Pergamon

0031-9422(95)00272-3

Phytochemistry, Vol. 40, No. 4, pp. 1065-1070, 1995
Copyright © 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0031-9422/95 $9.50 + 0.00

LIPIDS A N D FATTY ACIDS IN GUARD-CELL PROTOPLASTS FROM VICIA
FABA LEAVESTAKESHI SAKAKI,*t ATUSHI SATOH,* KIYOSHI TANAKA,3~KENJI OMASAJ~and KEN-ICHIRO SHIMAZAKI§* Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Minami-ku, Sapporo 005, Japan;:~Environmental Biology Division, The National Institute for Environmental Studies, Onogawa, Tsukuba 305, Japan; §Biological
Laboratory, College of General Education, Kyushu University, Ropponmatsu, Fukuoka 810, Japan(Received 10 January 1995)
Key Word Index--Vicia faba; Leguminosae; broad bean; guard-cell protoplasts; mesophyll-cell
protoplasts; polar lipids; neutral lipids; fatty acids.

Abstract--Guard-cell and mesophyll-cell protoplasts were isolated from Viciafaba leaves, and their lipids and fatty
acids quantitatively analysed. All the glycerolipids and fatty acids that occurred in mesophyll-cell protoplasts were
found in guard-cell protoplasts. On a total fatty acid basis, levels of chloroplast lipids (monogalactosyldiacylglycerol,
digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol) and phosphatidylglycerol were lower and their constituent fatty acids were more saturated in guard-cell protoplasts than in mesophyll-cell ones. In contrast, levels of
extrachloroplastic lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin)
were higher in guard-cell protoplasts than in mesophyll-cell protoplasts. These results suggest that extrachloroplastic
membrane systems, including mitochondria, are more developed than chloroplast membranes. In addition, guard-cell
protoplasts contained a large amount of triacylglycerols, a probable component of spherosomes, lipid droplets and/or
plastoglobuli, which are frequently observed in guard cells.

INTRODUCTION
Stomatal aperture, which is controlled by volume
changes in guard cells, regulates gas-exchange between
a plant and the environment. Stomatal opening whichcorresponds to the swelling of guard cells, is induced by
the accumulation of K+, its closing corresponding toshrinking of the cells, being caused by release of K +. To
understand the mechanisms of volume regulation in
guard cells, many studies have been carried out on thepumps and channels in the plasma membranes and on
the activities of photosynthesis and respiration that provide energy for this ion transport 1,1-3].
It is clear that the physiological and biochemical properties of guard cells are quite different from those of
mesophyll cells. Guard cells have a higher activity of
respiration than their mesophyll counterparts on a volume basis; respiration provides the main energy source
required for stomatal movement I-4, 5]. Furthermore,guard cells develop signal transduction systems and can
respond to various kinds of environmental stimuli, suchas light, growth regulators (e.g. abscisic acid and auxin),
air pollutants, C O 2 and Ca 2 + [1, 3].
Despite extensive investigations of the physiology andbiochemistry on guard cells, there are only a few reportson the chemical analysis of their cellular components.

tAuthor to whom correspondence should be addressed.

Recently, protein profiles of guard-cell protoplasts subjected to SDS-PAGE, which differs largely from that ofmesophyll-cell protoplasts, have been shown I-6]. With
respect to the lipid profile of guard cells, Sato [7] has
shown lipid biosynthesis from acetate in guard-cell protoplasts from Vicia; however, he failed to quantify eachlipid class because of the low yields obtained from theprotoplasts.
In order to achieve a better understanding of thestructural and functional characteristics of guard cells,
we have determined the content and composition of thefatty acids and glycerolipids in guard-cell and mesophyll-cell protoplasts isolated from leaves of Viciafaba.

RESULTSAND DISCUSSION

Evaluation of #uard-cell and mesophyll-cell protoplasts
Guard-cell protoplasts were isolated from Vicia epidermis peeled from 80-120 leaves. The isolated protoplasts yielded 5-9 × 106 cells in each experiment. Microscopic observations showed that no mesophyll cells andless than 1% of epidermal cells were found in the protoplast preparations on the basis of cell number (Fig. 1).
Mesophyil-cell protoplasts were isolated from 10-20
leaves of Vicia. There was no contamination in the protoplast preparations by other cell-types (Fig. 1).
Isolated guard-cell and mesophyll-cell protoplasts hadhigh activities of photosynthetic 02 evolution with the1065

T. SAKAKIet al.

1066
GCPs

MCPs

protoplasts on both volume and chlorophyll bases. Thissuggests that guard cells have more dense intracellular
membranes and/or richer storage lipids than mesophyll
cells on a volume basis.
The content of protein in guard-cell protoplasts was ca
one-sixth that of mesophyll-cell protoplasts on a cellbasis (Table 1); the concentration of protein in guard cells
was also higher than their mesophyll counterparts.

Composition of fatty acids in guard-cell protoplasts
30 Ixm

100 ~ mFig. 1. Photomicrographs of guard-cell protoplasts (GCPs) andmesophyll-cell protoplasts (MCPs) isolated from Vicia fabaleaves. Guard-cell protoplasts were observed under a differential
interference microscope. Note the difference in size between the
protoplasts.

rates of 200-250 and 100-150 #mol mg71 chlorophyll
hr-1, respectively, under red light illumination. Proton
pumping was elicited in guard-cell protoplasts by a pulseof blue light (200/~mol m - 2 sec- 1 for 30 sec) under the
background red light (800#molm -2 sec -1) and theirmagnitudes were ca 0.3 nmol/~g- 1 protein pulse- 1.
These values are close to those in activities and magnitudes as previously reported in these cell-types from
Vicia [8-10].

Content of fatty acids and protein in guard-cell protoplasts
The content of total fatty acids in guard-cell protoplasts was found to be one-quarter of that in mesophyllcell protoplasts on a cell basis (Table 1). Since guard-cell
protoplasts from Vicia have a much smaller volume(ca 1/20) and a much lower content of chlorophyll(ca 1/80) than mesophyll-ceU protoplasts on a cell basis[4], the content of total fatty acids is calculated to bericher in guard-cell protoplasts than in mesophyll-cell

Among the peaks detected in the gas chromatogram of
the fatty acid methyl esters obtained from guard-cell
protoplasts, palmitic acid (16:0), stearic acid (18:0), oleic
acid (18: 1), linoleic acid (18:2), ~-linolenic acid (18:3),
arachidic acid (20:0), icosenoic acid (20:1), icosadienoic
acid (20:2), icosatrienoic acid (20:3), behenic acid (22:0)
and iignoceric acid (24"0) were identified by comparison
with R~ on GC and the patterns of mass spectra of thestandard esters. We also identified two other peaks as
hexadecenoic acids (16: 1) from their mass spectra, thatwere further confirmed to be 3-trans-16" 1 and 9-cis-16:1
by comparison with the corresponding authentic standards. Furthermore, the total ion chromatogram from
GC-MS showed the occurrence of docosenoic acid
(22: 1), albeit in trace amounts, in guard-cell protoplasts.
Table 2 presents the molar composition of fatty acidsin the lipid extracts from guard-cell and mesophyll-cell
protoplasts. Two differences were found. Firstly, C16 andCls fatty acids were more saturated in guard-cell protoplasts than in mesophyll-cell ones. The amount of 18:3,
which is abundant in chloroplast galactolipids of mesophyll cells [11], was particularly low in guard-cell
protoplasts, but was mainly compensated for by increases in the amounts of 18:1 and 18:2. Secondly, thecontents of long-chain fatty acids, C2o, C22 and C24
were higher in guard-cell protoplasts. This is consistent
with the active biosynthesis of 20" 0 and 22: 0 from acetate in guard-cell protoplasts, as well as in epidermal-cellones I-7]. These fatty acids are probably involved in thesynthesis of cuticular wax components [12].

Table 1. Content of fatty acids and protein in guard-cell and mesophyll-cell protoplasts from
Vicia faba leaves

Volume (ml 10 -9 cells)*
Chl (mg 10-9 cells)*
Protein (rag 10-9 cells)
Total fatty acids (#mo110-9 cells)
Total fatty acids (#mot ml-t)
Total fatty acids (#mol mg-1 Chl)
Total fatty acids (#mol mg- 1 protein)

Guard-cell
protoplasts

Mesophyll-cell
protoplasts

2.14
2.0
220
440
206
220
2.0

49.0
158
1390
1760
35.9
11.1
1.3

Whole
leaves
m

m

m

ll.l
0.77

*Data from Ref. [4].
Content of total fatty acids represents the sum of 16: 0, 16: 1, 18: 0, 18 : l, 18: 2, 18: 3, 20:0, 20: 1,
20: 2, 20: 3, 22:0 and 24:0 in lipid extracts. Values are averages of results from 4-7 separate
samples in each case. Chl, chlorophyll.

1067

Lipids and fatty acids in guard-cell protoplasts from Vicia faba
Table 2. Fatty acid composition in total lipid extracts of guard-cell and mesophyll-cell protoplasts from
Vicia faba leaves
Fatty acid composition (mol %)Samples

16:0

16:1" 18:0

18:1

18:3 20:0

20:1

20:2

20:3

22:0 24:0

GCPs
MCPs
Whole leaves

15.6
8.2
9.3

1.3
4.5
4.1

11.7 31.1 29.9 1.5
4.5
9.6 70.9 0.3
6.0 13.7 63.9 0.4

1.5
0.2
0.3

0.5
<0.1
0.1

0.3
<0.1
< 0.1

0.8
0.2
0.3

5.6
1.6
1.7

18:2

0.3
<0.1
< 0.1

*Sum of 3-trans-16:1 and 9-cis-16: I (see text).
From GC-MS measurements, we also identified 22:1 in total lipids from guard-cell protoplasts (less than0.1%). Values are averages of results from 5-8 separate samples in each case. GCPs, guard-cell protoplasts;
MCPs, mesophyll-cell protoplasts.
A

Chloroplast lipids in guard-cell protoplasts
Figure 2 shows the content of polar and neutral lipids
in guard-cell and mesophyll-cell protoplasts; it is expressed on a total fatty acid basis (Table 1) in order tocompare the distribution of lipids. All the glycerolipids
that generally occur in green leaves [11] were detectedin both types of protoplasts, though their contents differed. The amounts of monogalactosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG) and sulphoquinovosyldiacylglycerol (SQDG), specific to chloroplast membranes, were lower in guard-cell protoplasts
than in mesophyll-cell ones. The content of phosphatidylglycerol (PG), which is associated mainly withchloroplasts [13], was also lower in guard-cell protoplasts. Since the content of total fatty acids was lower in
guard-cell protoplasts than in their mesophyll counterparts on a cell basis (Table l), these results suggest that
guard cells contain fewer numbers of chloroplasts and/or
poorer development of thylakoids. Electron microscopic
observations confirm these morphological characteristics
of guard cells [14]. Higher degrees of fatty acid saturation in these lipid classes (Table 3) might be related topoorly developed thylakoids. On a volume basis, however, the content of chloroplast lipids is calculated to be
similar or rather higher in guard-cell protoplasts than inmesophyll-cell ones (cfi Table 1 and Fig. 2A).

Extrachloroplastic lipids in ffuard-cell protoplasts
In contrast to the chloroplast lipids, contents of theextrachloroplastic lipids, phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylinositol(PI) and cardiolipin (CL), were found to be higher in
guard-cell protoplasts than in mesophyll-cell ones
(Fig. 2A). This suggests that guard cells contain extrachloroplastic organelle(s) in larger number and/or withmore highly developed membrane systems. The higher
amount of CL is noteworthy (Fig. 2A). Bligny and Douce[15] have reported that CL is restricted to inner membranes of mitochondria in cultured sycamore cells. This isalso probably the case with Vicia guard cells, since morphological observations show the occurrence of numerous mitochondria in guard cells to be a common feature
in various plant species, including Vicia [ 14]. In addition,

150!-

IlGcP+

~,
0

a
100 O

E

_[
50oo

~_
-~
0

I'+' r--7:.,c,+ |]11
I •~;W.ole/leaves]++"°°FI

I

+11
5ohm

d

++:ll

ii

o

O

O

<

Fig. 2. Content of individual (A) polar lipids, and (B) neutral
lipids in guard-cell protoplasts (GCPs) and mesophyll-cell protoplasts (MCPs) isolated from Vicia faba leaves. Mean value_+s.d. of results from three separate samples is given in eachcase. PA, phosphatidic acid; DG, sum of 1,2-diacylglycerol and1,3-diacylglycerol. See text for other abbreviations.

1068

T. SAKAKIet al.
Table 3. Fatty acid composition of major glycerolipids in guard-cell and mesophyll-cell protoplasts from Viciafaba
leavesFatty acid composition (mol %)LipidsSamples

16:0

16:1

18:0

18:1 18:2

18:3 20:0 20:1

20:2

20:3

22:0 24:0

MGDG

GCPs
MCPs
GCPs
MCPs
GCPs
MCPs
GCPs
MCPs
GCPs
MCPs
GCPs
MCPs
Whole leaves

2.1
1.2
1.4 0.4
7.9
1.2
9.4
tr
42.8 10.1"
14.4 41.4"
18.0
tr
18.8
tr
22.2
tr
24.3
1.6
16.3
3.1
11.6
4.8
20.6
7.8

1.0
0.3
5.2
1.3
3.2
1.4
5.3
3.3
5.0
3.7
3.5
2.3
5.5

5.2
0.9
2.7
1.0
11.0
4.3
22.1
21.9
16.9
17.2
16.1
10.3
15.9

79.4
95.0
76.5
86.8
13.3
29.7
18.5
28.2
12.1
17.0
26.8
52.1
23.5

0
0
0
0
0
0
0.3
0.2
1.0
0.6
0.3
0.5
0

0
0
0
0
0
0
0.2
0.2
0.7
0.4
0.2
0.1
0

0
0
0
0
0.1
0
0.5
0.4
1.0
0.5
1.1
0
0.3

DGDG
PG
PC
PE
TG

11.2
2.1
6.5
1.4
19.1
8.8
32.0
25.1
36.4
32.3
29.7
17.8
24.8

0
0
0
0
0.2
tr
1.5
0.8
1.2
1.1
1.7
0.2
0.8

0
0
0
0
0
0
1.4
0.6
2.9
1.1
1.0
0.3
0.4

0
0
0
0
0.2
0
0.2
0.5
0.6
0.2
0.4
0.1
0.4

*3-trans- 16:1.
GCPs, guard-cell protoplasts; MCPs, mesophyll-cell protoplasts; tr, trace (less than 0.1%).

the high respiratory activities of Vicia guard-cell protoplasts have been demonstrated, viz, a high rate of KCNsensitive 02 uptake and high activity of cytochromeoxidase [4]. Portions of PC, PE and PI may participate
in the membrane constituents of the mitochondria.
As shown in Table 3, fatty acid species and their composition of PC and PE in guard-cell protoplasts werecomparable to those in mesophyll-cell ones. However, wenote that amounts of long-chain fatty acids (C2o, C2~ andC24) were higher in these phospholipids of guard-cell
protoplasts; these acids were rarely detected in thechloroplast lipids of both protoplasts (Table 3). AbdulKarim et al. [16] have shown that acetate is actively
incorporated into long-chain fatty acids of PC and neutral lipids in leek epidermal cells. Murata et al. [17],however, have shown that these acids are restricted tophosphatidylserine in various organs of 18 plant species.
Thus, fatty acid analysis of phosphatidylserine will provide more detailed distribution of long-chain saturated
and unsaturated fatty acids in guard cells; we have confirmed the occurrence of this phospholipid in guard-cellas well as mesophyll-cell protoplasts (results not shown).
Recent investigations have shown that exogenous inositol triphosphate induces Ca 2÷ release in the cytoplas'm of guard cells from Commelina, triggering the closing of stomata [18, 19], and that 1,2-diacyiglyceroi (1,2DG) activates the plasma membrane H ÷ pump, leading
to stomatal opening [20]. If these compounds take partin the signal transduction of intact leaves as second
messengers, polyphosphoinositides should occur and be
actively turned over in guard cells by the analogy withthose in animal cells [21]. As shown in Fig. 2A, we founda considerable amount of PI in guard-cell protoplasts
from Vicia. However, we could not detect phosphatidylinositol mono- or bisphosphate (both lipids to beless than 0.5 nmol/~mol - 1 total fatty acid) on TLC plates
using sprays of primuline, H2SO4 (both for general lipid

detection) or molybdenum blue (phospholipid detection).
This is probably due to the limitations of the sensitivity of
our analytical techniques; radiotracer experiments will beneeded for more precise determination of these minor but
important phospholipids.
Neutral lipids in guard-cell protoplasts
Guard-cell and mesophyll-cell protoplasts were foundto contain larger amounts of triacylglycerol (TG) thanwhole leaves (Fig. 2B). Increases in TG in mesophyll-cell
protoplasts are probably due to the enhanced productionof TG during the isolation procedure [22]. We haverecently proposed that TG is produced in leaf cells undervarious stresses as a result of the enzymatic conversion
from M G D G via 1,2-DG and free fatty acid (FFA), withconcomitant production of oligogalactolipids, such astri- and tetragalactosyldiacylglycerol (TGDG and
TTGDG) [23, 24]. Since the procedure for protoplast
isolation appears to be a strong stress factor for plant
cells, we could expect TG in mesophyll-cell protoplasts tobe produced via this pathway [23]. As shown in Table 4,
T G D G and T T G D G did accumulate in mesophyll-cell
protoplasts but not in whole leaves, demonstrating theoperation of the pathway from M G D G to TG during
isolation of the protoplasts.
In contrast, we failed to find these oligogalactolipids inguard-cell protoplasts (Table 4). This suggests that TG
is the original lipid constituent in guard cells. Since
spherosomes, lipid droplets and plastoglobuli are often
observed in guard cells [14, 25], TG may at least be
partly located in these organelles.
Since TG generally function as an energy reserve inplant and animal cells, it is likely that TG in guard cells
is utilized for a substrate of mitochondrial respiration
through fl-oxidation. In accord with this, guard-cell protoplasts survive much longer than mesophyll-cell proto-

1069

Lipids and fatty acids in guard-cell protoplasts from ViciaJdba
Table 4. Relationships between contents of triacylglycerols (TGI and oligogalactolipids in guardcell protoplasts, mesophyll-cell protoplasts and whole leaves of Viciafaba
Lipid content(nmol #mol- 1 total fatty acid)
Samples

TG

MGDG

DGDG

TGDG

TTGDG

Guard-cell protoplasts*
Mesophyll-cell protoplasts*
Whole leaves

94.5
26.7
2.7

27.3
82.3
87.1

25.0
60.7
57.0

nd
2.4
nd

nd
2.7
nd

*Guard-cell protoplasts had blue light-dependent H+-pumping activity of 0.29 nmol pg-~ protein pulse ~. Mesophyll-cell protoplasts had photosynthetic O2-evolving activity of ca
100/tmol mg- ~ chlorophyll hr- 1.
Guard- and mesophyll-cell protoplasts were isolated from the same leaves. Values are averages ofresults from two separate samples in each case. nd, not detected (less than 0.2 nmol pmol ~ total
fatty acid).
plasts from the same plant species in the dark [26].
Recent investigations indicate that guard-cell chloroplasts have an extended longevity in yellow and senescentleaves, even when mesophyll chloroplasts are degraded
completely, and that stomata have a capacity to open
and close in these leaves [27]. Thus, it is possible thatTG acts as an effective energy source for respirationand provides energy for stomatal movement in senescing
leaves.
EXPERIMENTAL

Plant materials and protoplast preparation. Plants ofVicia faba were grown hydroponically from seeds ina greenhouse for 4-8 weeks [10]. Guard-cell and mesophyll-cell protoplasts were enzymatically isolated fromthe abaxial epidermis of 80-120 leaves [10] and theremaining 10-20 leaves devoid of epidermis [8], respectively. Isolated guard-cell and mesophyll-cell protoplasts
were washed twice with 0.4 M mannitol containing
1 mM CaC12 and 0.6 M mannitol containing 1 mM
CaCI2, respectively. Protoplasts were finally suspended
in 0.5 ml of the same composition of washing medium.
Lipid extraction. Immediately after protoplast prepn,
their suspensions (0.4 ml) were mixed with 4 ml of boiling
iso-PrOH, followed by boiling the mixt. until most of the
iso-PrOH had evapd. Whole leaves of Vicia faba weresimilarly treated with boiling iso-PrOH. Total lipids wereextracted from the boiled residues according to Ref. [28].
To examine the occurrence of polyphosphoinositides,
total lipids were extracted from guard-cell protoplasts
with CHC13-MeOH 3.4 N aq. HCI (6:6: 1) according toRef. [29], followed by the partitioning in aq. and organic
phases [28]. The organic phase containing polyphosphoinositides was recovered, concd under N2, dissolvedin 5 ml of CHCI3 containing 0.05% (w/v) butylated hydroxytoluene as an antioxidant and stored at - 20 ° untiluse [30].
Separation and quantification of lipids. Neutral lipids
were sepd by TLC on silica gel developed withhexane-Et20 HOAc (70:30: 1). Polar lipids, except for
oligogalactolipids and polyphosphoinositides, were sepd

by 2D TLC on silica gel using CHC13-MeOH-H20(65:25:4) in the first and C H C I 3 - M e 2 C O - M e O H H O A c - H 2 0 ( 1 0 : 4 : 2 : 2 : l ) in the second dimension;
CHCI3-MeOH-conc. NH3-isopropylamine (13:7:1:0.1)
in the second dimension was also used. Oligogalactolipids (TGDG and TTGDG) were sepd according to Ref.
[23], and polyphosphoinositides were sepd by TLC on
potassium oxalate-impregnated silica gel as described inRef. [31]. Spots were visualized and identified according
to Ref. [30]. In some expts, spots were charred by spraying with 50% HzSO4 and heating at 150 °. Fatty acid Me
esters from the individual lipids and the total lipid exts
were prepd according to Ref. [30] with 15:0 as int.
standard and quantified by F I D - G C [32]. When the Me
ester frs were contaminated with pigments, they were
purified by TLC before the analysis by GC [32].
To identify fatty acid species in the total lipids ofguard-cell protoplasts by GC-MS, fatty acid Me esters
were chromatographed on a fused silica capillary column(60 m × 0.25 mm i.d., SP-2330; Supelco) with He as carrier; the column temp. was prog. at 175-215 ° for
2 ° min- ~. MS were recorded at an ionization voltage of
70 eV.
Photosynthetic and proton-pumpin 9 activities. Rates ofphotosynthetic 02 evolution in guard-cell and mesophyll-cell protoplasts were determined according toRefs [4] and [8], respectively. Proton-pumping activities
in guard-cell protoplasts were measured as described inRef. [10].
Content of chlorophyll and protein. Chlorophyll was
extracted with 80% aq. Me2CO and determined spectrophotometrically [33]. Protein was measured according
to Ref. [34] with BSA as standard.

Acknowledgements--The authors thank Dr M. Yamada(Hokkaido Tokai University) for valuable discussions
during the course of the work.
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