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The tissue-cultured Lilium longiflorum cv. 'White heaven' was used in the experiments. Sterile lily seedlings were cultivated on MS medium in a standard culture room. Arabidopsis thaliana (Col-0) seeds were sterilized with 0.1% NaClO and 0.01% TritonX-100, and then sown on MS medium, followed by dark conditions at 4 °C for 3 d. After germination, the seedlings were transferred from MS plates to plastic pots of a sterile rooting mixture under controlled conditions (22/16 °C, 16-h/8-h light/dark).
Cloning of LlWRKY22 from lily
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We extracted total RNA from the heat-treated leaves by using an RNAprep Pure Kit (Tiangen, China). The leaves were sampled from the tissue-cultured 'White heaven' plants treated with 1-h heat stress at 37 °C. The cDNA was synthesized using a First Strand cDNA Synthesis Kit (R323-01, Vazyme, China). The open reading frame (ORF) of LlWRKY22 was isolated with special primers on the basis of the transcriptome data (Supplemental Table S1).
Abiotic stress treatments and gene expression assay of lily
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The 2-week-old, healthy, tissue-cultured lily seedlings with similar sizes were chosen for the treatments and gene expression assay. To analyze the expression patterns under high temperature conditions, the selected plants were exposed to 37 °C for various durations (0, 0.5, 1, 3, 6, 12 h) in a temperature-controlled incubator (Liance, China). For salt and mannitol treatments, different time course and concentration gradients were pre-tested, and then we transferred plants from the growth medium to 200 mM NaCl, 300 mM mannitol, or 10 μM ABA solution and put them at 22 °C for 3 h, deionized water served as control. Immediately after treatment, leaves were frozen in liquid nitrogen. Total RNA was extracted as described above, and reverse transcription was performed with a HiScript II kit (Vazyme, China). Real-time quantitative PCR (RT-qPCR) was applied to detect the expression levels. The 18S rRNA of lily was applied as an internal control. The primers are listed in Supplemental Table S2 for the RT-qPCR assay.
Isolation of LlWRKY22 promoter from lily
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Genomic DNA of lily leaves was extracted using a Plant Genprep DNA kit (Zomanbio, China). We isolated LlWRKY22 promoter through the Hi-tail PCR method[36,55]. The 1053-bp upstream fragment from the ATG of LlWRKY22 were cloned and identified from lily 'White heaven'.
Generation of LlWRKY22 transgenic Arabidopsis lines
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The LlWRKY22 ORF was cloned and inserted into pCAMBIA1300 vector driven by 35S promoter. Promoter of LlWRKY22 was cloned into pCAMBIA1391 vector containing a GUS (β-glucuronidase) reporter gene. The recombinant vectors were transformed, respectively, into 5-week-old Arabidopsis plants using the floral-dip method. The transformed seeds were screened by adding 30 mg·L−1 hygromycin to MS medium. We used RT-PCR to identity all transgenic lines, and selected three T3-generation homozygous lines for gene functional analysis. The primers used for vector constructions are shown in Supplemental Table S3.
GUS activity assay of LlWRKY22 promoter transgenic plants
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The LlWRKY22 transgenic seeds were sown on MS medium, and put in dark conditions at 4 °C for 3 d. After 5 d of germination, the seedlings were transplanted to vertically oriented MS medium for an additional 10 d. For heat stress, we put the seedlings at 37 °C for 3 h, and the untreated seedlings was used as a control. For NaCl, mannitol, and ABA treatments, the plants were submerged in 200 mM NaCl, 400 mM mannitol and 10 μM ABA solution for 3 h, and the controlled seedlings were cultured in sterile water. After treatment, the seedlings were collected and submerged in GUS staining solution for incubation at a temperature of 37 °C for 12 h. For the removal of chlorophyll, 70% ethanl was used.
Thermotolerance test of transgenic seedlings
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The thermotolerance test was performed similar to that described in previous studies[34,37,56]. Briefly, seedlings were vernalized on MS medium and grown at 22 °C in a standard culture room as previously described. For heat stress, the 5-day-old seedlings were treated with 1-h 45 °C treatment, and we recorded the survival rate after a period of 7-day of recovery under normal growth conditions.
Abiotic stress treatments of transgenic seedlings
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To investigate the effects on germination, different time courses and concentration gradients of salt, mannitol and ABA were pre-tested and appropriate treatments were selected. Wild-type and transgenic seeds were sown onto MS medium containing NaCl (0, 100, 200 mM), mannitol (0, 200, 400 mM), or ABA (0, 0.5, 1.0 μM), and their germination rates were recorded daily. To investigate the effects of NaCl, mannitol, and ABA on root growth, the 5-day-old seedlings were transferred to MS medium containing NaCl (200 mM), mannitol (300 mM), or ABA (5.0 μM). After 10 d of growth, a picture was taken and the root length of each plant was recorded.
Gene expression assay of transgenic Arabidopsis seedlings
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Gene expression analysis was performed with 5-day-old transgenic and wild-type seedlings. Their RNA extractions were performed as described above. The expression level of detected genes was determined by RT-qPCR. AtActin2 was used as a normalization control. The primers used for RT-qPCR are listed in Supplemental Table S2.
Statistical analysis
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GraphPad Prism 7.00 was used to analyze the experimental data and draw the diagrams. Data for P-value determinations and inference were analyzed by Student's t-test at a significant level of 0.05 or 0.01.
Data availability
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The data underlying this article will be shared on reasonable request to the corresponding author.
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About this article
Cite this article
Li T, Zhou T, Liang J, Zhang D, Teng N, et al. 2022. Overexpression of lily LlWRKY22 enhances multiple abiotic stress tolerances in transgenic Arabidopsis. Ornamental Plant Research 2:17 doi: 10.48130/OPR-2022-0017
Overexpression of lily LlWRKY22 enhances multiple abiotic stress tolerances in transgenic Arabidopsis
- Received: 05 September 2022
- Accepted: 30 September 2022
- Published online: 28 October 2022
Abstract: In our previous study, a heat-induced differentially expressed WRKY-IIe gene LlWRKY22 is isolated from lily (Lilium longiflorum), which acts as a positive role in thermotolerance, but whether it is involved in other stress responses is unknown. Here, the expression of LlWRKY22 was indicated to be positively influenced by heat, salt, or mannitol treatments, and its promoter activity was also enhanced after heat, salt, or mannitol treatments. In addition, LlWRKY22 responded to ABA treatment, which activated its expression and also increased the promoter activity. Overexpression of LlWRKY22 in Arabidopsis contributed to growth defects and early flowering. Simultaneously, compared with the wild type, the ABA sensitivity in transgenic lines was increased in both the germination stage and late growth stage. Further analysis showed that LlWRKY22 overexpression elevated the thermotolerance of transgenic plants and induced the expression of AtDREB2A, AtDREB2B, AtDREB2C, and AtJUB1. The salt and mannitol tolerances of the overexpression lines were also improved. Overall, our results illustrated that LlWRKY22 is affected by heat, salt, and osmotic stresses, and positively regulates heat, salt, and osmotic tolerances, which reveals that it acts as a generalist character responding to different abiotic stresses. And further to that, the regulatory pathway of LlWRKY22 also involves in ABA signaling.
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Key words:
- Lilium longiflorum /
- LlWRKY22 /
- Abiotic stress /
- ABA /
- Thermotolerance