Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus

doi:10.21203/rs.3.rs-3227294/v1

Download PDF

Research Article

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus

https://doi.org/10.21203/rs.3.rs-3227294/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 30 Apr, 2024

Read the published version in Gene →

Version 1

posted

You are reading this latest preprint version

AmCIP is a dehydrin-like protein which involved in abiotic stress tolerance in xerophytes evergreen woody plant A. mongolicus. AmCIP could be induced in the cotyledon and radicle during cold acclimation. To further elucidate the regulation of the upstream region of the gene, we isolated and characterized the promoter of AmCIP. Herein, a 1048 bp 5’-flanking region of AmCIP genomic DNA was isolated and cloned by Genome walking from A. mongolicus and the segment sequence was identified as “PrAmCIP” promoter. Analysis of the promoter sequence revealed the presences of some basic cis-acting elements, which were related to various environmental stresses and plant hormones. GUS histochemical staining showed that PrAmCIP was induced by 4℃, 55℃, NaCl, drought and ABA, whereas it could hardly drive GUS gene expression under normal conditions. Furthermore, we constructed three deletion fragments and genetically transformed them into Arabidopsis thaliana. GUS histochemical staining showed that the MYCATERD1 element of the CP7 fragment(-122~-1)may be a key element in response to drought. In conclusion, we provide an inducible promoter, PrAmCIP, which can be applied to the development of transgenic plants under abiotic stresses.

Ammopiptanthus mongolicus

PrAmCIP promoter

Abiotic stress

GUS-activity

Deletion analysis

Dehydrins (DHNs) belongs to the late embryonic abundance proteins (LEA), which are usually abundantly expressed in the late stage of seed growth and development (Koag et al. 2009). Dehydrins can be classified into five subfamilies based on their conserved motifs: Kn, SKn, KnS, YnKn, and YnSKn (Close 1996). It has been shown that different types of dehydrins can respond to different external factors, for example, YnSKn dehydrins can be induced by salt and drought (Wang et al, 2014), and KnS, Kn, SKn and YnKn can be induced by cold stress (Graether and Boddington 2014). Dehydrins play important functions in ROS detoxification systems, biotic and abiotic stresses (Riyazuddin et al. 2021; Yang et al. 2012; Cui et al. 2020). Nowadays, dehydrin genes have been isolated and identified in different plants, such as rice (Verma et al. 2017; Kuma et al. 2018; Ganguly et al. 2020), wheat (Brini et al. 2007; Saibi et al. 2015), tomato (Guo et al. 2017). Most studies have shown that dehydrin genes are respond to biotic and abiotic stresses (Abdul Aziz et al. 2021).

As functional elements that participate in gene expression and regulation, promoters serve as transcriptional regulatory centers that control temporal and spatial gene expression patterns. Currently, the promoters of several dehydrin genes have been reported to have many cis-acting elements in the promoter region for both adversity and hormone response (Zhu et al. 2014; Qin and Qin 2016; Lv et al. 2017). DREB/CBF transcription factors are able to enhance plant resistance to cold stress by binding to CRT sequences in the dehydrin promoter region and thereby regulating the expression of dehydrin genes (Vazquez Hernandez et al. 2017). H3K4me3 positively regulates the binding of OsbZIP23 to the dehydrin promoter by modification thereby enhancing the expression of the dehydrin gene under drought stress (Zong et al 2020). Despite of significant progress in the analysis of the structure of dehydrin gene promoters, revealing the specific regulation and function of dehydrin gene promoters in different plants is an actual challenge.

Ammopiptanthus mongolicus as the only evergreen broad-leaved shrub in the deserts of eastern China, can survive in -30°C or even lower in the winter and is an ideal shrub for the investigation in the cold resistance mechanism (Xu et al. 2002; Liu et al. 2006; Liu et al. 2013). In previous studies, we have identified a cDNA, AmCIP, encoding a novel 15-mer K-segment of HKEGLVDKIKDKVHG similar to the consensus sequence EEKKGIMDKIKELPG of typical dehydrins protein from a cold-acclimated A. mongolicus seedling (Liu et al. 2006). We found that AmCIP enhanced cold resistance not only in transformed E.coli but also in transgene tobacco (Shi et al. 2016). However, little is known about the promoter information of AmCIP and the functional elements.

In this work, we isolated and characterized the promoter sequence of PrAmCIP and also analyzed its activity in heterologous systems under various stress conditions. Furthermore, the key regulatory element, which was necessary for promoter core sequence has been confirmed. Our results will not only provide the basis for the identification of AmCIP gene expression regulatory networks, but also provide a favorable tool for plant genetic engineering.

Plant material and growth conditions

A. mongolicus seeds were obtained from Alashan Desert in Inner Mongolia, China. Healthy seeds were sterilization with 75% ethanol for 30 s and then with 10% sodium hypochlorite for 15 min. After rinsed thoroughly with distilled water three times, the seeds were germinated and grown on the 200 mL pots containing hormone-free Murashige and Skoog medium, which was autoclaved at 121°C for 20 min. The seedlings were incubated with a photoperiod of 16 h light and 8 h dark in a culture room.

Isolation and amplification of the PrAmCIP promoter

Two-week-old cotyledons of A. mongolicus were used for promoter cloning. Genomic DNA was extracted and used as templates for Genome Walking PCR amplification (Xu et al. 2002). For PCR reaction, the AmCIP gene specific primers CSP1, CSP2, CSP3 (sequences shown in Table S1) and the adaptor primers AP1 (Siebert et al. 1995) were used. Amplification procedures are described in Figure S2. The nucleotide sequence of the AmCIP was registered in the GenBank nucleotide sequence databases with the accession number AY590122. Putative functional cis-acting elements of the PrAmCIP promoter were identified by the PLACE (http://www.dna.affrc.go.jp/PLACE/) and PLANTCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database.

Construction of reporter plasmids

To construct reporter plasmids fusing the PrAmCIP promoter or its derivatives with β-glucuronidase (GUS) reporter gene cassette, PCR was performed to generate sequential PrAmCIP promoter as Hind III and Xba I segment at corresponding restriction sites just upstream of the GUS gene in the binary vector pCP-GUS binary vector to analyze the expression pattern of the induced promoter in vivo. Three expression vectors containing various lengths of PrAmCIP promoters were individually obtained and designated CP2, CP4, and CP7. All primers information were listed in Table S2. Then the CaMV35S promoter of pBI121 was separately replaced by the above released segments. Escherichia coli JM109 were used in all construction experiments and Agrobacterium tumefaciens LBA4404 and GV3101 were used for the purpose of plant transformation. All constructs were verified by sequencing. In addition, the CaMV35S promoter was the positive control and wild-type tobacco/ wild-type Arabidopsis the negative control.

Arabidopsis and tobacco transformation

The recombinant plasmid was transfected into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. PrAmCIP promoter::GUS chimeric construct was transferred to Tobacco (N. tabacum cv. K326) by the leaf discs method (Horsch 1985) and Arabidopsis was transformed by floral dip method (Clough and Bent 1998). Transformants were screened for integration of the intact PrAmCIP promoter::GUS chimeric gene into the genome DNA on MS agar plates containing 50 µg/ml kanamycin further confirmed by PCR.

Identification of transgenic plants and PCR analysis

PCR analysis was carried out using the primer pair: CIP_GUSSF:5‘-GTCACTCATTACGGCAAAGT-3'// CIP_GUSSR: 5‘-CAGCAGCAGTTTCATCAATC-3'. Cycling conditions were 94°C, 5 min; 35 cycles of 94°C, 30 s; 55°C, 30 s; 72°C, 1 min followed by a final extension of 72°C for 10 min. The total genome DNA was isolated from leaves of kanamycin-resistant plants using the Tiangen plant DNeasy kit and was used as a template. PCR products were analyzed on 1% (w/v) agarose gel containing ethidium bromide. Kanamycin-resistant T1 transgenic seedlings were transplanted into soil and allowed to self-fertilize and produce T2 generation.

Histochemical GUS staining

For GUS histochemical staining assays, two-week-old transgenic tobacco plants containing PrAmCIP promoter::GUS fusion constructs were vacuum infiltrated in a solution containing 50 mM NaP buffer (pH 7.0), 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 0.1% Triton X-100, 10 mM Na₂EDTA, 20% methanol and 10 mg/ml X-Gluc (5-bromo-4-chloro-3-indole-β-D-glucuronide cyclohexyl ammonium salt) for several minutes and incubated overnight at 37°C (Jefferson et al. 1987). The GUS-positive plant individual’s tissues were followed by 70% ethanol fixation and then examined with a light microscope (Leica) at a low magnification and photographed with a digital camera (Jefferson 1987). GUS blue stained tissues and plants assessment represent the typical results of at least three independent transgenic lines for each construct.

Protein extraction and fluorometric GUS assays

GUS activity of transgenic tobacco in seedling protein extracts was compared quantitatively by monitoring cleavage of the β-glucuronidase substrate 4-methyl-umbelliferyl β-D-glucuronide (MUG) for the fluorescent assay (Bradford, 1976). Briefly, two hundred milligrams of 3-week-old tissues from T2 transgenic plant were ground in a mortar and pestle pre-cooled with liquid nitrogen to powder. The powder was transferred to a microcentrifuge tube containing 0.6 ml of extraction buffer (50 mM NaHPO₄ (pH 7.0), 10 mM Na₂EDTA, 0.1% Triton X-100, 0.1% (w/v) Sarcosyl, 10 mM β-Mercaptoethanol), and then performed with the method of Vortex oscillation and subsequently placed on ice for 10 min, centrifuged in a microfuge at 13, 000 g for 10 min at 4°C and at last the supernatant was transferred to a clean tube on ice. The total protein concentrations of the extracts were measured by Bradford method. Fluorescence intensity was measured on a GENios (Tecan, Männedorf, Switzerland) with emission at 465 nm and excitation at 340 nm. Fluorescence units were plotted against 4-MU concentration to create a standard curve (R² > 0.99). The reaction consisted of 20 µl sample extract and 480µl assay buffer (GUS extraction buffer containing 1M MUG). The reaction was incubated at 37°C water bath in the dark. After 20 min, 100 µl reaction mixes was added to the 900 µl stop buffer (0.2 M Na₂CO₃); mixed, and 200 µl mixes above was transferred into each well of an opaque 96-well plate. GUS activity was expressed as nmol of 4-methylumbelliferone (4-MU) produced per minute per milligram of soluble protein, which has been normalized to protein concentration in each of the tissue homogenates.

Abiotic stress and hormone treatment

Histochemical GUS staining assay was used to characterize the PrAmCIP promoter. T2 transgenic tobacco plants were transferred to either normal MS agar plates or MS agar plates supplemented with 200 mM D-mannitol, 200 mM NaCl, or 20% PEG, respectively for 5 days. Besides, for cold stress and heat shock stress, plants growing on MS agar plates were exposed to 4°C and 40°C for 2 days, respectively. In fluorometric GUS assays, transgenic tobaccos were transferred to the MS agar plates supplemented with 0 mM, 50 mM, 100 mM, 150 mM and 200 mM NaCl. To induce low-temperature stress, the plants were transferred to an incubator at 4°C with 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 36 h, 48 h, 1 week or 2 weeks, respectively. Seedlings of transgenic plants without any induction were used as controls. All the above treatments were carried out under a growth regime of 16/8 h light/dark at 25 ± 1°C unless otherwise mentioned.

Data analysis

The results of GUS activity measurements were all expressed as mean values with ± SD. The standard deviations (SD) of the experimental data are showed as Error bars in figures. Each assay was repeated at least three times. The data presented were collected from at least three independent lines for each construct. The least significant difference (LSD) was performed at 1% probability level with the IBM SPSS 19.0 software package (IBM Corporation, Armonk, NY, USA).

Isolation and sequence analysis of the PrAmCIP promoter

Based on our previous study, the 5’-flanking region of the AmCIP gene was isolated and amplified from genomic DNA of seedlings in A. mongolicus via Genome Walking PCR amplification (Fig. S1). Sequencing resulted in a final fragment of 1158bp, sequence alignment demonstrated that a 110 bp sequence of overlapping compared with AmCIP gene DNA sequence has been obtained by BioEdit (Hall, 1999). In addition, sequence analysis of the 1048 bp 5’ UTR region showed that the transcription start site (TSS) of AmCIP is located probably 67 bp upstream of its ATG codon, and predicted as “A” via TSSP-TCM (Shahmuradov et al. 2005), according to the general rules for a transcription start site (Joshi, 1987). A putative ‘TATA’ box was found and located at -29 bp from the putative transcription start site (Fig. 1). This study also revealed that the A + T percentage was 70.6% (35.3% of A; 35.3% of T), while G + C percentage was 29.4% (11.7% of G and 17.7% of C), which is consistent with the composition character of the promoter sequence (Aozasa et al. 2001). We named this 1048 bp promoter as PrAmCIP (GenBank: AY590122)

Cis-elements in PrAmCIP promoter sequence

Analysis of the PrAmCIP promoter sequence using PLACE and PlantCARE databases has been performed in this work. Meanwhile, a mass of regulatory motifs involved in the activation of abiotic stress and hormone-responsive (ABA, GA, IAA, CTK and MeJA) genes were predicted and located in the sequence of PrAmCIP promoter.

The analysis showed that a TATA-box was detected in the site − 29 upstream of transcription start site, which is the most basic cis-acting element in eukaryotic promoter region. It often determines the selection of the gene transcription start, as one site of the junction of RNA polymerase. Certainly, the CAAT-box was also found in -332 bp, it may be the other site of junction of RNA polymerase; it mainly controls the transcription start frequency. In addition, several environmental-related and hormone-related stress cis-acting elements has been also examined, such as ABRE/EBOX, which is responsive to ABA signal cis-element and identified in plant promoters. An ACGT core characteristic sequence of G-boxes exists in this cis-acting element, which is mainly used in recognizing the sequences of leucine zipper (bZIP) transcription factors. The homologue cis-acting elements sequences of DRE could be induced by low temperature or salt (Kazuko and Kazuo 1994). Furthermore, the homologue sequences of DREC/LTRE, GT1-motif, MYBCORE, MYCATERD1, GGATA, MYC, CANNTG, NTB-motif, ACTTTA, TATCCA-motif, TATCCA, and WBOX were also generally typical cis-acting elements in activating the transcription and expression of resistant genes responding to abiotic stresses and all kinds of hormonal stresses, respectively (Table 1). More interestingly, most of these function cis-regulatory elements could be mainly induced by cold and drought stresses.

Vector construction of transgenic plants and PCR identification

To study the function of PrAmCIP, we constructed the reporter plasmid fusing the PrAmCIP promoter and derivatives with GUS reporter gene. But beyond that, we also performed a comparative transgenic analysis of four different PrAmCIP promoter deletion segments to drive GUS fusion constructs (CP2, CP4, and CP7). The schematic illustrations of the 35S-GUS and PrAmCIP Promoter -GUS binary vectors were showed in Fig. 2. After two weeks, there are lots of resistant shoots appearing on leaf disc edges, constantly. Kan-resistant groups were transferred to the regeneration medium, and multiple green shoots have been produced. After another two weeks, the transformed plants grew vividly and formed strong root systems. A total of 30 transgenic plant lines were obtained from the independent Kan resistant shoots. We collected the seeds of T1 and T2 generation transgenic plants and validated the positive groups of by PCR (Fig. 3). The results confirmed that foreign promoter DNA sequence had been integrated into the genome of three individual positive transgenic plants.

Histochemical analysis of GUS activities in transgenic tobacco seedlings

In order to better analyze activity and inducibility of the cis-acting elements in plant, a GUS staining experiment was performed in transgenic tobacco seedlings. It is interesting that no GUS staining was observed in young seedlings of the transgenic tobacco carrying the construct PrAmCIP-GUS under the normal growth conditions. However, intense GUS staining was found in the cotyledons, young leaves and roots after various biotic stresses treatment. More importantly, the GUS expression level of transgenic tobacco seedlings showed diversity and specificity under abiotic stress (Fig. 4). Clearly, PrAmCIP promoter could be induced in response to low temperature (4°C), heat shock (55°C), D-Mannitol, NaCl, 20% PEG, and ABA.

Fluorometric analysis of GUS activities in transgenic tobacco

To further verify the functions of PrAmCIP promoter in response to drought, NaCl, and cold stresses, GUS assay was performed from transgenic tobacco seedlings at different temperatures and concentrations (Fig. 5), and the GUS activity increased gradually with the increase of mannitol concentration and reached a maximum value of 101.4 nmol 4MU/min/g, which was 6.14 times higher than of the control.

Similarly, PrAmCIP was also induced by NaCl, and GUS viability was slowly increasing with increasing NaCl concentration. Under 200 mM NaCl treatment, GUS viability reached a maximum value of 40.2 nmol 4 MU/min/g, which was 2.47 times higher than of the control.

However, unlike mannitol and NaCl, PrAmCIP was more strongly induced by 4°C. Under cold stress, GUS activity increased gradually from 0 to 36 h and reached a maximum at 36 h, 245 nmol 4 MU/min/g, which was 14.26-fold higher than that the control group. But the GUS viability tended to decrease with the prolongation of the cold treatment. In all treatments, GUS activity was at a stable level in both the negative control (wild-type tobacco) and the positive control (35S), proving the reliability of our data.

The GUS activity driven by three promoter deletion segments

To further clarify the key action elements in the PrAmCIP promoter in response to drought and cold stress, three 5' deletion fragments of different lengths were designed based on the known distributional features of cis-acting elements in the PrAmCIP promoter sequences, and each of them was transfected into Arabidopsis thaliana and subjected to GUS histochemistry staining using the T2 generation of Arabidopsis thaliana (Fig. 6).

We observed that a cis-element (LTRE) involved in cold response exists the region between − 1048 and − 778 of PrAmCIP promoter. The results clearly showed that PrAmCIP, CP2 and CP4 fragments were able to drive GUS gene expression under treatments subjected to cold stress, and the staining results were not significantly different, whereas CP7 could not be induced by cold stress (Fig. 6). This result suggests that the LTRE element may function in the PrAmCIP promoter region in response to cold stress, but is not required for the promoter to respond to cold stress, and that MYC may be a key element in response to cold stress.

Based on the results of GUS staining, we found that CP2, CP4, CP7 were able to be induced by drought. Segment construct CP7 is the shortest sequence, which only contains one MYCATERD1 element. MYCATERD1 is the major cis-acting regulatory elements involved in drought stress response (Tran et al. 2004). Accordingly, GUS activity signal still could be detected in construct CP7 under drought treatment but not under the cold (Fig. 6).

Previous research has shown that AmCIP is a KnS-type dehydrin-like protein, in A. mongolicus and plays important roles in abiotic stress responses, especially in cold/ dehydration conditions (Liu et al. 2006; Shi et al. 2016). Herein, the 1048 bp promoter sequence of AmCIP was amplified from A. mongolicus genome by Genome Walking. Transcription start sites (TSS) are essential for the regulation of RNA molecules (Kapranov 2009), the TSS of the AmCBL1 and AmDUF1517 gene promoters are located between − 80 and − 60 bp upstream of the ATG codon (Guo et al. 2010; Zhou et al. 2020). The TSS of YnSKn-type wzy2 gene is “A”, located 75 bp upstream of the ATG codon (Zhu et al. 2014), and our study showed that the TSS of AmCIP gene was located 66 bp upstream of the ATG codon, also “A”.

Although KnS-type dehydrin are expressed in response to cold and dehydration, KnS dehydrin gene promoters typically contain GATA core motifs and do not have cis-acting elements typical of cold or drought response (Zolotarov and Strömvik 2015). However, our study revealed the presence of some cis-acting elements of cold stress and drought response in the AmCIP promoter region, such as DREB/LTRE, MYC, MYBCORE. DRE or C-repeats element is able to bind to the CBF/DREB or ERF transcription factor, thereby increasing the cold stress capacity of plants, and it was found that one or more of these elements were present in the promoter region of some dehydrin genes, such as PpDHN1, VviDHN2, TdCor410b (Wisniewski et al. 2006; Vazquez-Hernandez et al. 2017; Eini et al. 2013). MYC elements can be bound by ICE, which regulates the expression of CBF genes, and finally leads to the up-regulation of dehydrin genes under cold stress (Peng et al. 2014). In addition, we predicted a number of light-responsive elements and hormone-responsive elements in the AmCIP promoter region (Table 1). These results imply that AmCIP gene is regulated by multiple factors.

Current research shows that the promoter of the dehydrin gene is an inducible promoter, which can only activate gene expression in response to hormonal and abiotic stresses (Zhu et al. 2014; Qin and Qin 2016; Lv et al. 2017). Our results similarly demonstrated that PrAmCIP is also a inducible promoter, PrAmCIP was able to drive GUS gene expression in tobacco under 4°C, 55°C, drought, high salt, and ABA treatments, in contrast, was barely functionalunder normal conditions (Fig. 4). GT-1motif, LTRE, MYBCORE and MYCATERD1 are key core components in response to salt, cold and drought, respectively (Terzaghi et al. 1995; Baker et al. 1994; Urao et al. 2003; Tran et al. 2004), and they are also present in the PrAmCIP region.

Li et al. (2023) found that LTRE1HVBLT49 at -526 bp to -429 bp in the promoter region of PLkF3H2 was a key component in response to low temperature. In our result, CP2 and CP4 were able to be induced by cold stress as well as PrAmCIP, whereas CP7 did not respond to cold stress (Fig. 7), suggesting that the DREB/LTRE elements present in the PrAmCIP sequences are not required for PrAmCIP to respond to cold stress, and that the MYC elements may be the key elements for PrAmCIP to respond to cold stress. In addition, we found that CP7 was able to respond to drought stress but not to cold stress, implying that the MYCATERD1 element in the − 122bp~-1 region of PrAmCIP may play an important role in response to drought stress (Fig. 7).

In summary, we identified an inducible promoter PrAmCIP, which can respond to drought and cold stress, and these results provide a theoretical basis for explaining the expression and regulatory mechanism of AmCIP. Meanwhile, this inducible promoter can be used for the development of abiotic tolerant transgenic plants.

Supplementary Information The online version contains supplementary material available at

Author contributions

BG analyzed data and drafted the manuscript. KD, RCL, QRL, WWZ participated in the sample preparation and performed experiments. YZC and CFL planned and designed the research and approve the final manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (31270737), Beijing Municipal Natural Science Foundation (6112016, KZ20150020021), and the National Science Technology Support Program (2011BAI13B06), 111 Project (B13007) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13047).

Data Availability All data generated or analysed during this study are included in this published article (and its additional files). Requests for material should be made to the corresponding authors.

Conflict of Interest The authors declare that the research was conduct-ed in the absence of any potential conflict of interest.

Informed consent Informed consent was not required as no human or animals was involved.

Research involving human and animal rights Our study has no re-search involved human participants or animals.

Abdul Aziz M, Sabeem M, Mullath SK, Brini F, Masmoudi K (2021) Plant group II LEA proteins: intrinsically disordered structure for multiple functions in response to environmental stresses. Biomolecules 11(11):1662. https://doi.org/10.3390/biom11111662
Abe H, Yamaguchi Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997) Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell 9(10):1859–1868. https://doi.org/10.1105/tpc.9.10.1859
Aozasa N, Shiraishi H, Nakanishi Matsui M, Kobayashi A, Sekimizu K, Kubo T, Natori S (2001) Activation of the Sarcophaga lectin gene promoter by (A + T)-stretch binding protein. Eur J Biochem 268(8):2506–2511. https://doi.org/10.1046/j.1432-1327.2001.02133.x
Baker SS, Wilhelm KS, Thomashow MF (1994) The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24(5):701–713. https://doi.org/10.1007/BF00029852
Baumann K, De Paolis A, Costantino P, Gualberti G (1999) The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants. Plant cell 11(3):323–334. https://doi.org/10.1105/tpc.11.3.323
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1006/abio.1976.9999
Brini F, Hanin M, Lumbreras V, Amara I, Khoudi H, Hassairi A, Pagès M, Masmoudi K (2007) Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana. Plant Cell Rep 26(11):2017–2026. https://doi.org/10.1007/s00299-007-0412-x
Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17(8):1043–1054. https://doi.org/10.1101/gad.1077503
Close TJ (1996) Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97(4):795–803. https://doi.org/10.1111/j.1399-3054.1996.tb00546.x
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x
Cui H, Wang Y, Yu T, Chen S, Chen Y, Lu C (2020) Heterologous expression of three Ammopiptanthus mongolicus dehydrin genes confers abiotic stress tolerance in Arabidopsis thaliana. Plants (Basel) 9(2):193. https://doi.org/10.3390/plants9020193
Eini O, Yang N, Pyvovarenko T, Pillman K, Bazanova N, Tikhomirov N, Eliby S, Shirley N, Sivasankar S, Tingey S, Langridge P, Hrmova M, Lopato S (2013) Complex regulation by Apetala2 domain-containing transcription factors revealed through analysis of the stress-responsive TdCor410b promoter from durum wheat. PLoS ONE 8(3):e58713. https://doi.org/10.1371/journal.pone.0058713
Fusada N, Masuda T, Kuroda H, Shimada H, Ohta H, Takamiya K (2005) Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5'-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber. Plant Mol Biol 59(4):631–645. https://doi.org/10.1007/s11103-005-0579-x
Ganguly M, Roychoudhury A, Sengupta DN, Datta SK, Datta K (2020) Independent overexpression of OsRab16A and AtDREB1A exhibit enhanced drought tolerance in transgenic aromatic rice variety Pusa Sugandhi 2. J Plant Biochem Biot 29(3). https://doi.org/10.1007/s13562-020-00565-w
Graether SP, Boddington KF (2014) Disorder and function: a review of the dehydrin protein family. Front Plant Sci 5:576. https://doi.org/10.3389/fpls.2014.00576
Guilley H, Dudley RK, Jonard G, Balàzs E, Richards KE (1982) Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30(3):763–773. https://doi.org/10.1016/0092-8674(82)90281-1
Guo L, Yu Y, Xia X, Yin W (2010) Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus. BMC Plant Biol 10:18. https://doi.org/10.1186/1471-2229-10-18
Guo X, Zhang L, Zhu J, Liu H, Wang A (2017) Cloning and characterization of SiDHN, a novel dehydrin gene from Saussurea involucrata Kar. et Kir. that enhances cold and drought tolerance in tobacco. Plant Sci 256:160–169. https://doi.org/10.1016/j.plantsci.2016.12.007
Gusmaroli G, Tonelli C, Mantovani R (2002) Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. Gene 283(1–2):41–48. https://doi.org/10.1016/s0378-1119(01)00833-2
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95–98 http://dx.doi.org/10.1021/bk-1999-0734.ch008
Hattori T, Totsuka M, Hobo T, Kagaya Y, Yamamoto-Toyoda A (2002) Experimentally determined sequence requirement of ACGT-containing abscisic acid response element. Plant Cell Physiol 43(1):136–140. https://doi.org/10.1093/pcp/pcf014
Horsch RB (1985) A simple and general method for transferring genes into plants. Science 227(4691):1229–1231. https://doi.org/10.1126/science.227.4691.1229
Huang GT, Ma SL, Bai LP, Zhang L, Ma H, Jia P, Liu J, Zhong M, Guo ZF (2012) Signal transduction during cold, salt, and drought stresses in plants. Mol Biol Rep 39(2):969–987. https://doi.org/10.1007/s11033-011-0823-1
Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405. https://doi.org/10.1007/BF02667740
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907. https://doi.org/10.1002/j.1460-2075.1987.tb02730.x
Joshi CP (1987) An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15(16):6643–6653. https://doi.org/10.1093/nar/15.16.6643
Kapranov P (2009) From transcription start site to cell biology. Genome Biol 10(4). https://doi.org/10.1186/gb-2009-10-4-217. )-217
Koag MC, Wilkens S, Fenton RD, Resnik J, Vo E, Close TJ (2009) The K-segment of maize DHN1 mediates binding to anionic phospholipid vesicles and concomitant structural changes. Plant physiol 150(3):1503–1514. https://doi.org/10.1104/pp.109.136697
Kumar A, Smita S, Sahu N, Sharma V, Shankaracharya Vidyarthi A, Pandey DM (2009) In silico analysis of motifs in promoters of differentially expressed genes in rice (Oryza sativa L.) under anoxia. Int J Bioinform Res Appl 5(5):525–547. https://doi.org/10.1504/IJBRA.2009.028681
Kumar M, Lee SC, Kim JY, Kim SJ, Aye SS, Kim SR (2014) Over-expression of dehydrin gene, OsDhn1, improves drought and salt stress tolerance through scavenging of reactive oxygen species in rice (Oryza sativa L). J Plant Biol 57(6):383–393. https://doi.org/10.1007/s12374-014-0487-1
Li C, Jiang W, Jiang X, Gai Y (2023) Cloning and functional analysis of the PLkF3H2 promoter in Larix kaempferi. https://doi.org/10.1007/s11240-023-02467-w. Plant Cell Tiss Organ Cult
Lipiec J, Doussan C, Nosalewicz A, Kondracka K (2013) Effect of drought and heat stresses on plant growth and yield: a review. Int Agrophys 27(4):463–477. http://dx.doi.org/10.2478/intag-2013-0017
Liu M, Lu C, Shen X, Yin W (2006) Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus. DNA Seq 17(5):342–349. https://doi.org/10.1080/10425170600908484
Liu M, Shi J, Lu C (2013) Identification of stress-responsive genes in Ammopiptanthus mongolicus using ESTs generated from cold- and drought-stressed seedlings. BMC Plant Biol 13:88. https://doi.org/10.1186/1471-2229-13-88
Lv A, Fan N, Xie J, Yuan S, An Y, Zhou P (2017) Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway. Front in Plant Sci 8:748. https://doi.org/10.3389/fpls.2017.00748
Mantri N, Basker N, Ford R, Pang E, Pardeshi V (2015) The role of micro-ribonucleic acids in legumes with a focus on abiotic stress response. Plant Genome 6(3):841–856. https://doi.org/10.3835/plantgenome2013.05.0013
Pang T, Ye CY, Xia X, Yin W (2013) De novo sequencing and transcriptome analysis of the desert shrub, Ammopiptanthus mongolicus, during cold acclimation using Illumina/Solexa. BMC Genomics 14:488. https://doi.org/10.1186/1471-2164-14-488
Peng PH, Lin CH, Tsai HW, Lin TY (2014) Cold response in Phalaenopsis aphrodite and characterization of PaCBF1 and PaICE1. Plant Cell Physiol 55(9):1623–1635. https://doi.org/10.1093/pcp/pcu093
Porto MS, Pinheiro MP, Batista VG, dos Santos RC, Filho PdeA, de Lima LM (2014) Plant promoters: an approach of structure and function. Mol Biotechnol 56(1):38–49. https://doi.org/10.1007/s12033-013-9713-1
Qin YX, Qin F (2016) Dehydrins from wheat x Thinopyrum ponticum amphiploid increase salinity and drought tolerance under their own inducible promoters without growth retardation. Plant Physiol Bioch 99:142–149. https://doi.org/10.1016/j.plaphy.2015.12.011
Rajam MV, Chandola N, Saiprasad-Goud PSD, Kashyap V, Choudhary ML, Sihachakr D (2007) Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants. Biol Plant 51:135–141. https://doi.org/10.1007/s10535-007-0026-8
Riyazuddin R, Nisha N, Singh K, Verma R, Gupta R (2022) Involvement of dehydrin proteins in mitigating the negative effects of drought stress in plants. Plant Cell Rep 41(3):519–533. https://doi.org/10.1007/s00299-021-02720-6
Rupert PB, Daughdrill GW, Bowerman B, Matthews BW (1998) A new DNA-binding motif in the Skn-1 binding domain-DNA complex. Nat Struct Biol 5(6):484–491. https://doi.org/10.1038/nsb0698-484
Saibi W, Feki K, Ben Mahmoud R, Brini F (2015) Durum wheat dehydrin (DHN-5) confers salinity tolerance to transgenic Arabidopsis plants through the regulation of proline metabolism and ROS scavenging system. Planta 242(5):1187–1194. https://doi.org/10.1007/s00425-015-2351-z
Shahmuradov IA, Solovyev VV, Gammerman AJ (2005) Plant promoter prediction with confidence estimation. Nucleic Acids Res 33(3):1069–1076. https://doi.org/10.1093/nar/gki247
Shi J, Liu M, Chen Y, Wang J, Lu C (2016) Heterologous expression of the dehydrin-like protein gene AmCIP from Ammopiptanthus mongolicus enhances viability of Escherichia coli and tobacco under cold stress. Plant Growth Regul 79:71–80. https://doi.org/10.1007/s10725-015-0112-4
Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23(6):1087–1088. https://doi.org/10.1093/nar/23.6.1087
Terzaghi WB, Cashmore AR (1995) Light-regulated transcription Annu RevPlant Physiol. Plant Mol Biol 46(1):445–474. http://dx.doi.org/10.1146/annurev.pp.46.060195.002305
Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16(9):2481–2498. https://doi.org/10.1105/tpc.104.022699
Urao T, Yamaguchi Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5(11):1529–1539. https://doi.org/10.1105/tpc.5.11.1529
Vazquez Hernandez M, Romero I, Escribano MI, Merodio C, Sanchez Ballesta MT (2017) Deciphering the role of CBF/DREB transcription factors and dehydrins in maintaining the quality of table grapes cv. autumn royal treated with high CO2 levels and stored at 0°C. Front Plant Sci 8:1591. https://doi.org/10.3389/fpls.2017.01591
Verma G, Dhar YV, Srivastava D, Kidwai M, Chauhan PS, Bag SK, Asif MH, Chakrabarty D (2017) Genome-wide analysis of rice dehydrin gene family: Its evolutionary conservedness and expression pattern in response to PEG induced dehydration stress. PLoS ONE 12(5):e0176399. https://doi.org/10.1371/journal.pone.0176399
Wang Y, Xu H, Zhu H, Tao Y, Zhang G, Zhang L, Zhang C, Zhang Z, Ma Z (2014) Classification and expression diversification of wheat dehydrin genes. Plant Sci 214:113–120. https://doi.org/10.1016/j.plantsci.2013.10.005
Wisniewski ME, Bassett CL, Renaut J, Farrell R Jr, Tworkoski T, Artlip TS (2006) Differential regulation of two dehydrin genes from peach (Prunus persica) by photoperiod, low temperature and water deficit. Tree Physiol 26(5):575–584. https://doi.org/10.1093/treephys/26.5.575
Wu Y, Wei W, Pang X, Wang X, Zhang H, Dong B, Xing Y, Li X, Wang M (2014) Comparative transcriptome profiling of a desert evergreen shrub, Ammopiptanthus mongolicus, in response to drought and cold stresses. BMC Genomics 15(1):671. https://doi.org/10.1186/1471-2164-15-671
Xu P, Hu RY, Ding XY (2006) Optimized adaptor polymerase chain reaction method for efficient genomic walking. Acta Biochim Biophys Sin 38(8):571–576. https://doi.org/10.1111/j.1745-7270.2006.00194.x
Xu S, An L, Feng H, Wang X, Li X (2002) The seasonal effects of water stress on Ammopiptanthus mongolicus in a desert environment. J Arid Environ 51(3):437–447. https://doi.org/10.1006/jare.2001.0949
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6(2):251–264. https://doi.org/10.1105/tpc.6.2.251
Yang Y, He M, Zhu Z, Li S, Xu Y, Zhang C, Singer SD, Wang Y (2012) Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress. BMC Plant Biol 12:140. https://doi.org/10.1186/1471-2229-12-140
Zhang ZL, Xie Z, Zou X, Casaretto J, Ho TH, Shen QJ (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol 134(4):1500–1513. https://doi.org/10.1104/pp.103.034967
Zhou L, Hao Y, Lu G, Wang P, Guo H, Cheng H (2020) Cloning and functional analysis of AmDUF1517 promoter from Ammopiptanthus mongolicus. J Biosci Bioeng 130(3):233–238. https://doi.org/10.1016/j.jbiosc.2020.04.014
Zhu W, Zhang D, Lu X, Zhang L, Yu Z, Lv H, Zhang H (2014) Characterisation of an SK n -type dehydrin promoter from wheat and its responsiveness to various abiotic and biotic stresses. Plant Mol Biol Rep 32(3):664–678. https://doi.org/10.1007/s11105-013-0681-1
Zolotarov Y, Strömvik M (2015) De novo regulatory motif discovery identifies significant motifs in promoters of five classes of plant dehydrin genes. PLoS ONE 10(6):e0129016. https://doi.org/10.1371/journal.pone.0129016
Zong W, Yang J, Fu J, Xiong L (2020) Synergistic regulation of drought-responsive genes by transcription factor OsbZIP23 and histone modification in rice. J Integr Plant Biol 62(6):723–729. https://doi.org/10.1111/jipb.12850

Table 1 is available in the Supplementary Files section.

Download PDF

Journal Publication

published 30 Apr, 2024

Read the published version in Gene →

Version 1

posted

You are reading this latest preprint version

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Isolation and amplification of the PrAmCIP promoter

Construction of reporter plasmids

Arabidopsis and tobacco transformation

Identification of transgenic plants and PCR analysis

Histochemical GUS staining

Protein extraction and fluorometric GUS assays

Abiotic stress and hormone treatment

Data analysis

Results

Isolation and sequence analysis of the PrAmCIP promoter

Vector construction of transgenic plants and PCR identification

Histochemical analysis of GUS activities in transgenic tobacco seedlings

Fluorometric analysis of GUS activities in transgenic tobacco

The GUS activity driven by three promoter deletion segments

Discussion

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1