Photosynthesis activates plasma membrane H+-ATPase via sugar accumulation in Arabidopsis ...

55 Pages·2016·3.34 MB·English

Plant Physiology Preview. Published on March 25, 2016, as DOI:10.1104/pp.16.00355
1 Short title: Photosynthesis activates H+-ATPase
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3 Correspondence author: Toshinori Kinoshita
4 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University,
5 Chikusa, Nagoya 464-8602, Japan
6 TEL/FAX: [phone]
7 E-mail: [email protected]
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9 Photosynthesis activates plasma membrane H+-ATPase via sugar
10 accumulation in Arabidopsis leaves
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12 Masaki Okumuraa, Shin-ichiro Inouea, Keiko Kuwatab, Toshinori
13 Kinoshitaa,b
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15 aDivision of Biological Science, Graduate School of Science, Nagoya
16 University, Chikusa, Nagoya 464-8602, Japan; bInstitute of Transformative
17 Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602,
18 Japan
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25 One sentence summary
26 Photosynthesis activates the plasma membrane H+-ATPase in Arabidopsis
27 mesophyll cells through C-terminus phosphorylation, and this activation is
28 mediated by photosynthetic sugars, including sucrose.
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30 Footnotes
31 This work was supported in part by Grants-in-Aid for Scientific Research from
32 the Ministry of Education, Culture, Sports, Science and Technology, Japan
33 (15H05956 and 15H04386 to T.K.) and by a Grant-in-Aid for JSPS fellows
34 (253307 to M.O.).
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36 Corresponding author; e-mail [email protected]
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38 The author responsible for the distribution of materials integral to the findings
39 presented in this article, in accordance with the Journal policy described in the
40 Instructions for Authors (http://www.plantphysiol.org), is: Toshinori Kinoshita
41 ([email protected]).
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43 Abstract
44 Plant plasma membrane H+-ATPase acts as a primary transporter via proton
45 pumping, and regulates diverse physiological responses by controlling
46 secondary solute transport, pH homeostasis, and membrane potential.
47 Phosphorylation of the penultimate threonine and the subsequent binding of
48 14-3-3 proteins in the C-terminus of the enzyme are required for H+-ATPase
49 activation. We showed previously that photosynthesis induces phosphorylation
50 of the penultimate threonine in the non-vascular bryophyte Marchantia
51 polymorpha. However, (i) whether this response is conserved in vascular plants
52 and (ii) the process by which photosynthesis regulates H+-ATPase
53 phosphorylation at the plasma membrane remain unresolved issues. Here, we
54 report that photosynthesis induced the phosphorylation and activation of
55 H+-ATPase in Arabidopsis leaves via sugar accumulation. Light reversibly
56 phosphorylated leaf H+-ATPase, and this process was inhibited by
57 pharmacological and genetic suppression of photosynthesis.
58 Immunohistochemical and biochemical analyses indicated that light-induced
59 phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We
60 also show that the phosphorylation status of H+-ATPase and photosynthetic
61 sugar accumulation in leaves were positively correlated, and that sugar
62 treatment promoted phosphorylation. Furthermore, light-induced
63 phosphorylation of H+-ATPase was strongly suppressed in a double mutant
64 defective in ADP-glucose pyrophosphorylase (AGPase) and triose
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65 phosphate/phosphate translocator (TPT) (adg1-1 tpt-2); these mutations
66 strongly inhibited endogenous sugar accumulation. Overall, we show that
67 photosynthesis activated H+-ATPase via sugar production in the mesophyll cells
68 of vascular plants. Our work provides new insight into signaling from
69 chloroplasts to the plasma membrane ion transport mechanism.
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70 Introduction
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72 Photosynthesis is the planet’s essential biochemical reaction. It converts the
73 photon flux of sunlight into the chemical energy required by nearly all organisms
74 on Earth. Plants produce carbohydrates and oxygen from carbon dioxide and
75 water through the photosynthetic process. Photosynthetic sugars are used not
76 only as energy sources but also as signaling molecules in plant life cycles
77 (Smeekens, 2000; Rolland et al., 2002, 2006; Lastdrager et al., 2014). Plant
78 sugar levels are influenced by biotic and abiotic stresses (Roitsch, 1999; Roitsch
79 and González, 2004). Plants sense these stresses by monitoring sugar levels
80 and adapt to ever-changing environments by regulating their metabolism, growth,
81 and development. Plant growth and development are also controlled by plant
82 hormones, and sugar signaling is closely coordinated with plant hormone
83 signaling (León and Sheen, 2003; Ljung et al., 2015).
84 Plasma membrane H+-ATPase, a crucial enzyme for plant life, acts as
85 a primary transporter in fungi and plants. It actively transports H+ to extracellular
86 spaces using the energy provided by ATP hydrolysis. This mechanism regulates
87 pH homeostasis and membrane potential, and creates a driving force for a
88 variety of solute transport processes operating via secondary transporters
89 (Palmgren, 2001). H+-ATPase is responsible for diverse physiological processes,
90 including nutrient uptake in roots, stomatal opening, phloem loading, and cell
91 expansion (Palmgren, 2001; Duby and Boutry, 2009). H+-ATPase is kept in a
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92 low-activity state by its C-terminus autoinhibitory domain; it is activated through
93 phosphorylation of a penultimate threonine and subsequent binding of 14-3-3
94 proteins to its phosphorylated C-terminus in response to physiological stimuli,
95 such as blue light in guard cells, the plant hormone auxin, and sucrose in
96 elongating tissues (Fuglsang et al., 1999; Kinoshita and Shimazaki, 1999;
97 Svennelid et al., 1999; Maudoux et al., 2000; Niittylä et al., 2007; Takahashi et
98 al., 2012). This process is probably the primary mechanism by which H+-ATPase
99 is activated.
100 Photosynthesis regulates ion transport across the plasma membrane
101 (Spanswick, 1981; Marten et al., 2010). Light-induced hyperpolarization of the
102 plasma membrane has been particularly well studied in a diversity of plants,
103 including Chara corallina and Vallisneria spiralis (Prins et al., 1980; Mimura and
104 Tazawa, 1986). Photosynthesis-dependent membrane hyperpolarization is
105 believed to result from activation of the plasma membrane H+-ATPase. In V.
106 gigantia, H+-ATPase is likely involved in photosynthesis-dependent membrane
107 hyperpolarization in the mesophyll cells (Harada et al., 2002). Our recent studies
108 showed that photosynthesis induces the phosphorylation of the penultimate
109 threonine of H+-ATPase in the non-vascular bryophytes Marchantia polymorpha
110 and Physcomitrella patens (Okumura et al., 2012a, 2012b). Although it is
111 supposed that photosynthesis induces activation of the H+-ATPase in large plant
112 species, it remains unknown whether photosynthesis-dependent
113 phosphorylation of H+-ATPase is conserved in vascular plants, and the
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114 signaling mechanism by which photosynthesis controls the phosphorylation
115 status of H+-ATPase is unclear.
116 In this study, we show that light illumination induces phosphorylation of
117 H+-ATPase in the mesophyll cells of Arabidopsis thaliana in a
118 photosynthesis-dependent manner. Furthermore, we demonstrate that (i)
119 exogenous and endogenous sugars induce phosphorylation of H+-ATPase and
120 (ii) the defect of carbohydrate production suppresses light-induced
121 phosphorylation of H+-ATPase. Our investigation shows that photosynthetically
122 produced sugar activates H+-ATPase through phosphorylation.
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123 Results
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125 Photosynthesis activates plasma membrane H+-ATPase in Arabidopsis
126 leaves via phosphorylation
127 To determine whether H+-ATPase is phosphorylated by light in the vascular plant
128 Arabidopsis, we illuminated detached dark-adapted leaves with white light. The
129 phosphorylation status of the penultimate threonine of H+-ATPase was detected
130 by immunoblot analyses using the antibody against the phosphorylated
131 penultimate threonine of H+-ATPase (anti-pThr) (Hayashi et al., 2010).
132 Illumination of dark-adapted leaves induced the phosphorylation of H+-ATPase
133 with no changes in the quantity of H+-ATPase (Fig. 1A). We also measured the
134 ATP hydrolytic activity of H+-ATPase in microsomal membranes isolated from
135 light-treated and dark-adapted leaves. ATP hydrolytic activity was 1.5-fold higher
136 in light-treated leaves than in dark-adapted leaves (Fig. 1B).
137 We subsequently examined the time courses of H+-ATPase
138 phosphorylation and dephosphorylation. The phosphorylation status of
139 H+-ATPase reached a peak after 30 min of illumination (Fig. 1C). The
140 phosphorylation status of the H+-ATPase gradually decreased after the
141 illumination period ended, and returned to the original status after ca. 120 min
142 (Fig. 1D). Thus, the H+-ATPase in Arabidopsis leaves was reversibly
143 phosphorylated and activated by light.
144 To determine whether photosynthesis regulates the phosphorylation of
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145 H+-ATPase, we examined the effect of inhibitors of photosynthetic electron
146 transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and
147 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on the
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148 light-induced phosphorylation of H+-ATPase. DCMU and DBMIB at a
149 concentration of 10 µM strongly inhibited light-induced phosphorylation of
150 H+-ATPase (Fig. 2A and Supplemental Fig. S1). To genetically corroborate our
151 findings, we examined the light-induced phosphorylation of H+-ATPase in the
152 yellow variegated2 (var2-2) mutant of Arabidopsis, which has variegated leaves
153 with no photosynthetic activity in its white sections (Fig. 2B; Kato et al., 2007;
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