Simultaneous in situ detection of alkaline phosphatase activity and polyphosphate in arbuscules within arbuscular mycorrhizal roots
Rintaro Funamoto A , Katsuharu Saito B , Hiroshi Oyaizu A , Masanori Saito C and Toshihiro Aono A DA Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
B Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Nagano 399-4598, Japan.
C Department of Environmental Chemistry, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan.
D Corresponding author. Email: uaono@mail.ecc.u-tokyo.ac.jp
Functional Plant Biology 34(9) 803-810 https://doi.org/10.1071/FP06326
Submitted: 8 December 2006 Accepted: 6 June 2007 Published: 30 August 2007
Abstract
Inorganic phosphate (Pi) metabolism in arbuscules of arbuscular mycorrhizal (AM) fungi is not well understood, although recent research has revealed that host plants absorb Pi around arbuscules with mycorrhiza-specific transporters. Therefore, we analysed the localisation of polyphosphate (polyP) and alkaline phosphatase (ALP) activity in arbuscules, which could be indicators of Pi metabolism. We developed a dual-labelling method for polyP and ALP activity, i.e. first labelling with fluorescent probes 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI) and then labelling with enzyme-labelled fluorescence (ELF97). The dual-labelling method made it possible to observe polyP and ALP activity signals simultaneously in mycorrhizal roots. The dual-labelling method revealed that ALP activity was mainly observed in mature arbuscules where polyP was rarely observed. The expression of the AM fungal ALP gene was suppressed in the knockdown plants of an AM-inducible Pi-transporter, and there was much polyP in arbuscules that showed low ALP activity. These topological observations suggest that there may be some relationships between polyP metabolism and ALP activity in arbuscules, and that these are, in part, controlled by Pi uptake by plants via the AM-inducible Pi-transporter.
Additional keywords: Lotus japonicus, Glomus intraradices, fluorescent probe, dual-labelling method, RNA interference.
Acknowledgements
The authors thank D. Maeda and S. Hata (Kyoto University) for the kind gift of RNAi constructs and detailed technical advice on RNAi techniques. This study was supported, in part, by the Japan Society for the Promotion of Science (No. 17780244) (T.A.), and Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of the Bio-oriented Technology Research Advancement Institution, Japan (H.O. and T.A.).
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