically expressed in cold stress according to the results of two-dimensional electrophoresis of proteins. We cloned and characterized CbADH1 from C. bungeana. Multiple alignment result showed that the N-terminal domain of CbADH1 existed 43 unique amino acids, and the subcellular localization was affected, suggesting different biological functions.²

The classic alcohol dehydrogenase (ADH, alcohol: NAD⁺ oxidoreductase, EC1.1.1.1) is a Zn-binding enzyme that acts as a dimer and relies on a NAD (P) co-factor to interconvert ethanol and acetaldehyde (as well as other short linear alcohol/aldehyde pairs).³ ADH activity is subject to various stresses,⁴ including biotic and abiotic stress.^5,6 However, the function mechanism is not clear. ADH is related to the fermentative metabolism reduces acetaldehyde to ethanol, regenerates NAD+ and 2ATP, and protects cells from cytoplasmic acidosis.⁷ Ethanol enhances ATPase synthesis in different species.^8,9 ATPase-catalyzed proton pumping may contribute to chilling tolerance in plants.¹⁰ ADH1 plays a key role in maintaining the stability of the membrane structure for the enhancement of cold resistance in plants.

We also discussed Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis, the results showed that soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine) changed accordingly.¹¹

In this study, we proved CbADH1 significantly improved cold shock response through physiological and biochemical indicators, indicating CbADH1 has strong antifreeze function and great application prospect.

Results

Screening of homozygous lines overexpressing CbADH1

In order to further study the function of CbADH1, CbADH1 fused with pEarlyGate101-YFP vector was introduced into wild-type Arabidopsis thaliana (Col.) by dipping flower, and the homozygous lines expressing CbADH1 were obtained. Quantitative PCR was used to identify the changes of CbADH1 gene expression in homozygotes. As shown in Figure 1, the gene expression was increased in the four homozygous lines we selected. Among them, the gene expression in 1–6 and 9–4 lines increased four-fold and eight-fold, respectively, and these two lines were used in subsequent antifreeze tests (Figure 1).²

Figure 1. Relative expression level of CbADH1 in homozygous seedlings. Data presented are the mean values of triplicate independent experiments ± SD, and asterisks indicate the significant differences in the expression level compared to WT.

(*p < .05, **p < .01, t-test, Tukey)².

The seedlings of wild-type and transgenic plants of 3-week size were cultured at 4 °C for 7 d and the control lines were cultured in medium grew normally for 7 d, and the freezing resistance experiment was carried out at the same time.

CbADH1 improves the cold tolerance of plant independent of cold acclimation

As shown in Figure 2, transgenic plants are more freeze-resistant than wild-type plants. The half lethal temperature (LT50) of non-cold acclimated (Figure 2(a)), WT lines were −4.05 °C, while the LT50 of transgenic lines was −5.97 °C. Under cold acclimation (Figure 2(b)), the LT50 of transgenic lines also decreased by about one degree (−7.01 °C) compared with −6 °C of wild type lines. This result has been mentioned in our previous study.² These results suggest that CbADH1 can enhance the cold tolerance of plants, which is independent or partly dependent on cold acclimation.

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 to WT.

(*p < .05, **p < .01, t-test, Tukey).²

In order to find the functional difference of ADH1 gene of C. bungeana (CbADH1) and of A. thaliana (AtADH1) under cold stress, CbADH1 (including 43 unique amino acids), CbADH1△ and AtADH1 were overexpressed in tobacco and Arabidopsis thaliana, respectively. After plants were cultured for four weeks in room temperature, the freezing resistance of wild and transgenic homozygous lines was analyzed by sudden cold treatments. The transgenic lines of Arabidopsis thaliana were treated with sudden cold treatment at −10 °C for 3 h and the transgenic tobacco at −4 °C for 3 h. The survival rate of plants was counted after recovering at 23 °C for 7 d. The results showed that both AtADH1 and CbADH1 could enhance the freezing resistance of Arabidopsis thaliana and tobacco plants. Compared with AtADH1, the 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 tolerance in plants.

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).

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).

Discussion

According to our previous work, we cloned the CbADH1 gene of C. bungeana. The results of multiple sequence alignment showed that it contained three conserved domains of ADH1 family. Evolutionary analysis also showed high homology with the ADH1 gene of other species.² 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 very conservative. However, the 5‘ terminal deleted a specific sequence encoding 43 amino acids,² and we marked a triangle △ here. It could form different complexes because of the existence of 5‘ terminal special sequence so that they show different activity in transgenic plants. The 22nd serine is the unique phosphorylation site of cAMP and cGMP-dependent protein kinase in CbADH1, which may affect its localization. And the specific functions needed to be further studied. At the same time, the 5‘ flanking sequence of 4450 bp was cloned.² The upstream 2000 bp sequence of ATG was analyzed by PLACE¹² website. It was found that there are many cis-acting elements related to plant growth and development and abiotic stress response.

The expression pattern analysis showed that CbADH1 was highly expressed in young leaves and in flowers as well as in cotyledons and hypocotyls after 12 d of germination.² This expression pattern is similar to that of Arabidopsis thaliana.⁴ In addition, GUS activity occurs in pollen grains, suggesting that CbADH1 may be involved in pollinator attraction, seed propagation and defense against animal predation in alpine environments.³

The transcriptional level of CbADH1 increased in different abiotic stress,² which may be related to the stress-responsive binding elements in its promoter region. Previous studies showed that both biological and abiotic stress induced the expression of ADH1, but most of the studies focused on hypoxia stress, such as the study of water flooding stress in rice,¹³ metabolic pathway of adh1 mutant in Chlamydomonas reinhardtii under hypoxia,¹⁴ cold tolerance in diploid strawberry,¹⁵ suggesting that ethanol dehydrogenase could be used as a molecular marker for the study of cold tolerance. The ADH enzyme activity in C. bungeana increased under cold stress, which was consistent with the results of previous studies.¹⁶ It was found that the expression of ADH1 and enzyme activity increased under cold stress compared with the control group.

The semi-lethal temperature was lower in CbADH1 transgenic lines than the wild type without cold acclimation (Figure 2(a)), so CbADH1 could be used as a candidate gene for improving cold tolerance of plants. However, under cold acclimation, the semi-lethal temperature of CbADH1 transgenic lines was only scanty margin than that of the wild type (Figure 2(b)), suggesting that cold acclimation had little effect on the role of CbADH1 in cold stress. The transcription data¹⁷ showed that there was no homolog of CBFs in Chorispora bungeana, suggesting that cold acclimation had no or very weak effect on C. bungeana. The results showed that CbADH1 significantly enhanced the tolerance of transgenic plants under sudden cold stress (Figures 3 and 4). It provides theoretical support for the application of genes in Chorispora bungeana as the candidate genes for freezing resistance in crop genetic improvement.

Materials and methods

Experiment on freezing resistance of Arabidopsis transgenic lines

The transgenic lines of Arabidopsis thaliana were infected with the constructed subcellular localizing vector pEarlyGate101 into the wild-type plants of Arabidopsis thaliana in antifreeze experiment. The homozygous lines were screened by a herbicidal agent for antifreeze test. All seeds were sown on half-strength Murashige and Skoog (MS) germination medium,¹⁸ supplemented with 1% sucrose and 0.8% agar at pH 5.7.

The T-DNA mutant adh1–2 with BASTA resistance, and the open reading frames of CbADH1 and AtADH1 were fused to pMDC83 by Gateway system. GV3101 was introduced into Arabidopsis thaliana wild type and mutant lines. The homozygous strains were screened on MS medium containing 50 mg/L hygromycin. Over-expressed CbADH1 and AtADH1 transgenic homozygous lines were cultured for 4 weeks and then treated with cold stress. The treatment condition was 0 °C for 1.5 h, then decreased by 1 °C per hour until −10 °C for 3 h, then restored to 4 °C for 12 h. The plants were recovered in room temperature for 7 d. The phenotype was observed and photographed, and the survival rate was counted.

Establishment of transgenic tobacco system and freezing resistance experiment

Tobacco transformation experiment was carried out by leaf disk method.¹⁹ CDs regions of CbADH1 and AtADH1 were fused to pMDC83 and introduced into tobacco. First, agrobacterium is activated in 50 ml of LB (Kan + Gen + Rif), 28 °C for 8 min, and re-suspended 1/2 MS (add 2% sucrose, 50 uM acetosyringone), and the OD600 is 0.8. Then, the leaves of tobacco seedlings were cultured for four weeks, and the perforator was used to form a small leaf disc. The leaves were immersed in the bacterial solution and shook at 28 °C avoiding light (70–80 rpm) 30 min. The leaves were sandwiched on the ultra-clean worktable, and the aseptic filter paper was used to dry the bacterial solution on the surface of the leaves and moved to the 1/2 MS containing 1 mg/L 6-BA for 2 d. Then transferred to the screening medium for 1 month (1/2 MS 2% sucrose 0.6% Agar 2 mg/L 6-BA 500 mg/L car 50 mg/L hyg 0.5 mg/L NAA), and car (carboxybenzyl) was to inhibit bacteria. Finally, the plantlets were transferred to the medium (1/2 MS with 2% sucrose 0.5% Agar 500 mg/L car 50 mg/L hyg) for 20 d. Rooting was induced on rooting medium (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 screened on the MS medium containing hygromycin.

Wild-type and over-expressed CbADH1 and AtADH1 transgenic homozygous lines were cultured for 4 weeks and then treated with cold treatment. The treatment conditions were 4 °C for 0.5 h, then from 0 °C to −4 °C for 3 h decreased by 1 °C every half hour. After 4 °C 12 h, the recovery in room temperature for 7 d. The phenotype was observed and photographed, and the survival rate was counted.

Electrolyte leakage tests and lethal temperature calculation

Electrolyte leakage tests were performed as previously described 45. The third to fifth leaves from 18-d-old plants were added to capped test tubes (15 ml) and placed in a bath-type programmable freezer (XT5201-D31-R40C, XuTemp, China). The petiole was placed facing downward into the bottom of the tube (without damaging the leaves). Water was added to the bottom of the tube until the petiole was fully submerged. Samples were kept on ice for 30 min. The plants were exposed to freezing temperatures ranging from 0 to −20 °C (0, −4, −6, −8, −10, −12, −14, −16, −18, and −20 °C). Tubes were placed on ice after removal from the bath and until removal of all tubes was completed. Leaves were then immersed in 10 ml distilled water and placed on a shaker for 2 h at 4 °C. Electrolyte leakage was determined as the ratio of before and after sample boiling via a conductivity meter (DDSJ-308, Leici, China). The freezing temperature that caused a 50% electrolyte leakage (TEL50) was calculated from plotted data of relative electrolyte leakage as the log EC50 value of sigmoidal curve fitting to the leakage values using SPSS PASW Statistics 18.0 (IBM SPSS software, USA).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the National Natural Science Foundation of China (No. 31872682); the Fundamental Research Funds for the Central Universities (lzujbky-2018-110 and lzujbky-2018-kb05). Also thank Core Facility of School of Life Science, Lanzhou Unviersity for technical support.

Additional information

Funding

This work was supported by the Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau (No. 2017-ZJ-Y12); the National Natural Science Foundation of China (No. 31872682); the Fundamental Research Funds for the Central Universities (lzujbky-2018-110 and lzujbky-2018-kb05).