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Expression Profile Analysis of Hypoxia Responses in Arabidopsis Roots and Shoots

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Abstract

Physiological and molecular adaptation mechanisms enable plants to improve their survival under harsh conditions, including low oxygen levels caused by flooding. When Arabidopsis was exposed to hypoxia, we observed hyponastic response, shoot elongation, leaf chlorosis, and inhibited growth. To understand this response, we used a specialized complementary DNA microarray from our laboratory to examine the time-dependent profiles of gene expression in Arabidopsis roots and shoots. From this, we identified 282 hypoxia-responsive genes. These included novel genes for a zinc finger protein, WRKY family transcription factor, and glycosyl hydrolase as well as those previously identified as hypoxia-related genes including alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and phosphofructokinase. Cluster analysis of these profiles suggested that the hypoxia response occurs in two distinctive phases: early and late. The early response to imposed stress (hours 1, 3, and 8) includes increased expression of fermentation-related genes and transcription factors, such as by members of the C2H2 zinc finger family and WRKY family. The late response (hours 24 and 72) involves the down-regulation of genes that function in secondary metabolic pathways and up-regulation of transcription factors that are mostly related to the ethylene-responsive element binding protein family. Mutants of Arabidopsis defective in sucrose synthase1 (SUS1), the At1g05060 gene (with unknown function), ADH, and the WRKY33 were more sensitive to hypoxic stress, evidence of the importance of these genes in that response. The genes presented here allow us to deepen our understanding of the mechanism for this stress response and, eventually, will aid in the development of more flood-tolerant crops.

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Acknowledgments

This research was supported by grants from the National Research Foundation of Korea (NRF; project no. 20100016253), the Research Cooperating Program for Agricultural Science & Technology Development, RDA (project no. 201004010300390010700), the Korea Research Foundation, funded by the Korean Government (MOEHRD; project no. 2006-0508-6-7), and the National Research Foundation of Korea (NRF), funded by the Korea Government (MEST; project no. 2011-0006244).

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Authors and Affiliations

  1. Graduate Department of Life and Pharmaceutical Sciences and the Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, 120-750, South Korea

    Ji Hye Hwang, Yoon-Hi Choy & Dong-Hee Lee

  2. Department of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, 151-742, South Korea

    Mi Ok Lee & Choo Bong Hong

  3. Institute for Scientifically Able Youth, Kyungwon University, Seongnam, 461-701, South Korea

    Young-Mie Ha-Lee

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  1. Ji Hye Hwang
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Correspondence to Dong-Hee Lee.

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Supplemental Table

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Supplemental Figure 1

Molecular characterization of sus1, At1g05060 knockout mutant, adh, and wrky33 used in this study. a Schematic illustration of T-DNA insertion site in the knockout mutants. Genomic organization of SALK_014303 line carrying T-DNA insertions in the SUS1 (At5g20830), genomic organization of SALK_034347 line carrying T-DNA insertions in At1g05060 gene with an unknown function, genomic organization of SALK_066824 line carrying T-DNA insertions in the ADH (At1g77120), and genomic organization of SALK_064436 line carrying T-DNA insertions in the WRKY33 (At2g38470). Triangle indicates the position of T-DNA insertion; black box, exon; gray box, untranslated region; the line, intron. b RT-PCR analysis of each gene in WT and T-DNA insertion mutant lines. Total RNA was isolated from whole tissue of 2-week-old plants that had been exposed to hypoxia (0.1% O2/99.9% N2) for 1 h. RT-PCR was performed with each gene-specific primer (Supplemental Table 1), and UBQ10 was used as a control (DOC 212 kb)

Supplemental Figure 2

Confirmation of expression of the five hypoxia-responsive genes by RT-PCR. Two-week-old plants were exposed to hypoxia (5% O2/95% N2) for 1–72 h in darkened vacuum chamber at 23°C. The isolated total RNA (2 μg) in roots was reverse transcribed and used for PCR analysis. The transcripts for the target genes (ADH, PDC, SUS1, ETR2, and UBQ10) were detected using specific primers (Supplemental Table 1). The UBQ10 was used as a control. Relative expression level was a log2 ratio by microarray data (DOC 227 kb)

Supplemental Figure 3

Expression level of SUS1, At1g05060 gene, ADH, and WRKY33 under hypoxic stress. Relative expression level was a log2 ratio from microarray data (DOC 64 kb)

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Hwang, J.H., Lee, M.O., Choy, YH. et al. Expression Profile Analysis of Hypoxia Responses in Arabidopsis Roots and Shoots. J. Plant Biol. 54, 373–383 (2011). https://doi.org/10.1007/s12374-011-9172-9

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