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Article

Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco

by
Lingmin Zhao
1,†,
Yanfang Zhang
1,†,
Ying Shao
2,
Linan Xing
1,
Mingran Ge
1 and
Xiuwen Huo
1,*
1
Horticulture Department, Faculty of Horticulture and Plant Protection Science, Inner Mongolia Agricultural University, Hohhot 010019, China
2
Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot 010031, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2022, 13(12), 2296; https://doi.org/10.3390/genes13122296
Submission received: 5 October 2022 / Revised: 25 November 2022 / Accepted: 5 December 2022 / Published: 6 December 2022
(This article belongs to the Section Plant Genetics and Genomics)
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Abstract

:
β-amylase (BAM) plays an important role in plant development and response to abiotic stresses. In this study, 5 DoBAM members were identified in yam (Dioscorea opposita Thunb.). A novel β-amylase gene BAM1, (named DoBAM1), was isolated from yam varieties Bikeqi and Dahechangyu. The open reading frame (ORF) of DoBAM1 is 2806 bp and encodes 543 amino acids. Subcellular localization analysis indicates that DoBAM1 localizes to the cell membrane and cytoplasm. In the yam variety Dahechangyu, the starch content, β-amylase activity, and expression of DoBAM1 were characterized and found to all be higher than in Bikeqi. DoBAM1 overexpression in tobacco is shown to promote the accumulation of soluble sugar and chlorophyll content and to increase the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and β-amylase. Under cold treatment, we observed the induced upregulation of DoBAM1 and lower starch content and malondialdehyde (MDA) accumulation than in WT plants. In conclusion, these results demonstrate that DoBAM1 overexpression plays an advanced role in cold tolerance, at least in part by raising the levels of soluble sugars that are capable of acting as osmolytes or antioxidants.

1. Introduction

Yam (Dioscorea opposita Thunb.) belongs to the genus Dioscorea and the family Dioscoreaceae. In addition to its significance as a traditional starchy staple food, many Dioscorea species have been fostered and used extensively for nutritional and medicinal aims [1]. Yam is cultivated in many temperate and tropical regions and is one of the ten most significant rhizome crops in the world [2]. The tubers of this crop have high edible value, are highly nutritious, and provide additional health benefits. The secondary metabolites abundant within yam tubers can reduce blood lipid levels and inhibit cholesterol, fat absorption, cancer cell growth, and neurodegeneration [3,4]. Starch, as the most abundant component of yam tubers, contributes greatly to the value of all yam-tuber-related foodstuffs [5,6].
Starch is the main energy-storage carbohydrate and is closely associated with abiotic stresses and secondary metabolism pathways in plants [7,8,9]. β-amylase (BAM) is a critical enzyme regulating starch metabolism, with distinctive roles in the breakdown of starch and the synthesis of sugar [8]. BAMs (β-amylases; EC 3.2.1.2) are members of the glycosylhydrolase family 14 (PF01373). The important role of these enzymes is conferred by two typical catalytic sites at the N-terminus that are central to hydrolyzing the 1,4-α-glucosidic linkages within starch [10,11]. Hence, BAMs are thought to positively regulate the accumulation of soluble sugars. In addition, soluble sugars are produced during the degradation of starch, and the synthesis of soluble sugars is transferred from chloroplasts to the cytoplasm, which can then participate in the energy metabolism pathway [12,13]. In potato, the activity of BAM increases four- to five-fold at low temperatures, causing sweetness in tubers [14]. In banana, MaBAMs participate in the regulation of fruit-ripening, and the expression levels of several MaBAMs change markedly under several stress conditions, including low-temperature stress conditions [15]. Studies in perennial plants, such as pear, blueberry, orange, and tea, have shown that the expression level of BAM and activity of BAMs can both be increased under cold stress conditions [16,17,18].
In plants, cold stress can lead to a range of metabolic changes. In plants, cold conditions affect particular physiological, biochemical, and molecular processes, enable tolerance to cold stress, and determine geographic distribution [19,20]. Under cold stress, reactive oxygen species (ROS) accumulate as the balance between ROS production and ROS scavenging is disrupted, which may lead to cell membrane damage and osmotic imbalance [21,22]. Cold stress also counteracts the defense pathway and can lead to increased accumulation of osmolytes or osmoprotectants, such as proline and soluble sugars, via the upregulation of genes related to metabolism [23,24,25]. There is mounting evidence showing that soluble sugars are of great importance in ROS scavenging and in enhancing cold tolerance [26,27]. For example, carbohydrates in potato leaves undergo significant changes during the early phases of cold stress, leading to the accumulation of soluble sugars [28]. In wheat, defense against low-temperature stress is achieved through antioxidant enzyme activity [29].
Chinese yam is unique because it is native to temperate regions and can tolerate much colder temperatures than its relatives [30]. Both Dahechangyu and Bikeqi are early-maturing varieties that grow in the Inner Mongolia Autonomous Region, north of the Yangtze River Basin in China, and are widely cultivated. Continuous crop damage, pests, and diseases in Southern China severely restrict the development of traditional yam production areas in the country. Therefore, in 2009, China’s Ministry of Finance and Ministry of Agriculture officially proposed the implementation of a “south to north migration of yam” and “north to west expansion” strategy (200903022) [31]. Thus far, areas in Northwest China, such as Yulin City of Shaanxi Province [32] and Xinjiang Autonomous Region [33], have been widely planted. The western region of Inner Mongolia has become an emerging yam-producing area; it is a cold and arid region with abundant land resources, sufficient light, an arid climate, low air humidity, a large temperature difference between day and night, a loose soil structure, and fewer pests and diseases, which are ideal for yam cultivation: the yams produced here are also of higher quality. Nevertheless, the annual frost-free period is only 100–150 days, and the average temperature throughout the year is 2–14 °C. The growth cycle of yam lasts 180–200 days from early April to mid-October [1]. Therefore, the low temperature is a key factor limiting the development of the yam industry. Under such conditions, we studied DoBAM1, a key gene regulating cold stress, as a candidate for molecular breeding aimed at extending the yam growth cycle through the breeding of mid- to late-maturing varieties for cultivation in northern regions.
In this work, we isolated a novel BAM gene called DoBAM1 from the yam varieties Bikeqi and Dahechangyu. We overexpressed DoBAM1 in tobacco and assessed the phenotype and physiological changes to determine the characteristics that improve cold tolerance in addition to characterizing the expression of stress-response genes. We found a correlation between increased BAM activity and the higher accumulation of soluble sugars after cold treatment. This study provides a theoretical basis for further investigations on the role of BAM in development and stress tolerance in yam.

2. Materials and Methods

2.1. Plant Materials, Cultivation, and Stress Treatments

Two yam cultivars with different starch content were used as the experimental materials. Dahechangyu (DHCY) is a high-starch yam cultivar, while Bikeqi (B1) is a low-starch yam cultivar. The two cultivars were planted in a completely randomized block design at the Germplasm Resource Nursery of Inner Mongolia Agricultural University (40°29′28.01″ N and 111°47′07.69″ E, Hohhot, China). Yams of each cultivar were randomly sampled in the morning at five different developmental stages: after 105 d (T1), 120 d (T2), 135 d (T3), 150 d (T4), and 165 d (T5). Micropropagated plantlets of DHCY were selected on Murashige and Skoog 1962 (MS) culture media containing 7.0 g/L agar and 30 g/L sucrose (pH 6.0). Plants grown in bottles for thirty days were selected based on uniformity of growth and treated in a low-temperature incubator at 4 °C. Whole leaves were then sampled at 0, 1, 6, 12, and 24 h time points. The tubers, stems, and young leaves of yams were stored at −80 °C for further use. Three biological replicates of samples were used for the purposes of RNA extraction and physiological index assay.

2.2. Determination of Physiological Parameters

DHCY and B1 yam cultivars grown in the field for different sampling periods were harvested, heated at 80 °C for approximately 24 h, and then ground into powder to obtain a constant dry weight (DW). The dried samples (100 mg) were used to determine the starch content, as described by Ning Li et al. [34]. The fresh samples (1 g) were used to measure the β-amylase enzyme activity using a β-amylase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.3. DoBAM1 Expression Analysis

Total RNA was extracted from tubers, stems, and young leaves in the field and leaves under 4 °C treatment for the synthesis of the first-strand cDNA. DoBAM1-specific primers, DoBAM1-qF1/R1, were designed to determine expression levels (Table S1). The PCR thermal cycle conditions were as follows: initial denaturation at 95 °C for 30 s; 45 cycles of denaturation at 95 °C for 5 s; annealing at 60 °C for 30 s. Melting-curve analysis involved 1 cycle of 95 °C for 5 s, 60 °C for 1 min, and 95 °C for 15 s. In addition, all data were reckoned using the 2−ΔΔCt method [35]. The actin gene was used as an internal control (Table S1) [36].

2.4. Isolation and Cloning of the DoBAM1 Gene

The gene BAM1 (Unigene0000684) was screened according to the transcriptome results obtained for yam via our previous high-throughput sequencing study [37,38]. Total RNA was isolated from tuber using a MiniBEST Plant RNA Extraction Kit (Takara, Beijing, China). The rapid amplification of cDNA ends (RACE) method (SMARTer® 5′RACE & 3′RACE Kit, Takara, Kusatsu, Japan) was used to clone the full-length sequence of BAM1 cDNA. Sequencing results were compared and spliced with the conserved region sequence via DNAMAN 6.0. The 5′/3′RACE clone primer sequences DoBAM1-5′R1/3′F1 and the open reading frame (ORF) primers DoBAM1-ORF-F/R were designed using Primer Premier 5.0 (Table S1). The PCR thermal cycle conditions were as follows: initial denaturation at 94 °C for 10 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. Subsequently, we inserted the pGM-T vector (TIANgel, Beijing, China) for sequencing.

2.5. Sequence Analysis of DoBAM1

The full-length cDNA of DoBAM1 was analyzed using the online BLAST tool of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/, accessed on 25 August 2022). The accession numbers of the Arabidopsis thaliana (A. thaliana) BAM genes were referred from the research of Fulton et al. [12,39], and the full-length coding sequences (CDS) of genes were downloaded from TAIR (https://www.arabidopsis.org/, accessed on 28 August 2022). BAM protein sequences of rice (Oryza sativa), water yam (Dioscorea alata), and potato (Solanum tuberosum) were downloaded from the Phytozome v13 database. DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA) was used for the multiple alignment analysis of A. thaliana and yam sequences. Based on this, the phylogenetic analysis of the BAM gene family members from yam and other species was conducted with the MEGA6 software package using the neighbor-joining method and 1000 bootstrap replications [40]. The ProtParam tool (http://web.expasy.org/protparam/, accessed on 26 August 2022) was used to calculate the theoretical molecular weight (kDa) and isoelectric point (pI). (http://web.expasy.org/protparam/, accessed on 26 August 2022).

2.6. Subcellular Localization of DoBAM1

The ORF of DoBAM1 was amplified using the specific primers DoBAM-SF/R with restriction enzyme cutting sites and was then inserted into the CaMV-35S-GFP vector (Table S1). Following sequence confirmation, the 35S::DoBAM1-GFP and CaMV-35S-GFP (control) plasmids were transferred into Agrobacterium tumefaciens EHA 105 (Weidi Biotechnology, Shanghai, China). The empty CaMV-35S-GFP vector was used as a control. The vectors were transferred into tobacco (Nicotiana tabacum L.) leaves [17], which were then cultured for 2–3 days. Infected parts of leaves were then cut and used for fluorescent signal detection under a laser confocal microscope (C2-ER; Nikon, Tokyo, Japan).

2.7. Generation and Identification of Transgenic Plants

To construct the pPZP221–DoBAM1 expression vector, the DoBAM1 CDS with the BamHⅠ and KpnⅠ restriction sites was amplified using the specific primers DoBAM-ZF/R (Table S1) and was inserted into the pPZP221-35S-NOS expression vector. The Agrobacterium tumefaciens strain EHA101 (Weidi Biotechnology, Shanghai, China) was transformed into wild-type (WT) tobacco via the freeze–thaw method. Transgenic plants were grown on 1/2 MS containing 50 mg/L gentamicin and 250 mg/L cefotaxim. DNA and RNA of transgenic and WT tobacco leaves were isolated using a Hi-DNAsecure Plant Kit (TIANGEN, Beijing, China). Finally, we selected three positive transgenic lines (BAM4, BAM5, and BAM12) for further gene-functional verification.

2.8. Cold Treatment of Transgenic Tobacco and Analysis of Physiological Characteristics

For the cold tolerance assay, 3-week-old WT and transgenic tobacco in 1/2 MS were transferred to individual pots containing substrate soil for a period of 2 weeks. Following the method described by Guo [41] as a reference, we carried out treatments at 4 °C (T1) and −2 °C (T3) in addition to 2 °C (T2) for 2 h to determine the cold tolerance of transgenic tobacco. To measure physiological changes, the healthy, uniform-sized plants from the WT and transgenic lines were selected for the establishment of lines for further analysis. Starch content was then determined using the method of Wang et al. [42]. Soluble sugar and chlorophyll content was assessed using the method described by Su [43] and Chow [44]. SOD, POD, CAT, and BAM activity, and MDA content, were all measured according to the kit manual (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Histochemical staining with 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium chloride (NBT) was used to examine the in situ accumulation of hydrogen peroxide (H2O2) and superoxide (O2¯) under cold stress [45,46].

2.9. Statistical Analysis

All data were based on three independent samples. Data and graphs were processed using Excel 2019. The statistical analysis was performed using the SPSS 21.0 software package. Differences among treatments were identified by data analysis using t-tests and one-way ANOVA, and Duncan’s multiple comparisons were used for sample comparisons at significance levels of p < 0.05 or p < 0.01.

3. Results

3.1. Analysis of Starch Content and Amylase Activity in Tubers of Yam Cultivars

Tubers from the two yam cultivars were harvested at five ripening stages to investigate the changes in starch content and β-amylase activity. As the tubers developed, the starch content of both DHCY and B1 cultivars increased and peaked at T3. However, at each stage of development, the starch content in DHCY was significantly higher than in B1 (Figure 1A). We also found a significant difference in β-amylase activity between the two yam cultivars in the T1–T3 period, although there was a decrease in both cultivars after the T2 stage (Figure 1B).

3.2. Expression Patterns of DoBAM1

The transcripts of DoBAM1 reached their highest levels at the T2 stage. The expression of DoBAM1 was lowest at T5 (Figure 2A). The results of tissue-specific expression analysis show that the levels of DoBAM1 were highest in leaves (Figure 2B). The expression levels were lowest in B1 when considering tubers and lowest in DHCY when considering stems. The transcript level of DoBAM1 first increased and then decreased during the twenty-four hours of cold treatment, but it remained higher throughout than the value at the initial zero-hour time point (Figure 2C). To conclude, the results indicate that DoBAM1 is responsive to cold stress.

3.3. Isolation and Characteristics of DoBAM1

In a previous study, we obtained and analyzed the BAM gene (GenBank Accession No. MT683336) from the transcriptome database (unpublished data) of Dioscorea opposita Thunb. The complete ORF of 2806 bp is flanked by a 1091 bp 5′-untranslated region (UTR) and a 42 bp 3′-UTR (Figure 3). The cDNA, designated as DoBAM1, contains an intact ORF and encodes a polypeptide of 543 amino acids with a calculated molecular mass of 622.92 kD and an isoelectric point of 5.60. As predicted by SWISS-MODEL, the secondary structure of the DoBAM1 protein contains 37.57% helices, 12.52% extended strands, and 6.54% turns. To analyze the evolutionary relationship of DoBAM1, a phylogenetic tree was constructed using a total of 32 BAM protein sequences from yam, Arabidopsis, water yam, potato, and rice (Figure 4). The BAM proteins were divided into four clades. DoBAM1 exhibited the highest homology with DaBAM5. The multisequence alignment revealed that the Glyco_hydro_14 domain and the catalytic residues Glu-186 and Glu-380 were highly conserved across yam and Arabidopsis BAM amino acids (Figure 5).

3.4. Subcellular Localization of DoBAM1

To further understand the function of DoBAM1 in the regulation of transcription, we constructed a DoBAM1–GFP fusion vector, as well as a GFP fusion expression empty vector and a control. These were then transformed into tobacco leaves, and GFP fluorescence was visualized under confocal microscopy. We observed that the DoBAM1–GFP was located in the cell membrane and cytoplasm, while fluorescence corresponding to expression from the control vector was distributed throughout the whole cell (Figure 6).

3.5. Regeneration and Identification of Transgenic Tobacco with DoBAM1 Gene

Overexpression of DoBAM1 from the recombinant vector pPZP221–DoBAM was used to research the cold stress tolerance of DoBAM1 in transgenic tobacco (Figure 7A). The transgenic tobacco exhibited increased growth under control conditions as well as higher tolerance of cold treatment than the WT plant (Figure 7B). The relative expression of DoBAM1 in the three overexpressing lines was significantly higher than in the untreated transgenic plants (Figure 7C).

3.6. Physiological Characteristics of Tobacco Overexpressing DoBAM1 under Cold Stress

DoBAM1 is highly affected by cold stress, so we measured the physiological characteristics of transgenic and WT plants to understand the mechanisms of its ability to enhance cold tolerance. Our results show that β-amylase activity was significantly higher in the three transgenic tobacco lines than in WT under the different cold treatments (Figure 8A). However, the starch content was significantly lower than in WT (Figure 8B). We found that the overexpression of DoBAM1 induced the accumulation of soluble sugars under cold treatment and that the soluble sugar content was significantly higher than in the untreated transgenic plants (Figure 8C). Similarly, the chlorophyll content was significantly higher in the three overexpressing lines than that in the untreated transgenic tobaccos (Figure 8D).
Abiotic stress induces a high level of ROS production, which leads to cellular damage [47]. ROS scavenging is therefore essential to protect plants [48,49]. Under the different cold treatments, we examined the accumulation of H2O2 and O2− in tobacco leaves by means of in situ histochemical staining using DAB and NBT. The stained leaves exhibited conspicuous differences under the different cold stressors. Serried plaques appeared less frequently in the leaves of transgenic tobacco, as evidenced by the DAB and NBT staining (Figure 9A–C). These results indicate that overexpressing DoBAM1 decreases ROS accumulation under cold stress conditions. In plants, SOD, CAT, and POD activity and MDA content are all involved in the process of O2− being reduced to H2O2, which enhances plant cold resistance [50,51]. We noticed that the DoBAM1-overexpressing lines displayed significantly higher SOD, POD, and CAT activities than WT (Figure 10A–C). However, the MDA content was significantly lower than in the WT plants (Figure 10D). These results indicate that the overexpression of DoBAM1 strengthens the antioxidant capacity toward maintaining sustain the dynamic balance of ROS under cold stress conditions.

4. Discussion

Plants have developed the capacity to tolerate freezing temperatures to survive under conditions of cold stress [11]. It has been confirmed in numerous studies that starch is important in regulating their tolerance to stresses such as high salinity and low temperatures by releasing energy from sugar, obtaining metabolites produced from sugar, and through starch breakdown [9]. Starch breakdown can be used to provide energy and compensate for insufficient photosynthesis, and the metabolites formed by the synthesis and breakdown of sugars act as osmoprotectants under stress conditions [52]. A large spectrum of enzymes is implicated in the degradation of starch, including BAMs [17]. Recently, BAMs have been isolated and cloned in multiple plant species, such as A. thaliana [53], Musa acuminata (banana) [15], Chenopodium quinoa Willd. (quinoa) [54], Ziziphus jujuba Mill. (jujube) [55], Oryza sativa. (rice) [56], and others. In this study, we cloned the full-length sequence of BAM1 from yam and named it DoBAM1. According to the phylogenetic analysis, a total of 5 BAMs are present in yam, and these are divided into four groups, suggesting that BAMs are evolutionarily conserved among different plants. DoBAM1 can be classified into the Group I category together with DaBAM5 from Dioscorea alata L. and OsBAM6 from Oryza sativa. Multiple sequence alignment has shown that DoBAMs and AtBAMs share similar amino acids related to substrate binding and hydrolytic catalysis [57]. In this study, subcellular localization indicated that DoBAM1 is mainly distributed in the cytoplasm and cell membrane, a finding not wholly consistent with previous findings [58]. In yam tubers, we found that the expression of DoBAM1 and β-amylase was higher in the high-starch yam cultivar DHCY than in the low-starch cultivar B1. Based on yam growth in our region, DHCY in our experiment was treated with cold stress at 4 °C, and it was shown that the transcript expression levels of DoBAM1 were significantly higher than those of the control. In light of the above, we speculate that DoBAM1 also plays a crucial role in cold stress resistance. This supposition is confirmed by means of functional identification using DoBAM1 transgenic tobacco.
Sugar metabolism and carbon partitioning are used to maintain the balance required for plant growth and also represent two effective methods to respond to abiotic stress conditions [59]. Soluble sugars, acting as antioxidants, have been shown to ameliorate ROS-derived oxidative stresses [27,60]. In our study, the three selected DoBAM1-overexpressing lines (BAM4, BAM5, and BAM12) exhibited a more robust phenotypic morphology, with higher BAM activity, higher soluble sugar levels, and significantly lower starch content than wild tobacco under cold-stress conditions. This indicates that the materiality of sugars derived from DoBAM1 is involved in starch hydrolysis in the cold-tolerance process. Plants have evolved efficient scavenging mechanisms, including both enzymatic and non-enzymatic systems [61]. As one of the most important enzymes in abiotic stress, SOD can catalyze O2¯ conversion to O2 and H2O2, which are further scavenged by the coordinated action of POD with other factors [62,63,64]. In this study, the DAB and NBT staining results show that the accumulation of H2O2 and O2− is greatly decreased in DoBAM1-overexpressing lines, compared with WT plants. Under different cold treatments, the activities of SOD, CAT, and POD were significantly higher in the DoBAM1-overexpressing plants than in WT. Numerous previous studies have confirmed that lower MDA content is indicative of enhanced abiotic stress tolerance [65,66,67]. In the present study, the MDA content was lower in DoBAM1-overexpressing lines than in WT. To conclude, our results demonstrate that DoBAM1 overexpression enhances soluble sugar accumulation and regulates ROS homeostasis in response to cold stress, in line with the findings of previous studies [62,68]. The results of this study indicate that DoBAM1 may be a reference by which to investigate the molecular mechanisms involved in cold tolerance, and it represents a potential gene resource for improving the abiotic resistance of yam and other crops.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/genes13122296/s1: Table S1: Related primer sequences of the DoBAM1 gene.

Author Contributions

Conceptualization, X.H. and Y.Z.; methodology, L.Z., L.X. and M.G.; investigation, L.Z., L.X., M.G. and Y.S.; data curation, L.Z. and L.X.; writing—original draft preparation, L.Z. and Y.Z.; writing—review and editing, L.Z., Y.Z. and X.H.; project administration, Y.Z. and X.H.; funding acquisition, X.H. and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number 31860558; Inner Mongolia Autonomous Region Science and Technology Plan Project, grant number 2020GG0044; and Inner Mongolia Autonomous Region Natural Science, grant number 2022MS03052.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Janina, E.; Natalie, L. An underutilized orphan tuber crop-Chinese yam: A review. Planta 2020, 252, 58. [Google Scholar]
  2. Tafadzwanashe, M.; Vimbayi, G.P.C.; Sithabile, H.; Festo, M.; Sean, M.; Luxon, N.; Albert, T.M. Prospects of orphan crops in climate change. Planta 2019, 250, 695–708. [Google Scholar]
  3. Nurmuhammat, A.; Raziya, A.; Sajida, A.; Parida, H.Z.O.; Dolkun, M.; Anake, K. Aqueous extract of Dioscorea opposita thunb. normalizes the hypertension in 2K1C hypertensive rats. BMC Complement. Altern. Med. 2014, 14, 36. [Google Scholar]
  4. Zhang, Z.S.; Wang, X.M.; Liu, C.B.; Li, J.F. The degradation, antioxidant and antimutagenic activity of the mucilage polysaccharide from Dioscorea opposita. Carbohydr. Polym. 2016, 150, 227–231. [Google Scholar] [CrossRef] [PubMed]
  5. Zhang, B.; Guo, K.; Lin, L.S.; Wei, C.X. Comparison of structural and functional properties of starches from the rhizome and bulbil of Chinese yam (Dioscorea opposita Thunb.). Molecules 2018, 23, 427. [Google Scholar] [CrossRef] [Green Version]
  6. Zhang, C.Q.; Chen, X.; Ding, K. Structural characterization of a galactan from Dioscorea opposita Thunb. and its bioactivity on selected Bacteroides strains from human gut microbiota. Carbohydr. Polym. 2019, 218, 299–306. [Google Scholar] [CrossRef]
  7. Zhu, S.; Wang, Y.J.; Wei, H.; Xu, J.; Wang, Z.M.; Xu, J.L.; Yuan, Z.H. Enhanced accumulation of carbohydrate and starch in Chlorella zofingiensis induced by nitrogen starvation. Appl. Biochem. Biotechnol. 2014, 174, 2435–2445. [Google Scholar] [CrossRef]
  8. Zhao, L.Y.; Gong, X.; Gao, J.Z.; Dong, H.Z.; Zhang, S.L.; Tao, S.T.; Huang, X.S. Transcriptomic and evolutionary analyses of white pear (Pyrus bretschneideri) β-amylase genes reveals their importance for cold and drought stress responses. Gene 2019, 689, 102–113. [Google Scholar] [CrossRef]
  9. Matthias, T.; Diana, S. Starch as a determinant of plant fitness under abiotic stress. New Phytol. 2017, 214, 943–951. [Google Scholar]
  10. Jonathan, D.M.; Jillian, S.B.; Lauren, E.P. Arabidopsis β-amylase2 is a K+-requiring, catalytic tetramer with sigmoidal kinetics. Plant Physiol. 2017, 175, 1525–1535. [Google Scholar]
  11. Sun, S.H.; Fang, J.B.; Lin, M.M.; Qi, X.J.; Chen, J.Y.; Wang, R.; Li, Z.; Li, Y.K.; Abid, M. Freezing Tolerance and Expression of β-amylase Gene in Two Actinidia arguta Cultivars with Seasonal Changes. Plants 2020, 9, 515. [Google Scholar] [CrossRef] [PubMed]
  12. Daniel, C.F.; Michaela, S.; Tabea, M. β-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active β-amylases in Arabidopsis chloroplasts. Plant Cell 2008, 20, 1040–1058. [Google Scholar]
  13. Samuel, C.Z.; David, T.; Nicole, S. Plastidial α-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol. 2004, 135, 849–858. [Google Scholar]
  14. Tom, H.N.; Uta, D.; Mark, S. A β-amylase in potato tubers is induced by storage at low temperature. Plant Physiol. 1997, 113, 503–510. [Google Scholar]
  15. Miao, H.; Sun, P.; Miao, Y. Genome-wide identification and expression analysis of the β-amylase genes strongly associated with fruit development, ripening, and abiotic stress response in two banana cultivars. Front. Agric. Sci. Eng. 2017, 3, 346–356. [Google Scholar] [CrossRef] [Green Version]
  16. Lee, J.H.; Yu, D.J.; Kim, S.J. Intraspecies differences in cold hardiness, carbohydrate content and β-amylase gene expression of Vaccinium corymbosum during cold acclimation and deacclimation. Tree Physiol. 2012, 32, 1533–1540. [Google Scholar] [CrossRef] [Green Version]
  17. Peng, T.; Zhu, X.F.; Duan, N. Ptr BAM1, a β-amylase-coding gene of Poncirus trifoliata, is a CBF regulon member with function in cold tolerance by modulating soluble sugar levels. Plant Cell Environ. 2014, 37, 2754–2767. [Google Scholar] [CrossRef]
  18. Hao, X.Y.; Yue, C.; Tang, H. Cloning of β-amylase gene (CsBAM3) and its expression model response to cold stress in tea plant. Acta Agron. Sin. 2017, 43, 1417–1425. [Google Scholar] [CrossRef]
  19. Anna, D.; Dietz, K.J. Reactive oxygen species and the redox-regulatory network in cold stress acclimation. Antioxidants 2018, 7, 169. [Google Scholar]
  20. Chu, M.X.; Li, J.J.; Zhang, J.Y. AtCaM4 interacts with a Sec14-like protein, PATL1, to regulate freezing tolerance in Arabidopsis in a CBF-independent manner. J. Exp. Bot. 2018, 69, 5241–5253. [Google Scholar] [CrossRef] [Green Version]
  21. Ron, M. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [Google Scholar]
  22. Gad, M.; Nobuhiro, S.; Sultan, C.Y. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 2010, 33, 453–467. [Google Scholar]
  23. Silva-Ortega, C.O.; Ochoa-Alfaro, A.E.; Reyes-Agüero, J.A.; Aguado-Santacruz, G.A.; Jiménez-Bremont, J.F. Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiol. Biochem. 2008, 46, 82–92. [Google Scholar] [CrossRef]
  24. Andreas, T.; Christophe, C.; Essaïd, A.B. Physiological and molecular changes in plants grown at low temperatures. Planta 2012, 235, 1091–1105. [Google Scholar]
  25. Julieta, V.C.; Anabella, F.L.; Matias, D.Z. Novel perspectives for the engineering of abiotic stress tolerance in plants. Curr. Opin. Biotechnol. 2014, 26, 62–70. [Google Scholar]
  26. Bolouri-Moghaddam, M.R.; Le Roy, K.; Xiang, L.; Rolland, F.; Van den Ende, W. Sugar signalling and antioxidant net-work connections in plant cells. FEBS J. 2010, 277, 2022–2037. [Google Scholar] [PubMed]
  27. Els, K.; Darin, P.; Jaco, V. Plant sugars are crucial players in the oxidative challenge during abiotic stress: Extending the traditional concept. Plant Cell Environ. 2013, 36, 1242–1255. [Google Scholar]
  28. Orzechowski, S.; Sitnicka, D.; Grabowska, A.; Compart, J.; Fettke, J.; Zdunek-Zastocka, E. Effect of short-term cold treatment on carbohydrate metabolism in potato leaves. Int. J. Mol. Sci. 2021, 22, 7203. [Google Scholar] [CrossRef]
  29. Li, X.; Jiang, H.; Liu, F.; Cai, J.; Dai, T.; Cao, W.; Jiang, D. Induction of chilling tolerance in wheat during germination by pre-soaking seed with nitric oxide and gibberellin. Plant Growth Regul. 2013, 71, 31–40. [Google Scholar] [CrossRef]
  30. POWO. Plants of the World Online. In Facil; Gard, R.B., Ed.; Kew. Publ: Richmond, UK, 2019. [Google Scholar]
  31. Wei, B.; Shen, Z. Chinese yam (Dioscorea opposita Thunb.) Notillage Ecological Cultivation Technique. Agric. Sci. Technol. 2015, 16, 2629–2631. [Google Scholar]
  32. Fu, Y.; Sun, Y.; Lin, Y. Yam variety introduction trial in the wind-sand grassland area of Yulin. Northwest Hortic. (Gen.) 2021, 3, 61–63. [Google Scholar]
  33. Keranmu, A. A Floristic Analysis on the Medicinal Plants in the West Tianshan Mountains; Xinjiang University: Urumqi, China, 2015. [Google Scholar]
  34. Li, N.; Zhang, S.J.; Zhao, Y.J. Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize. Planta 2011, 233, 241–250. [Google Scholar] [CrossRef] [PubMed]
  35. Kenneth, J.L.; Thomas, D.S. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar]
  36. Shao, Y.; Zhang, Y.F.; Guo, S.C.; Zhao, L.M.; Sun, X.H.; Huo, X.W. Enhanced Carbohydrate Metabolism and Salinity Tolerance of Tobacco Plants Overexpressing a Vacuolar H+-ATPase Subunit B2 (DoVHAb2) Gene from Yam (Dioscorea opposita Thunb.). J. Biobased Mater. Bioenergy 2021, 15, 504–513. [Google Scholar] [CrossRef]
  37. Gao, Y.L.; Zhang, Y.F.; Wang, J.X.; Sun, X.H.; Suo, N.N.; Yang, F.; Huo, X.W. Transcriptome Analysis Reveals the Accumulation Mechanism of Starch in Chinese Yam (Dioscorea opposite Thunb.) Tubers Using RNA-Seq and Iso-Seq. Sci. Adv. Mater. 2022, 14, 305–318. [Google Scholar] [CrossRef]
  38. Zhang, Y.F.; Guo, S.C.; Shao, Y.; Zhao, L.M.; Xing, L.N.; Ge, M.R.; Huo, X.W. Transciptome Analysis Molecular Mechanism of Starch Synthesis During Tuber Development in Chinese Yam (Dioscorea opposita). J. Biobased Mater. Bioenergy 2021, 15, 589–597. [Google Scholar] [CrossRef]
  39. Liang, G.P.; He, H.H.; Nai, G.J. Genome-wide identification of BAM genes in grapevine (Vitis vinifera L.) and ectopic expression of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato. BMC Plant Biol. 2021, 21, 156. [Google Scholar] [CrossRef]
  40. Koichiro, T.; Glen, S.; Daniel, P. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar]
  41. Guo, X.; Zhang, L.; Dong, G.; Xu, Z.; Li, J.; Liu, N.; Wang, A.; Zhu, J. A novel cold-regulated protein isolated from Saussurea involucrata confers cold and drought tolerance in transgenic tobacco (Nicotiana tabacum). Plant Sci. 2019, 289, 110246. [Google Scholar] [CrossRef]
  42. Wang, J.; Wang, F.; Zhang, G.; Xin, Y.; Zhang, H.; Liu, H. Comparison of Five Methods for Starch Measurement in Tobaccos. Mol. Plant Breed. 2019, 17, 1673–1678. [Google Scholar]
  43. Su, L.Y.; Dai, Z.W.; Li, S.H. A novel system for evaluating drought–cold tolerance of grapevines using chlorophyll fluorescence. BMC Plant Biol. 2015, 15, 82. [Google Scholar] [CrossRef] [Green Version]
  44. Pak, S.C.; Simon, M.L. A method for routine measurements of total sugar and starch content in woody plant tissues. Tree Physiol. 2004, 24, 1129–1136. [Google Scholar]
  45. Zhao, L.N.; Liu, F.X.; Xu, W.Y. Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana. Plant Biotechnol. J. 2009, 7, 550–561. [Google Scholar] [CrossRef]
  46. Hans, T.C.; Zhang, Z.G.; Wei, Y.D. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 1997, 11, 1187–1194. [Google Scholar]
  47. Kalaivani, K. Nadarajah. ROS Homeostasis in Abiotic Stress Tolerance in Plants. Int. J. Mol. Sci. 2020, 21, 5208. [Google Scholar]
  48. Zhong, C.; Mohan, R. Role of reactive oxygen species in modulating cross tolerance in plants via flavonoids. In Priming-Mediated Stress and Cross-Stress Tolerance in Crop Plants; Academic Press: Cambridge, MA, USA, 2020; pp. 203–214. [Google Scholar]
  49. Yin, X.M.; Huang, L.F.; Zhang, X. OsCML4 improves drought tolerance through scavenging of reactive oxygen species in rice. J. Plant Biol. 2015, 58, 68–73. [Google Scholar] [CrossRef]
  50. Wasif, N.; Farooq, A.; Tehseen, G. Profiling of polyphenolics, nutrients and antioxidant potential of germplasm’s leaves from seven cultivars of Moringa oleifera Lam. Ind. Crops Prod. 2016, 83, 166–176. [Google Scholar]
  51. Sarvajeet, S.G.; Narendra, T. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48, 909–930. [Google Scholar]
  52. Julia, K.; Claudia, J. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J. Exp. Bot. 2012, 63, 1593–1608. [Google Scholar]
  53. Martina, Z.; Gian, L.B.; Claudia, P. β-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress. J. Exp. Bot. 2016, 67, 1819–1826. [Google Scholar]
  54. Zhang, D.L.; Wang, Y.; Jia, B.C. Genome-Wide Identification and Expression Analysis of the β-Amylase Gene Family in Chenopodium quinoa. DNA Cell Biol. 2021, 40, 936–948. [Google Scholar] [CrossRef] [PubMed]
  55. Ma, Y.P.; Han, Y.R.; Feng, X.R. Genome-wide identification of BAM (β-amylase) gene family in jujube (Ziziphus jujuba Mill.) and expressi.on in response to abiotic stress. BMC Genom. 2022, 23, 438. [Google Scholar] [CrossRef] [PubMed]
  56. Tomojiro, K.; Yasuo, O.; Sueharu, H. Characterization of recombinant β-amylases from Oryza sativa. Biosci. Biotechnol. Biochem. 2011, 75, 793–796. [Google Scholar]
  57. Matthias, T.; Mario, C.; Tiago, M. The evolution of functional complexity within the β-amylase gene family in land plants. BMC Evol. Biol. 2019, 19, 1–18. [Google Scholar]
  58. Sun, S.H.; Hu, C.G.; Qi, X.J. The AaCBF4-AaBAM3. 1 module enhances freezing tolerance of kiwifruit (Actinidia arguta). Hortic. Res. 2021, 8, 97. [Google Scholar] [CrossRef] [PubMed]
  59. Vaughan, H.; Nathalie, D.; Ana, C. Photosynthesis at low temperatures. Plant Cold Hardiness 2002, 299, 161–179. [Google Scholar]
  60. Xiong, L.M.; Zhu, J.K. Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ. 2002, 25, 131–139. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Zhu, J.K. Abiotic stress signaling and responses in plants. Cell 2016, 167, 313–324. [Google Scholar] [CrossRef] [Green Version]
  62. Zhao, L.Y.; Yang, T.Y.; Xing, C.H. The β-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble sugar accumulation and ROS homeostasis in response to cold stress. Plant Sci. 2019, 287, 110184. [Google Scholar] [CrossRef]
  63. Wang, L.; Yao, L.N.; Hao, X.Y. Transcriptional and physiological analyses reveal the association of ROS metabolism with cold tolerance in tea plant. Environ. Exp. Bot. 2019, 160, 45–58. [Google Scholar] [CrossRef]
  64. Ruth, G.A.; Neval, E.; Lenwood, S.H. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 2002, 53, 1331–1341. [Google Scholar]
  65. Adriano, S.; Antonio, S.; Maria, N. Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. Int. J. Mol. Sci. 2015, 16, 13561–13578. [Google Scholar]
  66. Zhu, H.; Jiang, Y.N.; Guo, Y. A novel salt inducible WRKY transcription factor gene, AhWRKY75, confers salt tolerance in transgenic peanut. Plant Physiol. Biochem. 2021, 160, 175–183. [Google Scholar] [CrossRef]
  67. El-Esawi, M.A.; Al-Ghamdi, A.A.; Ali, H.M. Overexpression of AtWRKY30 transcription factor enhances heat and drought stress tolerance in wheat (Triticum aestivum L.). Genes 2019, 10, 163. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Zhu, H.; Yang, X.; Wang, X. The sweetpotato β-amylase gene IbBAM1. 1 enhances drought and salt stress resistance by regulating ROS homeostasis and osmotic balance. Plant Physiol. Biochem. 2021, 168, 167–176. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Changes in (A) starch content and (B) β-amylase activity in yam tubers. * and ** indicate a significant difference between the DHCY and B1 at p <0.05 and p < 0.01, respectively.
Figure 1. Changes in (A) starch content and (B) β-amylase activity in yam tubers. * and ** indicate a significant difference between the DHCY and B1 at p <0.05 and p < 0.01, respectively.
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Figure 2. (A) Changes in DoBAM1 expression during tuber expansion. (B) Expression of DoBAM1 in different tissues of yam. (C) Expression levels in yam leaves during time course of cold stress. * and ** indicate a significant difference at p < 0.05 and p < 0.01, respectively.
Figure 2. (A) Changes in DoBAM1 expression during tuber expansion. (B) Expression of DoBAM1 in different tissues of yam. (C) Expression levels in yam leaves during time course of cold stress. * and ** indicate a significant difference at p < 0.05 and p < 0.01, respectively.
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Figure 3. PCR amplification of DoBAM1 of full-length cDNA. (a) M1: DL2 000 bp DNA Marker; 1: 5′RACE PCR; (b) M2: 2000 bp DNA Marker; 2: 3′RACE PCR; (c) M3: DL5 000 bp DNA Marker; 3: ORF full-length PCR.
Figure 3. PCR amplification of DoBAM1 of full-length cDNA. (a) M1: DL2 000 bp DNA Marker; 1: 5′RACE PCR; (b) M2: 2000 bp DNA Marker; 2: 3′RACE PCR; (c) M3: DL5 000 bp DNA Marker; 3: ORF full-length PCR.
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Figure 4. Phylogenetic analysis of DoBAM1 using 5 DoBAMs from yam, 7 AtBAMs from Arabidopsis, 4 DaBAMs from water yam, 6 StBAMs from potato, and 10 OsBAMs from rice. Red indicates IV group, green indicates I group, blue indicates II group, and purple indicates III group. The red data indicate DoBAM1.
Figure 4. Phylogenetic analysis of DoBAM1 using 5 DoBAMs from yam, 7 AtBAMs from Arabidopsis, 4 DaBAMs from water yam, 6 StBAMs from potato, and 10 OsBAMs from rice. Red indicates IV group, green indicates I group, blue indicates II group, and purple indicates III group. The red data indicate DoBAM1.
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Figure 5. Multiple sequence alignment of DoBAM amino acid family. Blue shading indicates highly conservative substitutions. The two red boxes highlight the catalytic residues Glu-186 and Glu-380.
Figure 5. Multiple sequence alignment of DoBAM amino acid family. Blue shading indicates highly conservative substitutions. The two red boxes highlight the catalytic residues Glu-186 and Glu-380.
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Figure 6. Subcellular localization of the DoBAM1 fusion protein in cells of tobacco leaves. The 4 panels from left to right indicate the GFP fluorescence, chloroplast autofluorescence, bright field, and merged images, respectively. The horizontal scale bar = 20 μM.
Figure 6. Subcellular localization of the DoBAM1 fusion protein in cells of tobacco leaves. The 4 panels from left to right indicate the GFP fluorescence, chloroplast autofluorescence, bright field, and merged images, respectively. The horizontal scale bar = 20 μM.
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Figure 7. Cold tolerance testing of transgenic tobacco overexpressing DoBAM1. (A) PCR detection of DoBAM1 gene tobacco. (B) Phenotypes of overexpressed DoBAM1 tobacco and WT plants under control and cold treatments. (C) Transcript levels of DoBAM1 under different conditions. M1 = DL2000 bp DNA marker; + = plasmid pPZP221–DoBAM1 as positive control; − = water as negative control; WT = wild type; 5–9 = different transgenic lines. ** indicate a significant difference at p < 0.01 compared with WT, respectively.
Figure 7. Cold tolerance testing of transgenic tobacco overexpressing DoBAM1. (A) PCR detection of DoBAM1 gene tobacco. (B) Phenotypes of overexpressed DoBAM1 tobacco and WT plants under control and cold treatments. (C) Transcript levels of DoBAM1 under different conditions. M1 = DL2000 bp DNA marker; + = plasmid pPZP221–DoBAM1 as positive control; − = water as negative control; WT = wild type; 5–9 = different transgenic lines. ** indicate a significant difference at p < 0.01 compared with WT, respectively.
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Figure 8. (A) β-amylase activity, (B) starch content, (C) soluble sugar, and (D) chlorophyll content in the DoBAM1-overexpressing lines and WT under different cold treatments. * and ** indicate a significant difference at p < 0.05 and p < 0.01 compared with WT, respectively.
Figure 8. (A) β-amylase activity, (B) starch content, (C) soluble sugar, and (D) chlorophyll content in the DoBAM1-overexpressing lines and WT under different cold treatments. * and ** indicate a significant difference at p < 0.05 and p < 0.01 compared with WT, respectively.
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Figure 9. DAB and NBT staining of transgenic plant leaves is shown for plants subjected to cold treatments of (A) 4 °C for 2 h; (B) 2 °C for 2 h; (C) −2 °C for 2 h.
Figure 9. DAB and NBT staining of transgenic plant leaves is shown for plants subjected to cold treatments of (A) 4 °C for 2 h; (B) 2 °C for 2 h; (C) −2 °C for 2 h.
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Figure 10. (A) SOD activity, (B) CAT activity, (C) POD activity, and (D) MDA content in the transgenic lines and WT plants under different conditions. * and ** indicate a significant difference at p < 0.05 and p < 0.01 compared with WT, respectively.
Figure 10. (A) SOD activity, (B) CAT activity, (C) POD activity, and (D) MDA content in the transgenic lines and WT plants under different conditions. * and ** indicate a significant difference at p < 0.05 and p < 0.01 compared with WT, respectively.
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Zhao, L.; Zhang, Y.; Shao, Y.; Xing, L.; Ge, M.; Huo, X. Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco. Genes 2022, 13, 2296. https://doi.org/10.3390/genes13122296

AMA Style

Zhao L, Zhang Y, Shao Y, Xing L, Ge M, Huo X. Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco. Genes. 2022; 13(12):2296. https://doi.org/10.3390/genes13122296

Chicago/Turabian Style

Zhao, Lingmin, Yanfang Zhang, Ying Shao, Linan Xing, Mingran Ge, and Xiuwen Huo. 2022. "Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco" Genes 13, no. 12: 2296. https://doi.org/10.3390/genes13122296

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