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Prolyl-4-hydroxylase (AtP4H1) mediates and mimics low oxygen response in Arabidopsis thaliana

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Abstract

Proline hydroxylation is an important phenomenon of a living cell. Prolyl-4-hydroxylases (P4H) responsible for this process have been characterized from animals, and one of its forms, HIF-P4H, is regarded as an oxygen sensor. In plants, P4H has been partially characterized from few species, and one of the Arabidopsis P4H (AtP4H1) has been shown to hydroxylate proline-rich peptides in vitro. In order to study its function in planta, we have overexpressed AtP4H1 in Arabidopsis. The AtP4H1oexp plants showed hypoxia-in-normoxia phenotype with strict requirement for carbon source for its growth, increased root hair, absence of trichome, and reduction in seed size. Genome-wide expression analyses suggest that expression of several genes related to hypoxia as well as plant growth and development are upregulated in AtP4H1oexp lines. Based on our studies on AtP4H1oexp lines, we speculate a direct role of AtP4H1 in hypoxia stress and in different stages of plant growth and development.

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Acknowledgement

Authors are thankful to Director, National Botanical Research Institute, Lucknow for providing necessary facilities for carrying out this work. The authors thankfully acknowledge the financial support provided by Council of Scientific and Industrial Research, Government of India.

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Authors and Affiliations

  1. Plant Gene Expression Laboratory, National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow, 226 001, India

    Mehar Hasan Asif, Prabodh Kumar Trivedi, Prashant Misra & Pravendra Nath

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  1. Mehar Hasan Asif
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  2. Prabodh Kumar Trivedi
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  3. Prashant Misra
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  4. Pravendra Nath
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Corresponding authors

Correspondence to Prabodh Kumar Trivedi or Pravendra Nath.

Electronic supplementary material

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Supplementary Fig. S1

Expression of OsP4H1 in response to hypoxia. Three-week-old rice (Oryza sativa L., Indica cultivar IR64) seedlings were connected to continuous nitrogen supply and samples were collected after 30 min. Numbers above the panel correspond to time of samples collection during nitrogen supply in hours. Expression of ubiquitin genes were used as internal controls for semi-quantitative RT-PCR analysis (PDF 5 kb)

Supplementary Fig. S2

a–c Expression of anaerobic induced genes in AtP4H1oexp lines. Expression of hypoxia responsive genes by qRT-PCR in 2-week-old wild-type and AtP4H1oexp line seedlings (a) Adh alcohol dehydrogenase; Pdc1 pyruvate decarboxylase1; Pdc2 pyruvate decarboxylase2; and Sus1 sucrose synthase. in planta ADH activity staining in wild-type and AtP4H1–3oexp line. Wild-type plants were connected to nitrogen supply for 30 min before staining. Minimum of 20 plants from WT and AtP4H1–3oexp lines were taken for staining and representative pictures are shown in the figure. In gel staining of the ADH activity of the above samples (c). Data are expressed as means ± SD of at least three independent replicates (PDF 50 kb)

Supplementary Fig. S3

The normalization of arrays using three independent methods, i.e., probe logarithmic intensity error (PLIER), robust multiarray average (RMA), and GCRMA using ArrayAssist software (Stratagene, La Jolla, CA, USA). To identify statistically significant differentially expressed genes, a combined criterion of twofold or more change and t test p < 0.05 was adopted. Only common probe sets which were present in three different analyses were considered significant (PDF 32 kb)

Supplementary Fig. S4

Pathways affected by AtP4H1 overexpression as analyzed using MapMan software. Total microarray data was utilized to generate the pathways. Genes for which expression has been affected in AtP4H1 overexpressing lines are shown in pathway. Glycolysis, TCA, and ETC (a) and sugar metabolism (b) pathways are shown in the figure. Of the total genes involved in the pathway up- and downregulated genes are shown by red and blue boxes. White boxes represent for which expression was unaffected in the AtP4H1 overexpressing lines (PDF 71 kb)

Supplementary Fig. S5

Expression profiles of anaerobic genes (a) and trichome-related genes (b) in AtP4H1oexp lines. The color scale (representing one signal log ratio) is shown at the bottom. Differentially expressed genes between our data and as shown by Loreti et al. (2005) were utilized to construct heat map for (a) using one signal log ratio (PDF 45 kb)

Supplementary Fig. S6

In silico analysis of AtP4H1 expression in wild-type plants. The expression profile in different tissue was analyzed using the Genevestigator Gene atlas tool. AtP4H1 expression is high in actively growing tissues, like callus, suspension cells, root elongation zone, and endodermis (PDF 23 kb)

Supplementary Fig. S7

In silico analysis of putative P4H target proteins. PPPPP was used as query to search AGI proteins using BLASTP program. A total of 100 hits were obtained of which GO functional characterization was carried out at TAIR (PDF 3 kb)

Table S1

List of primers used for RT-PCR and qRT-PCR analyses (DOC 34 kb)

Table S2

List of probe sets significantly downregulated twofold in AtP4H1 overexpressing lines (XLS 91 kb)

Table S3

List of probe sets significantly upregulated twofold in AtP4H1 overexpressing lines (XLS 124 kb)

Table S4

Differentially regulated genes associated with various functions (XLS 135 kb)

Table S5

List and fold change of up- and downregulated genes in AtP4H1oexp lines related to hypoxia. Differentially expressed genes between our data and as shown by Loreti et al. (2005) were utilized in the table (XLS 22 kb)

Table S6

List of differentially expressed genes in AtP4H1oexp lines involved in the trichome formation (DOC 26 kb)

Table S7

List of microarray experiments showing the maximum variation in AtP4H1 expression. The list was generated using the gene swinger tool on NASCArrays (http://affymetrix.arabidopsis.info/) (XLS 24 kb)

Table S8

Putative targets of AtP4H1 (XLS 45 kb)

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Asif, M.H., Trivedi, P.K., Misra, P. et al. Prolyl-4-hydroxylase (AtP4H1) mediates and mimics low oxygen response in Arabidopsis thaliana . Funct Integr Genomics 9, 525–535 (2009). https://doi.org/10.1007/s10142-009-0118-y

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