NaCl–induced Proteomes of Tonoplast from the Leaves of a Halophyte Mesembryanthemum crystallinum L.

概要

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NaCl–induced Proteomes of Tonoplast from the
Leaves of a Halophyte Mesembryanthemum
crystallinum L.
Hoang Thi Kim HONG
Biotechnology Department, College of Medicine and Pharmacy, Duy Tan University

NAGAI, Terutaka
Fucalty of Agriculture, Saga University

AGARIE, Sakae
Laboratory of Plant Production Physiology, Division of Agrobiological Sciences, Department of
Bioresource Sciences, Faculty of Agriculture, Kyushu University

https://doi.org/10.5109/6796251
出版情報：九州大学大学院農学研究院紀要. 68 (2), pp.111-121, 2023-09. 九州大学大学院農学研究院
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J. Fac. Agr., Kyushu Univ., 68 (2), 111–121 (2023)

NaCl–induced Proteomes of Tonoplast from the Leaves of a Halophyte
Mesembryanthemum crystallinum L.
Hoang Thi Kim HONG1,2, Terutaka NAGAI3 and Sakae AGARIE*
Laboratory of Plant Production Physiology, Division of Agrobiological Sciences, Department of Bioresource Sciences,
Faculty of Agriculture, Kyushu University, Fukuoka 819–0395, Japan
(Received May 9, 2023 and accepted May 18, 2023)
This study aims to examine the characteristics of vacuole ATPase (V–ATPase) activities and protein
expression in vacuole fractions isolated from the common ice plant (Mesembryanthemum crystallinum
L.) grown under control and salt–stressed conditions, using various centrifugation conditions of sucrose
density gradient. The protein samples were analyzed using two–dimensional electrophoresis (2DE) gels.
The results reveal that the vacuolar fractions collected at 20%, 30%, and 40% (w/w) sucrose density in the
0 mM, 100 mM, and 400 mM NaCl–stressed plants, respectively, exhibited the highest V–ATPase activities.
Using the differential display with 2DE gels, the proteins in the vacuole fractions were analyzed, and the
abundant proteins on each gel were identified based on their isoelectric points and molecular weights. The
Arabidopsis database and the ExPASy–TagIdent tool were used to estimate the proteins. The results show
165, 194, and 199 abundant proteins with 41, 51, and 53 different proteins relating to the vacuole fractions
among control samples (0 mM NaCl) and salt–stressed samples at 100 mM NaCl and 400 mM NaCl, respectively. Although the proteins on each 2DE gel varied among the samples, they were generally focused on
ten physiological function groups, including pump proteins, transporter proteins, metal channel proteins,
peptide transport proteins, channel proteins, stress response proteins, protease proteins, autophagic proteins, stress response proteins, and unknown proteins.
Key words: Common ice plant, Proteome, Salt stress, Tonoplast

uptake of malate into the vacuole follows the electrochemical gradient of H+ between the inner and outer vacuole membrane. Therefore, vacuole ATPase activity,
which establishes the H+ gradient, is increased at night
and decreased during the day. Conversely, Na+ is also
taken up into the vacuole using the H+ gradient created
by the vacuole membrane ATPase, highlighting the need
to maintain ATPase activity high during both day and
night to prevent salt absorption (Epimashko et al., 2004,
2006). The vacuole membrane is expected to host various H+ requiring reactions simultaneously under salt
stress–induced CAM conditions. In the ice plant, under
CAM conditions, stomata close during the day to minimize water loss, whereas they open at night to uptake
CO2. The CO2 converts to malic acid, which is metabolized during the day, helping the plant to release CO2 and
use it for photosynthesis, a fundamental difference
between the CO2 fixation mechanisms of CAM, C3, and
C4 plants.
This unique mechanism allows CAM plants to adapt
and survive in arid lands with soil salinity. The induction
of CAM is reported to change the molecular weight of
vacuole membrane proteins (Bremberger and Lüttge,
1992). Interestingly, the ice plant possesses two types of
vacuoles with contrasting functions, salt storing and
malate cycling, which can co–exist in the same cells of
leaves (Epimashko et al., 2004). Although the study provided experimental evidence of the physiological roles of
the two separate vacuoles in the same ice plant cell, little
is known about the protein characteristics and their
functions on each vacuole under salt stress conditions
via proteome analysis with the 2DE technique.
In recent years, proteomic technologies based on
2DE analysis have been increasingly applied to the study

I N T RODUCTION
The vacuole plays a crucial role in plant growth and
development, serving a multitude of functions such as
storage and transport, intracellular environmental stability, and response to adverse stress environments. Plant
cells possess up to 10,000 different proteins, yet vacuolar–located proteins account for only a fraction (1%) of
the plant’s total proteins (Martinoia et al., 2018). In contrast to other organelles, such as mitochondria and chloroplasts, the isolation of plant vacuoles via traditional
methods is challenging and often results in fragile vacuoles (Robert et al., 2007). Hence, large–scale isolation
and identification of vacuoles, as well as the qualitative
and quantitative analysis of subcellular pathways, will
greatly enhance our understanding of the plant system
(Tan et al., 2019).
The common ice plant serves as an excellent model
species for examining the functional genomics, metabolomics, and proteomics of crassulacean acid metabolism
(CAM) plants. When grown under non–stressed conditions, this species exhibits C3 photosynthesis and completes its entire life cycle without displaying net nocturnal CO2 uptake. However, under salinity or drought
stress conditions, plants exhibit the physiological features of CAM plants (Cushman et al., 2008). CAM is
induced under salt stress, leading to the accumulation of
malic acid and NaCl in vacuoles during the night. The
Biotechnology Department, College of Medicine and Pharmacy,
Duy Tan University, Danang 550000, Vietnam
2
Institute for Research and Training in Medicine, Biology and
Pharmacy, Duy Tan University, Danang, 550000, Vietnam
3
Fucalty of Agriculture, Saga University, Saga 840–8502, Japan
* Corresponding author (agarie@agr.kyushu–u.ac.jp)
1

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H. T. K. HONG et al.

of plant organelle proteins, such as chloroplasts and
mitochondria, in Arabidopsis thaliana and many other
C3 and C4 photosynthesis species. However, successful
application of 2DE analysis to plant vacuoles remains
rare. For example, two proteomic analyses of the tonoplast used a membrane fraction isolated by sucrose density gradient and chromatographic separation, as
reported by Sazuka et al. (2004) and Szponarski et al.
(2004), but their results showed significant contamination from other membranes (Tan et al., 2019). On the
other hand, previous researchers have reported the isolation of intact vacuoles and proteomic analysis from
suspension–cultured cells or vacuoles of A. thaliana,
including Shimaoka et al. (2004), Jaquinod et al. (2007),
Carter et al. (2004), and Ohnishi et al. (2018). However,
most of these analyses have used SDS–PAGE and
Western blot or LC–MS/MS, but not 2DE techniques for
vacuolar proteome analysis. In previous work, we successfully applied the 2DE technique for tonoplast proteome analysis of two CAM species, Kalanchoe pinata
and Ananas comosus (Lin et al., 2008), as well as
simultaneous chloroplast and mitochondria proteome
analysis from the leaves of the ice plant under well–
watered and drought–stressed conditions (Hong et al.,
2019).
In this study, we report the characteristics of V–
ATPase activity and 2DE proteomic analysis of ice plant
control samples (0 mM NaCl) and salt–stressed samples
treated with 100 mM NaCl and 400 mM NaCl. We isolated and purified the ice plant vacuolar fractions from
leaf samples with varying concentrations of sucrose to
obtain the highest ATPase activities. We then measured
vacuolar–ATPase activities and conducted one–dimensional SDS–PAGE analysis together with 2DE analysis on
highly intact vacuoles. We identified differences in protein expression on each 2DE map under control and salt
stress conditions. Finally, we present a detailed analysis
of these proteins and their roles in each vacuole’s accumulation of Na+ and malate in the same cell of ice plant
under salt stress conditions.
M AT ER I A LS A N D M ET HODS
Plant materials and growth conditions
Seeds of the wild–type of the common ice plant were
germinated according to the methods described by
Sunagawa et al. (2007). The seeds were sterilized in
2.0% sodium hypochlorite solution for 7 min. After sterilization, seeds were put onto a germination medium
(pH 5.7) containing 4.6 g L–1 MS salts (Murashige and
Skoog, 1962), 1 × B5 vitamins (Gamborg et al., 1968),
30 g/L sucrose and 10 g/L agar. Seedlings were grown in
a growth chamber at 25ºC with 16h/8h (light/dark) photoperiod under a cool–white fluorescent light (70–
80 µmol m–2 s–1). The plants were grown hydroponically
in a greenhouse under natural sunlight, oscillating
between around 20ºC to 30ºC. When plants reached the
growth stage at which the 4th–6th leaf appeared, NaCl was
added to the culture solution to a final concentration of
0 mM, 100 mM and 400 mM as salt–stress treatment, and

the pH was maintained at around 5.75.
Sample collection
To ensure the grow–up time was enough for ice
plant to fully convert to CAM plants, approximately 0.5 g
of each leaf sample was used to determine the pH value.
The leaf samples were collected at 10 a.m., washed,
dried, and finely ground with a mortar and pestle. Then,
the pH of the leaf extract was measured with pH test
paper (pH 0–14) (Whatman). Most ice plant treated
with 400 mM NaCl became CAM plants after at least
14 days of salt treatment and their leaf extract solution
showed pH less than 5, while the control and 100 mM
NaCl treated samples were still C3 plants with a high pH
value. Based on this test, the leaf of the control samples
and salt–treated samples were collected after 14 days of
NaCl treatment. ...

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