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Loss of Arabidopsis Halotolerance 2-like (AHL), a 3′-phosphoadenosine-5′-phosphate phosphatase, suppresses insensitive response of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant to abiotic stress

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Abstract

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Destruction of PAP phosphatase AHL suppresses atrzf1 phenotype in abiotic stress responses. AHL plays an intermediate role in the regulation of proline accumulation by PAP nucleotidase.

Abstract

Proline (Pro) metabolism is important for environmental responses, plant development, and growth. However, the role of Pro in abiotic stress process is unclear. Using atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for T-DNA tagging mutagenesis, we identified a suppressor mutant designated as proline content alterative 17 (pca17) that suppressed insensitivity of atrzf1 to abiotic stresses during early seedling growth. Pro content of pca17 was lower than that in both wild type (WT) and atrzf1 while complementary lines were less sensitive to abscisic acid (ABA) and abiotic stresses compared to WT. Thermal Asymmetric Interlaced (TAIL)-PCR of pca17 showed that T-DNA was inserted at site of At5g54390 (AHL for Arabidopsis Halotolerance 2-like) encoding 3′-phosphoadenosine-5′-phosphate (PAP) phosphatase. Under drought stress condition, products of sulfate metabolism such as PAP and adenosine monophosphate were significantly lower in pca17 than those in WT and atrzf1. Furthermore, pca17 showed significantly higher levels of several important drought parameters including malondialdehyde, ion leakage, and water loss than WT and atrzf1. Fluorescence signal of green fluorescent protein (GFP)-tagged AHL was quite strong in nuclei of the root and guard cells of transgenic seedlings. Additionally, AHL promoter-β-glucuronidase (GUS) construct revealed substantial gene expression in vasculature tissues and pollen. Collectively, these findings demonstrate that pca17 acts as a dominant suppressor mutant of atrzf1 in abiotic stress response by modulating proline and sulfate metabolism.

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Abbreviations

ABA:

Abscisic acid

GFP:

Green fluorescent protein

PAP:

3′-Phosphoadenosine-5′-phosphate

qPCR:

Quantitative real-time polymerase chain reaction

RT-PCR:

Reverse transcription-PCR

WT:

Wild-type

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Acknowledgements

This work was supported in part by a grant to C.S.K. from the Next-Generation BioGreen21 program (SSAC, PJ013171) funded by the Rural Development Administration and by the Basic Science Research Program funded by the Ministry of Education, Science and Technology of Korea (2018R1D1A1B07045242).

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Authors and Affiliations

  1. Department of Plant Biotechnology, Chonnam National University, Gwangju, 61186, Republic of Korea

    Da-Jeong Shin, Ji-Hee Min, Tinh Van Nguyen & Cheol Soo Kim

  2. Department of Food Science & Technology, Chonnam National University, Gwangju, 61186, Republic of Korea

    Young-Min Kim

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  1. Da-Jeong Shin
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Contributions

We thank Dr. Y-MK for technical assistance with HPLC analysis. CSK designed experiments and interpreted results. DJS, JHM, and TVN carried out experiments and interpreted the results.

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Correspondence to Cheol Soo Kim.

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Supplementary Table S1

Primers used for TAIL-PCR, qPCR, RT-PCR, and gene cloning (DOCX 18 KB)

Supplementary Fig. S1

Germination of pca17 mutant following abiotic stress. a Seeds of samples (WT, atrzf1, and pca17) were sown on MS medium and permitted to grow for 8 days. Photograph indicates that seedling growth and green cotyledons among WT, atrzf1, and pca17 plants were similar. b-d Seeds were sown on MS agar plates supplemented with 400 mM mannitol (b), 1 μM ABA (c), or 6 mM H2O2 (d). Complete emergence of the radicle indicates germination. It was scored on the indicated days. Data represent mean ± standard deviation values of three independent experiments (n = 50 of each). Error bars represent standard deviations. Different letters above bars indicate statistically significant difference (ANOVA, P < 0.05) (TIF 908 KB)

Supplementary Fig. S2

Cotyledon greening of pca17 mutant following abiotic stress and ABA treatment. Seeds were sown on MS agar plates supplemented with 400 mM mannitol (a), 1 μM ABA (b), or 6 mM H2O2 (c) and cultured for 6-8 days. Photographs were taken on day 6 for dehydration and ABA or on day 8 for oxidative stress. Decreased development and fewer green cotyledons were observed in pca17 compared with those in WT and atrzf1 under stress conditions (TIF 3074 KB)

Supplementary Fig. S3

Reduced tolerance of pca17 mutants to drought stress. a Drought sensitivity of pca17 mutants. Two-week-old WT, atrzf1, and pca17 plants were subjected to drought stress by withholding water for 10 days and then re-watering for 3 days. b Survival rates of WT, atrzf1 and pca17 plants were measured after re-watering. Error bars represent standard deviations. Different letters above bars indicate statistically significant difference (ANOVA, P < 0.05) (TIF 2683 KB)

Supplementary Fig. S4

Alignment of full-length deduced amino acid sequences of AHL and structural features of the AHL protein. a Sequences of Arabidopsis AHL (At5g54390), SAL1 (At5g63980), SAL2 (At5g64000), SAL3 (At5g63990), and SAL4 (At5g09290). Black and grey shading indicate identical and similar amino acids, respectively. Dashes indicate gaps introduced to optimize alignment. b Structure of the conserved motif region in AHL. Light gray, gray, and black boxes indicate active domain (41-310 aa), lithium-binding domain (144-310 aa), and substrate-binding domain (SBD) (257-310 aa), respectively (http://myhits.isb-sib.ch). c Phylogenetic tree depicting homology relationships among Arabidopsis thaliana AHL members (http://www.megasoftware.net). Branch length represents substitutions per site (TIF 1593 KB)

Supplementary Fig. S5

Abiotic stress response in complementation transgenic plants. aAHL expression levels in WT, atrzf1, pca17, three independent complementation transgenic lines (Com4, Com6, and Com9), three independent ahl RNAi (ri2-3, ri5-2, and ri6-7), and three independent AHL-overexpressing lines (OE2-2, OE5-1, and OE7-7) were confirmed by qPCR using RNA extracted from 8-day-old seedlings. Actin 1 (ACT1) was used as an internal qPCR control. Error bars indicate standard deviations from three independent experiments (n = 7 of each). Different letters above bars indicate statistically significant difference (ANOVA, P < 0.05). b-e Seeds were sown on MS agar medium without additive (b) or with 400 mM mannitol (c), 1 µM ABA (d), or 6 mM H2O2 (e) and grown for 8 days after germination. Photographs show that atrzf1, three independent complementation, and three independent AHL-overexpressing lines exhibit better development and green cotyledons than WT, pca17 and three independent ahl RNAi plants under dehydration, ABA, and oxidative stress conditions (TIF 2065 KB)

Supplementary Fig. S6

AHL expression in Arabidopsis plants under abiotic stress conditions. qPCR analyses showing expression levels of AHL, RAB18, and APX1 in response to abiotic stress. All quantifications were made in three independent RNA samples obtained from plants treated with 100 µM ABA (a), 400 mM mannitol (b), or 6 mM H2O2 (c) for the indicated times. Error bars indicate standard deviations from three independent experiments conducted. Each experiment was performed with total RNA obtained from ten pooled seedlings of each line (WT, atrzf1 and pca17). Arabidopsis Actin 1 was used as an internal control. Different letters above bars indicate statistically significant difference (ANOVA, P < 0.05) (TIF 302 KB)

Supplementary Fig. S7

Nuclear localization of AHL in transgenic Arabidopsis plants. a Five-day-old transgenic plants grown on MS agar medium were analyzed for GFP expression by confocal microscopy. AHL-GFP green fluorescent signal was observed in the nuclei of root cells. b Ten-day-old transgenic plants grown on MS agar medium were analyzed for GFP expression in guard cells by confocal microscopy. AHL-GFP green fluorescent signal was relatively strong in the nuclei of guard cells. GFP, green fluorescent protein; B/W, black and white. Scale bars = 200 µm. This experiment was performed in duplicate. Five individual seedlings were analyzed per experiment and similar results were obtained. c Immunoblot analysis of transgenic plants producing AHL-GFP fusions. Plant extracts were prepared from 10-day-old seedlings, 20 µL of cell lysate was loaded in each lane, and AHL-GFP (arrow) was detected with an anti-GFP monoclonal antibody. WT, wild-type plant; AHL-GFP, Arabidopsis expressing AHL-GFP. Molecular masses (kDa) of protein standards are shown on the left. Total proteins were separated by SDS-PAGE and electrophoretically transferred onto nylon membranes followed by Ponceau S staining (TIF 1595 KB)

Supplementary Fig. S8

AHL promoter-GUS expression pattern in transgenic Arabidopsis plants. a GUS staining for a 7-day-old seedling plant. b GUS staining results are shown in a flower of a 4-week-old transgenic plant. GUS expression was observed in the vascular system of leaves or hypocotyl, the anther, the filament, and the vein of the petal. c-f Ten-day-old seedlings of AHL promoter-GUS transgenic plants were subjected to H2O (c), 400 mM mannitol (d), 50 µM ABA (e), or 6 mM H2O2 (f). After 12 h of abiotic stress exposure, seedlings were stained for GUS expression. H2O-treated seedlings were used as the control (c). This experiment was repeated twice. Each GUS staining assay was analyzed from five individual seedlings per experiment and similar results were obtained (TIF 1116 KB)

Supplementary Fig. S9

Proline contents in WT, atrzf1, pca17 and AHL transgenic plants under drought stress. Light-grown 5-week-old plants were grown for 7 days without watering. Proline contents were measured from drought-treated leaves. Values of Pro content represent the average of three independent experiments. Each assay was performed with extracts obtained from ten seedlings of each plant. Error bars represent standard deviations. Different letters above bars indicate statistically significant difference (ANOVA, P < 0.05) (TIF 122 KB)

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Shin, DJ., Min, JH., Van Nguyen, T. et al. Loss of Arabidopsis Halotolerance 2-like (AHL), a 3′-phosphoadenosine-5′-phosphate phosphatase, suppresses insensitive response of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant to abiotic stress. Plant Mol Biol 99, 363–377 (2019). https://doi.org/10.1007/s11103-019-00822-0

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