Photochemistry and Photobiology, 20**, **: *–* Protein Phosphatase Activity and Acidic/Alkaline Balance as Factors Regulating the State of Phytochrome A and its Two Native Pools in the Plant Cell Vitaly Sineshchekov* 1 , Larissa Koppel 1 , Ekaterina Shor 1 , Galina Kochetova 1 , Paul Galland 2 and Mathias Zeidler 3 1 Biology Department, M.V. Lomonosov Moscow State University, Moscow, Russia 2 Department of Biology, Philipps-Universität Marburg, Marburg, Germany 3 Institute of Plant Physiology, Justus Liebig University, Giessen, Germany Received 1 May 2012, accepted 15 August 2012, DOI: 10.1111/j.1751-1097.2012.01226.x ABSTRACT Phytochrome A (phyA), the most versatile plant phyto- chrome, exists in the two isoforms, phyA′ and phyA′′, differ- ing by the character of its posttranslational modification, possibly, by phosphorylation at the N-terminal extension [Sineshchekov, V. (2010) J. Botany 2010, Article ID 358372]. This heterogeneity may explain the diverse modes of phyA action. We investigated possible roles of protein phosphatases activity and pH in regulation of the phyA pools’ content in etiolated seedlings of maize and their extracts using fluores- cence spectroscopy and photochemistry of the pigment. The phyA′/phyA′′ ratio varied depending on the state of develop- ment of seedlings and the plant tissue/organ used. This ratio qualitatively correlated with the pH in maize root tips. In extracts, it reached a maximum at pH  7.5 characteristic for the cell cytoplasm. Inhibition of phosphatases of the PP1 and PP2A types with okadaic and cantharidic acids brought about phyA′ decline and/or concomitant increase of phyA′′ in coleoptiles and mesocotyls, but had no effect in roots, revealing a tissue/organ specificity. Thus, pH and phosphory- lation status regulate the phyA′/phyA′′ equilibrium and con- tent in the etiolated (maize) cells and this regulation is connected with alteration of the processes of phyA′ destruc- tion and/or its transformation into the more stable phyA′′. Abbreviations: FR, far-red light; c 1 , extent of the phototrans- formation of the initial form of phytochrome into the first pho- toproduct; HIR, high-irradiance response; k a , k e , k max , wavelengths of the actinic and excitation light and of the maxi- mum of phytochrome fluorescence; LFR, low fluence response; lumi-R, the first photoproduct of the phototransfor- mation of phytochrome in its red form stable at 77–85 K; OA and CA, okadaic and cantharidic acid; phy, phytochrome; P tot , total phy content; Pr, red-light absorbing form of phy; Pfr, far-red-light absorbing form of phy; R, red light; PP1 and PP2A, protein phosphatases 1 and 2A; PKS1–PKS4, phytochrome kinase substrates 1–4; phyA and phyB, phyto- chromes A and B; phyA′ and phyA′′, subpopulations of phyA; VLFR, very low fluence response. INTRODUCTION One of the major steps forward in phytochrome (phy) research was the discovery of its structural and functional heterogeneity (see Ref. 1 for a review). The phytochrome family consists of a small number of phy members (phyA–phyE in Arabidopsis), the major ones being phyA and phyB. The light stable phyB shows classical characteristics of phy light sensing at low light fluences (the red-induced/far-red-reverted low fluence responses, LFR). phyA, which is the predominant phytochrome in etiolated seed- lings, whose major fraction is light labile, shows a quite different behavior: it promotes irreversible effects in the whole range of its absorption spectrum under very weak light (very low fluence responses [VLFR]) and under high fluence rates with the maxi- mum activity in the far-red region (high-irradiance responses, HIR). Along with these well established properties of phyA, it may perform as well the LFR functions characteristic for phyB (2–5). In particular, active phyA is translocated to the nucleus with the help of FHY1 and FHL, possibly via their red (R)/far- red (FR) light reversible phosphorylation (6–8). In the context of the different types of the phyA photoresponses, the differentia- tion between cytoplasmic and nuclear phyA functions is relevant (as reviewed in Ref. 9). Not all the phyA translocates to the nucleus, part of it remains active in the cytoplasm. Mediation of root phototropism by phyA together with phyB under R (10,11) and modification of gravi- and phototropism (12–15) can be attributed to the cytoplasmic fraction (16). These different functions and properties of phyA were explained by its heterogeneity (see Refs. 2,17–19). With the use of low-temperature fluorescence spectroscopy two phyA types, phyA′ and phyA′′, were detected in wild-type mono- and dicot- ous plants and their phy mutants. They differ by spectroscopic and photochemical properties, and their content depends on plant species and tissues and on physiological conditions. One of them, phyA′, predominates in growing tissues and is light labile. It has the emission (absorption) maximum at k max = 687 (673) nm at low temperatures, and is efficient in Pr ? lumi-R photoconversion at 77–85 K (Pr′ photochemical type). In con- trast, phyA′′ is a minor species more stable in the light and its concentration does not change significantly with tissue type. It has k max = 682–683 (668) nm and is ineffective in the Pr ? lumi-R conversion at low temperatures (Pr′′ photochemical *Corresponding author email: vsineshchekov@yahoo.com (Vitaly Sineshchekov) © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology 0031-8655/12 1