Plant Molecular Biology

, Volume 74, Issue 4, pp 423–435

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

  • Greg Clark
  • Michael Wu
  • Noel Wat
  • James Onyirimba
  • Trieu Pham
  • Niculin Herz
  • Justin Ogoti
  • Delmy Gomez
  • Arinda A. Canales
  • Gabriela Aranda
  • Misha Blizard
  • Taylor Nyberg
  • Anne Terry
  • Jonathan Torres
  • Jian Wu
  • Stanley J. Roux
Article

DOI: 10.1007/s11103-010-9683-7

Cite this article as:
Clark, G., Wu, M., Wat, N. et al. Plant Mol Biol (2010) 74: 423. doi:10.1007/s11103-010-9683-7

Abstract

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H2O2, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H2O2 signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H2O2 in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.

Keywords

Root hair Growth Extracellular nucleotide Apyrase Nitric oxide Reactive oxygen species 

Supplementary material

11103_2010_9683_MOESM1_ESM.tif (351 kb)
Fig. S1 a Threshold for root hair growth inhibition by applied ATPγS is between 100 and 150 μM. b Threshold for promotion of root hair growth by applied ATPγS is between 5 and 15 μM in pre-washed roots. Differentletters above the bars indicate mean values that are significantly different from one another (P < 0.05; n ≥ 30). The data above are representative of three or more biological repeats (TIFF 350 kb)
11103_2010_9683_MOESM2_ESM.tif (498 kb)
Fig. S2 a NONOate promotes root hair growth at 50 μM and higher concentrations. b cPTIO inhibits root hair growth at 20 μM and higher concentrations. c Treatment of seedlings with 1 μM cPTIO alone has no effect on the average root hair growth rate, but co-treatment with 200 μM ADPβS blocks the ability of 200 μM ADPβS to inhibit root hair growth. d ODQ inhibits root hair growth at 50 μM and higher concentrations. e Treatment of seedlings with 25 μM ODQ alone has no effect on the average root hair growth rate, but co-treatment with 20 μM ADPβS blocks the ability of 20 μM ADPβS to stimulate root hair growth in pre-washed roots. Differentletters above the bars indicate mean values that are significantly different from one another (P < 0.05; n ≥ 30). The data above are representative of three or more biological repeats (TIFF 497 kb)
11103_2010_9683_MOESM3_ESM.tif (498 kb)
Fig. S3 a A dose-response curve shows that treatment with various concentrations of H2O2 results in promotion of root hair growth at 0.1 μM and inhibition of growth at 10 μM. b Treatment with 1 μM DPI alone has no effect on the average root hair growth rate, but co-treatment with 20 μM ADPβS blocks the ability of 20 μM ADPβS to promote root hair growth after a wash treatment. Differentletters above the bars indicate mean values that are significantly different from one another (P < 0.05; n ≥ 30). The data above are representative of three or more biological repeats (TIFF 497 kb)
11103_2010_9683_MOESM4_ESM.tif (2.9 mb)
Fig. S4 Images of root hair tips of wild-type, nia1nia2 and atrbohD/F mutants untreated and treated with low and high ADPβS. a Wild-type control root hairs. b Wild-type root hairs of pre-washed roots treated with 20 μM ADPβS. c Wild-type root hairs treated with 200 μM ADPβS. dnia1nia2 control root hairs. enia1nia2 root hairs of pre-washed roots treated with 20 μM ADPβS. fnia1nia2 root hairs treated with 200 μM ADPβS. gatrbohD/F control root hairs. hatrbohD/F root hairs of pre-washed roots treated with 20 μM ADPβS. iatrbohD/F root hairs treated with 200 μM ADPβS. Scalebars = 100 μm (TIFF 2982 kb)
11103_2010_9683_MOESM5_ESM.tif (2.3 mb)
Fig. S5 Detection of DAF-FM 2D staining in wild-type and nia1nia2 root hairs untreated or treated with low or high [ATPγS]. a Fluorescence image of untreated wild-type root hairs (control). b Fluorescence image of pre-washed wild-type roots treated with 20 μM ATPγS. c Fluorescence image of wild-type root hairs treated with 200 μM ATPγS. d Wild-type root hairs viewed under light microscopy (control). e Root hairs of pre-washed wild-type roots treated with 20 μM viewed under light microscopy. f Wild-type root hairs treated with 200 μM ATPγS viewed under light microscopy. g Fluorescence image of untreated nia1nia2 root hairs (control). h Fluorescence image of pre-washed nia1nia2 roots treated with 20 μM ATPγS. i Fluorescence image of nia1nia2 root hairs treated with 200 μM ATPγS. jnia1nia2 root hairs viewed under light microscopy (control). k Root hairs of pre-washed nia1nia2 roots treated with 20 μM ATPγS viewed under light microscopy. lnia1nia2 root hairs treated with 200 μM ATPγS viewed under light microscopy. Scalebars = 100 μm (TIFF 2380 kb)
11103_2010_9683_MOESM6_ESM.tif (2.3 mb)
Fig. S6 Detection of H2DCFDA staining in wild-type and atrbohD/F root hairs untreated or treated with low or high [ATPγS]. a Fluorescence image of untreated wild-type root hairs (control). b Fluorescence image of root hairs from pre-washed wild-type roots treated with 20 μM ATPγS. c Fluorescence image of wild-type root hairs treated with 200 μM ATPγS. d Wild-type root hairs viewed under light microscopy (control). e Root hairs of pre-washed wild-type roots treated with 20 μM viewed under light microscopy. f Wild-type root hairs treated with 200 μM ATPγS viewed under light microscopy. g Fluorescence image of untreated atrbohD/F root hairs (control). h Fluorescence image of root hairs from pre-washed atrbohD/F roots treated with 20 μM ATPγS. i Fluorescence image of atrbohD/F root hairs treated with 200 μM ATPγS. jatrbohD/F root hairs viewed under light microscopy (control). katrbohD/F root hairs from pre-washed roots treated with 20 μM viewed under light microscopy. latrbohD/F root hairs treated with 200 μM ATPγS viewed under light microscopy. Scalebars = 100 μm (TIFF 2379 kb)
11103_2010_9683_MOESM7_ESM.tif (268 kb)
Fig. S7 a Pre-treatment and co-incubation of root hairs with cPTIO effectively blocks the DAF-FM 2D fluorescence induced by ATPγS. b Pre-treatment and co-incubation of root hairs with NAC effectively blocks the H2DCFDA fluorescence induced by ATPγS (TIFF 267 kb)

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Greg Clark
    • 1
  • Michael Wu
    • 1
  • Noel Wat
    • 1
  • James Onyirimba
    • 1
  • Trieu Pham
    • 1
  • Niculin Herz
    • 1
  • Justin Ogoti
    • 1
  • Delmy Gomez
    • 1
  • Arinda A. Canales
    • 1
  • Gabriela Aranda
    • 1
  • Misha Blizard
    • 1
  • Taylor Nyberg
    • 1
  • Anne Terry
    • 1
  • Jonathan Torres
    • 1
  • Jian Wu
    • 1
  • Stanley J. Roux
    • 1
  1. 1.Section of Molecular Cell and Developmental BiologyUniversity of TexasAustinUSA

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