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Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens

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Abstract

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens.

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Abbreviations

ABA:

Abscisic acid

ABI:

ABA-insensitive

ABRE:

ABA-response element

DRE:

Drought-responsive element

GUS:

β-Glucuronidase

JA:

Jasmonic acid

LEA:

Late embryogenesis abundant

MPK:

Mitogen-activated protein kinase

PP2C:

Protein phosphatase 2C

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Acknowledgments

We thank Dr. Tuan-hua David Ho and Dr. Jose Casaretto for providing DNA constructs of 35S-abi1-1 and Ubi-LUC, and Dr. Daisuke Takezawa for technical assistance with the freezing experiments and for his valuable advice. This work was supported by a JSPS Postdoctoral Fellowship for Research Abroad to Y.S., the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Young Scientists (B) to Y.S., and the Science Research Promotion Fund (2007) by the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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Authors and Affiliations

  1. Department of Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan

    Kenji Komatsu, Yuri Nishikawa, Tomohito Ohtsuka, Teruaki Taji, Shigeo Tanaka & Yoichi Sakata

  2. Department of Biology, Washington University, One Brookings Drive, St. Louis, MO, 63130-4899, USA

    Ralph S. Quatrano

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  1. Kenji Komatsu
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  2. Yuri Nishikawa
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  3. Tomohito Ohtsuka
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  4. Teruaki Taji
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  7. Yoichi Sakata
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Correspondence to Yoichi Sakata.

Additional information

Accession Numbers: PpABI1A-AB369256, PpABI1B-AB369255, pphn39k21-AB369257.

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Table S1

Primers used. Supplementary material 1 (DOC 53 kb)

Fig. S1

Comparison of methods for measurement of protonemata growth. a Wild type protonemata were grown for 2 weeks on BCDAT medium, which contained various concentration of ABA. b Growth of protonemal colonies shown in (a) was determined by the image analysis software (black bars), or chlorophyll contents (white bars). Values are means ± SE (n = 21). Scale bars: 10 mm. Supplementary material 2 (EPS 7582 kb)

Fig. S2

The exon–intron structures of the ABI1, PpABI1A and PpABI1B genes. Untranslated regions, coding regions, and introns are represented by open boxes, filled boxes and horizontal thick lines, respectively. The arrowhead indicates the position of the Gly residue mutated in the abi1-1 allele. Numbers show the nucleotide positions from the 5′ end of the cDNAs. Supplementary material 3 (EPS 565 kb)

Fig. S3

Comparison of the amino acid sequences of ABI1, PpABI1A and PpABI1B. Amino acid sequences of ABI1, PpABI1A and PpABI1B were aligned using the T-COFFEE program (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi). Identical amino acids are marked with asterisks (*), strongly similar amino acids are marked with two dots (:), and weakly similar amino acids are marked with one dot (·). Colors indicate alignment quality in a regional context. The PP2C domain is underlined in black. The arrowheads indicate two Gly residues in the catalytic domain of ABI1 that correlated with regulation of ABA signaling. Supplementary material 4 (EPS 3126 kb)

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Komatsu, K., Nishikawa, Y., Ohtsuka, T. et al. Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens . Plant Mol Biol 70, 327–340 (2009). https://doi.org/10.1007/s11103-009-9476-z

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