Plastid Osmotic Stress Activates Cellular Stress

Responses in Arabidopsis1[C][W][OPEN]
Margaret E. Wilson, Meera R. Basu, Govinal Badiger Bhaskara, Paul E. Verslues,
and Elizabeth S. Haswell *
Department of Biology, Washington University, Saint Louis, Missouri 63130 (M.E.W., M.R.B., E.S.H.); and
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11529 (G.B.B., P.E.V.)

Little is known about cytoplasmic osmoregulatory mechanisms in plants, and even less is understood about how the osmotic
properties of the cytoplasm and organelles are coordinately regulated. We have previously shown that Arabidopsis (Arabidopsis
thaliana) plants lacking functional versions of the plastid-localized mechanosensitive ion channels Mechanosensitive Channel of
Small Conductance-Like2 (MSL2) and MSL3 contain leaf epidermal plastids under hypoosmotic stress, even during normal
growth and development. Here, we use the msl2 msl3 mutant as a model to investigate the cellular response to constitutive
plastid osmotic stress. Under unstressed conditions, msl2 msl3 seedlings exhibited several hallmarks of drought or environmental
osmotic stress, including solute accumulation, elevated levels of the compatible osmolyte proline (Pro), and accumulation of the
stress hormone abscisic acid (ABA). Furthermore, msl2 msl3 mutants expressed Pro and ABA metabolism genes in a pattern
normally seen under drought or osmotic stress. Pro accumulation in the msl2 msl3 mutant was suppressed by conditions that
reduce plastid osmotic stress or inhibition of ABA biosynthesis. Finally, treatment of unstressed msl2 msl3 plants with exogenous
ABA elicited a much greater Pro accumulation response than in the wild type, similar to that observed in plants under drought or
osmotic stress. These results suggest that osmotic imbalance across the plastid envelope can elicit a response similar to that elicited
by osmotic imbalance across the plasma membrane and provide evidence for the integration of the osmotic state of an organelle
into that of the cell in which it resides.

Proper cytoplasmic osmolarity is essential for cell

function and viability, and multiple osmoregulatory
mechanisms serve to adjust cellular solute contents in
response to the extracellular environment (Zhu, 2002;
Wood, 2011). Many organisms respond to limited water
availability by increasing cellular solute content, thereby
driving water uptake or preventing water loss (Zhang
et al., 1999; Burg and Ferraris, 2008). In plant systems,
solute accumulation is required to maintain cell turgor,
an important force that underlies plant structure and
drives cell shape and expansion. Therefore, plants are
an excellent model system in which to study the basic
mechanisms of osmoregulation (Verslues and Sharma,
2010). Furthermore, osmoregulation is important in
plants exposed to drought and high salinity, which are
major stresses capable of reducing growth and yield of
crop species (Boyer, 1982). Despite their fundamental
1
This work was supported by the National Science Foundation
(grant nos. MCB0816627 and MCB1253103 to E.S.H.) and an Academia Sinica Career Development Award (to P.E.V.).
* Address correspondence to ehaswell@wustl.edu.
The author responsible for distribution of materials integral to the
ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Elizabeth S. Haswell (ehaswell@wustl.edu).
[C]
Some gures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OPEN]
Articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.114.236620

and applied importance, relatively little is known about

plant osmoregulatory mechanisms.
A key aspect of cellular osmotic adjustment is the
increased production of solutes that do not disrupt
normal biological processes (referred to as compatible
osmolytes). One of the most widely used compatible
osmolytes is the highly water-soluble a-amino acid Pro
(Burg and Ferraris, 2008; Verslues and Sharma, 2010;
Wood, 2011). Drought, salinity, and freezing, all of which
reduce water availability to the cell, have been shown to
induce Pro accumulation (in some cases, in excess of 100fold; Delauney and Verma, 1993; Yoshiba et al., 1997; Xin
and Browse, 1998). In the model owering plant Arabidopisis (Arabidopsis thaliana), stress-induced accumulation of Pro is primarily attributed to increased levels of
the biosynthetic enzyme pyrroline-5-carboxylate synthetase1 (P5CS1) and reduced expression of the catabolic
enzyme Pro dehydrogenase (PDH1) in most plant tissues
(Verbruggen and Hermans, 2008; Verslues and Sharma,
2010).
The mechanism by which P5CS1, PDH1, and other
Pro metabolic enzyme activities are regulated by hyperosmotic stress is not completely understood, but the
stress hormone abscisic acid (ABA) has long been implicated in the process. ABA accumulates to high levels
during drought stress and can induce P5CS1 expression, thereby increasing Pro levels (Verbruggen and
Hermans, 2008; Verslues and Sharma, 2010). However,
osmotic stress represses PDH1 expression in an ABAindependent manner and is capable of overriding the
Pro-dependent induction of PDH1 expression observed

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Wilson et al.

in the absence of stress (Miller et al., 2005; Verslues and

Sharma, 2010). Furthermore, ABA applied to unstressed
plants only induces a low level of Pro accumulation; full
induction requires both ABA and drought (low water
potential) stress (Verslues and Sharma, 2010). These
data indicate that the existence of a low water potentialspecic signal is needed for the cell to respond to ABA and
accumulate osmotically signicant levels of Pro (Sharma
and Verslues, 2010; Verslues and Sharma, 2010). The nature of this stress-specic signal has not been identied.
Even less is known about osmotic balance across
organellar membranes. Comparison with bacterial systems has been particularly useful in the case of plastids,
the plant-specic endosymbiotic organelles responsible
for photosynthesis as well as the production of fatty
acids, amino acids, and numerous other essential compounds (Lopez-Juez and Pyke, 2005). We have recently
characterized two Arabidopsis homologs of the bacterial mechanosensitive channel Mechanosensitive Channel of Small Conductance (MscS) that are localized to
the plastid inner envelope: MscS-Like2 (MSL2) and
MSL3. MSL2 and MSL3 likely serve as organellar osmotic
release valves, mediating the ux of osmolytes out of the
plastid stroma in response to increased membrane tension
under hypoosmotic swelling (Haswell and Meyerowitz,
2006; Veley et al., 2012). In msl2 msl3 mutants, where
stromal osmolytes cannot be released through such a
safety valve, nongreen plastids of the leaf epidermis
and root are enlarged and abnormally round (Haswell
and Meyerowitz, 2006). This phenotype is consistent
with the overaccumulation of stromal solutes driving
water uptake into the plastid and leading to abnormal
expansion of the plastid envelope, and it is suppressed
by a variety of conditions that increase cytoplasmic
osmolytes (Veley et al., 2012). Thus, the nongreen
plastids in the msl2 msl3 mutant are constitutively under hypoosmotic stress. Chloroplasts in the msl2 msl3
mutant are greatly enlarged and exhibit defective division site selection (Haswell and Meyerowitz, 2006;
Wilson et al., 2011) but do not appear round, and we
speculate that they have a second pathway for osmoregulation in addition to mechanosensitive ion
channels. Loss of MSL2 and/or MSL3 function also
results in whole-plant phenotypes, including leaves
with variegation, notching, and rumpled surfaces and
dwarng of the adult plant (Haswell and Meyerowitz,
2006; Jensen and Haswell, 2012). These data suggest
that organellar osmotic stress responses are integrated
into a larger cellular context and that msl2 msl3 mutants
might provide a powerful tool to study the cellular response to plastid hypoosmotic stress.
These observations led us to hypothesize the presence of a mechanism that serves to integrate the osmotic state of the plastid into cellular osmoregulation.
In msl2 msl3 mutants, where stromal solutes are likely
to be abnormally high relative to the cytoplasm, such a
mechanism might activate cellular osmotic adjustment
in an attempt to increase solutes (and thereby lower
water potential) in the cytoplasm. Consistent with this
idea, we show that osmotic imbalance in the plastid is
120

capable of inducing a cellular osmoregulatory response

similar to that induced by environmental stresses, such
as drought or salinity stress. These observations give us
a unique view of plant osmoregulation and suggest that
organelles can serve as osmosensors capable of detecting osmotic imbalance and activating cellular osmotic
stress pathways.
RESULTS
msl2 msl3 Mutant Seedlings Overaccumulate Solutes

We have previously documented the presence of

enlarged and abnormally round leaf epidermal plastids in the msl2-1 msl3-1 mutant background (Haswell
and Meyerowitz, 2006; Veley et al., 2012) and the msl2-3
mutant background (Jensen and Haswell, 2012). Although msl2-1 and msl3-1 are likely to be intermediate
loss-of-function alleles, msl2-3 is a null allele (Wilson
et al., 2011). Confocal laser scanning microscopy images of leaves expressing RecARED, a plastid-localized
version of dsRED (Clontech; Haswell and Meyerowitz,
2006), revealed that msl2-3 msl3-1 double mutants also
contain large, round nongreen plastids (Fig. 1). As expected, the nongreen plastid phenotype was more severe in the msl2-3 msl3-1 double mutant than the msl2-3
or msl2-1 msl3-1 mutants. Therefore, we chose to use leaf
epidermal plastids of msl2-3 msl3-1 double mutant plants
(hereafter referred to as msl2 msl3) as a model system for
exploring the cellular responses to plastid stress
To test the hypothesis that plastid osmotic stress can
elicit cellular osmotic adjustment, we measured the osmotic potential (CS) of cell sap extracted from wild-type,
msl2, and msl2 msl3 seedlings that had been transferred
to either control agar plates (20.25 MPa) or polyethylene
glycol (PEG)-infused plates (20.7 MPa) for a moderate
low water potential treatment in a nontranspiring system
(Verslues et al., 2006; Verslues, 2010). Note that the high
Mr solute PEG produces osmotic stress by increasing
extracellular osmolarity but is not taken up by plant cells
(Verslues et al., 2006). As shown in Figure 2, msl2 and
msl2 msl3 mutants had decreased CS (increased solute
content) compared with the wild type in both control
and 20.7-MPa treatments. The CS values of unstressed
msl2 and msl2 msl3 seedlings were 0.05 and 0.12 MPa,
respectively, lower than that of the wild type (Fig. 2A).

Figure 1. msl2 msl3 mutant seedlings contain large, round nongreen

plastids. Representative images of nongreen plastids in the leaf epidermis
of Arabidopsis seedlings expressing the fluorescent plastid marker
RecARED (pseudocolored red). Col-0, Columbia-0. Bar = 10 mm.
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Plastid Stress Activates Cellular Osmoregulation

Figure 2. msl2 msl3 mutant seedlings accumulate high levels of solutes. CS of mutant and wild-type seedlings grown under control (A) or
moderately low water potential (B) conditions. Data from three independent experiments are presented (n = 57). Error bars indicate SEM.
Significant difference from the wild type is indicated. *, P , 0.01
(Students t test). Col-0, Columbia-0.

According to the vant Hoff equation (Verslues, 2010),

these data indicate an increase in cellular solute concentrations of 22 mM over the wild type in the msl2
mutant and 50 mM over the wild type in the msl2 msl3
mutant. Similarly, under mild water stress treatment,
msl2 and msl2 msl3 seedlings contained cellular solutes
90 mM and 140 mM higher than the wild type, respectively (Fig. 2B). An msl2 msl3 mutant complemented with
the genomic version of MSL2 (MSL2g; Haswell and
Meyerowitz, 2006) had a CS value indistinguishable
from the wild type under either condition (Fig. 2). These
results show that msl2 and msl2 msl3 seedlings undergo
osmotic adjustment in the absence of extracellular osmotic stress and suggest that they exhibit enhanced osmotic adjustment in response to mild water stress.
msl2 msl3 Seedlings Accumulate Pro and Exhibit Altered
Expression of Pro Metabolism Genes

To determine if the compatible osmolyte Pro is increased in response to plastid osmotic stress, we quantied

Pro levels in the aerial tissue of wild-type, msl2, and

msl2 msl3 seedlings. As shown in Figure 3A, msl2 and
msl2 msl3 seedlings had 3.6-fold and 11.2-fold more Pro,
respectively, in their leaves than wild-type seedlings,
whereas the complemented line did not differ signicantly from the wild type. The observed Pro accumulation is unlikely to be a general effect of abnormal plastid
function or leaf morphology, because an Arabidopsis
mutant with impaired plastid biogenesis and mesophyll
development, variegated1 (var1; Sakamoto et al., 2002),
contained wild-type levels of Pro (Fig. 3A). Furthermore, Pro levels in a plastid division mutant with
greatly enlarged green and nongreen plastids, accumulation and replication of chloroplasts6-1 (arc6-1; Pyke et al.,
1994; Holzinger et al., 2008), were statistically indistinguishable from the wild type, indicating that increased
plastid volume alone is not sufcient to trigger Pro accumulation.
We hypothesized that Pro accumulation in msl2 and
msl2 msl3 seedlings might be associated with transcriptional up-regulation of Pro synthesis enzymes
and/or the down-regulation of Pro degradation enzymes, which has been established for stress-induced
Pro accumulation (Verbruggen and Hermans, 2008;
Verslues and Sharma, 2010). Compared with the wild
type, msl2 msl3 mutants exhibited a 4.6-fold increase
in P5CS1 transcripts (Fig. 3B). Immunoblots with a
P5CS1-specic antiserum (Kesari et al., 2012) conrmed a corresponding up-regulation of P5CS1 protein in msl2 msl3 seedlings (Fig. 3C). In addition, msl2
msl3 mutants showed a 3.8-fold decrease in PDH1
transcripts (Fig. 3B). In unstressed wild-type plants,
such elevated Pro levels would lead to the induction of
PDH1 expression (Verbruggen and Hermans, 2008;
Verslues and Sharma, 2010). In the msl2 msl3 mutant,
however, PDH1 expression was repressed, just as it
would be in a wild-type plant exposed to drought or
salt stress (Miller et al., 2005). Thus, msl2 msl3 mutants

Figure 3. msl2 msl3 mutant seedlings accumulate Pro and exhibit altered expression of Pro metabolism genes. A, Pro content of
mutant and wild-type seedlings. The average of three biological replicates (each performed in triplicate; n $ 25 seedlings) is
presented. Col-0, Columbia-0; FW, fresh weight. B, The qRT-PCR analysis of Pro metabolism gene expression normalized to the
expression of the internal reference gene ACTIN. The average of three biological replicates (each performed in triplicate; n $ 25
seedlings) is presented. A and B, Significant differences from the wild type are indicated. Error bars indicate SEM. *, P , 0.01
(Students t test). WT, Wild type. C, Immunoblot of P5CS1 protein levels in wild-type and mutant seedlings; 1.5-fold dilutions of
each protein extract were loaded, and the blot was detected with anti-P5CS1 (top). Both a-tubulin (middle) and Ponceau S
staining of Rubisco (bottom) were used for loading comparison because of the appearance of decreased levels of a-tubulin in
msl2 msl3 mutants. [See online article for color version of this figure.]
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121

Wilson et al.

exhibited a combination of Pro accumulation and Pro

metabolism gene expression that is characteristic of osmotic stress response, although the plants were not
exposed to any external osmotic stress.

Pro Accumulation and Leaf Morphology Defects in msl2

msl3 Seedlings Were Suppressed by Increased
Cytoplasmic Osmolarity

NaCl, Suc, and mannitol are readily taken up by

plant roots, and growth on media containing these
osmolytes can result in increased cellular solute levels
(Djardin et al., 1999; Inan et al., 2004). We have previously shown that these treatments lead to the suppression of the msl2-1 msl3-1 round plastid phenotype
by reducing the hypoosmotic stress experienced by the
plastid (Veley et al., 2012). As expected, when msl2 and
msl2 msl3 seedlings were grown on solid media supplemented with 0, 25, 50, or 82 mM NaCl, a clear positive
correlation between the amount of NaCl in the media

and the level of suppression of plastid size and shape

was seen (Fig. 4A). When grown on 82 mM NaCl, the leaf
epidermal plastids in the msl2 msl3 mutant appeared
similar to those plastids in the wild type (small and
ovoid), whereas 25 mM NaCl was sufcient to completely suppress the nongreen plastid phenotype of the
msl2 mutant. Growth on 50 and 82 mM NaCl-containing
media resulted in complete suppression of the leaf
phenotypes in msl2 and msl2 msl3 seedlings, respectively (Fig. 4A).
We next quantied Pro levels in plants grown on
these salt-containing media to determine if reducing
plastid osmotic stress could suppress the Pro accumulation phenotype. Growth on NaCl-containing media produced the expected effect of inducing Pro accumulation
in the wild-type seedlings. However, when msl2 msl3
seedlings were grown on 25 mM NaCl, Pro levels decreased dramatically from 16.7-fold to only 3.8-fold
over the wild type (Fig. 4B). Even at 82 mM NaCl, where
in wild-type plants, Pro levels were 10-fold higher than
in unstressed plants, Pro levels in the msl2 msl3 mutant

Figure 4. Pro accumulation and leaf morphology defects in msl2 msl3 seedlings are suppressed by increased cytoplasmic
osmolarity. A, Representative images of whole seedlings and confocal micrographs of nongreen plastids in seedlings grown on
media supplemented with indicated concentrations of NaCl. Col-0, Columbia-0. Bars in seedling images = 5 mm; bars in
plastid images = 10 mm. B, Pro content of seedlings grown on the indicated concentrations of NaCl. The average of three
biological replicates (each performed in triplicate; n $ 25 seedlings) is presented. Significant differences from the wild type are
indicated. *, P , 0.01 (Students t test). FW, Fresh weight. C, Effect of the pgm-1 allele on Pro content in the msl2 msl3
background. The average of two biological replicates (each performed in triplicate; n $ 25 seedlings) is presented. Statistical
groups a and b were determined using one-way ANOVA and Tukeys test (P , 0.01). B and C, Error bars indicate SEM. D,
Confocal laser scanning microscopy images of nongreen plastids of plants grown on the indicated concentrations of NaCl.
Scale bar = 10 mm.
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Plastid Stress Activates Cellular Osmoregulation

were lower than when grown without NaCl. Thus, intermediate levels of NaCl could suppress the high Pro
levels observed in the msl2 msl3 mutant background,
likely by reducing the hypoosmotic stress experienced
by plastids lacking a mechanosensitive mechanism for
solute release.
Plants harboring a lesion in the PHOSPHOGLUCOMUTASE (PGM) gene contain elevated levels of soluble sugars during the day, and we have previously
shown that the pgm-1 allele is capable of suppressing
the plastid phenotype of the msl2-1 msl3-1 mutant
(Veley et al., 2012). We found that the pgm-1 allele also
completely suppressed the elevated Pro levels in the
msl2 msl3 mutant background; msl2 msl3 pgm-1 seedlings contained levels of Pro that were statistically indistinguishable from the wild type (Fig. 4C). Plastid
size was also reduced in the msl2 msl3 pgm-1 mutant,
although the addition of 25 mM NaCl to media was
required to produce leaf epidermal plastids similar in
size and shape to the wild type (Fig. 4D). Thus, complete suppression of the plastid morphology phenotype
of the msl2 msl3 mutant was not required to suppress
the Pro accumulation phenotype. This result can be
explained if Pro accumulation is only induced under
extreme plastid osmotic stress, and the effect of the pgm-1
lesion (a 2-fold increase in cytoplasmic sugars; Veley
et al., 2012) is enough to reduce plastid size and prevent
activation of the osmotic stress response pathway but not
sufcient to return plastids to a fully wild-type shape.
When taken together, the data presented in Figure 4
provide strong evidence that the Pro accumulation and
leaf morphology defects in the msl2 msl3 mutant result
from plastid osmotic stress.
Plastid Osmotic Stress Results in Elevated Levels of ABA
and ABA Hypersensitivity

Because the stress hormone ABA participates in Pro

accumulation in response to extracellular osmotic stress
(Verslues and Bray, 2006), we hypothesized that it
might also play a role in the induction of Pro accumulation in response to plastid osmotic stress. We found
that the ABA content in msl2 msl3 seedlings was 3.5
times higher than that of wild-type seedlings, whereas
the msl2 mutant showed a modest but statistically signicant 1.4-fold increase in ABA content (Fig. 5A). We
further analyzed the expression of three genes that encode key components of ABA biosynthesis (ABSCISIC ACIDDEFICIENT1 [ABA1], NINE-CIS-EPOXYCARTOTENOID
DIOXYGENASE3 [NCED3], and ABSCISIC ALDEHYDE
OXIDASE3 [AAO3]; Seo and Koshiba, 2002) and two
well-documented ABA-response genes (RESPONSIVE TO
DESSICATON29A [RD29A] and COLD-REGULATED15A
[COR15A]; Yamaguchi-Shinozaki and Shinozaki, 1994;
Shinozaki et al., 2003) in wild-type and mutant plants.
Although no signicant differences in transcript levels
of ABA1 and AAO3 were observed, NCED3 was dramatically induced in msl2 and msl2 msl3 seedlings (3- and
36-fold increases over wild-type levels, respectively

Figure 5. msl2 msl3 mutants exhibit elevated levels of ABA and induced expression of ABA biosynthesis and ABA response genes.
A, ABA levels in wild-type and mutant seedlings. The average of three
biological replicates of $30 seedlings each is presented. Error bars
indicate SD. Col-0, Columbia-0; FW, fresh weight. B, The qRT-PCR
analysis of ABA biosynthesis and stress response gene expression. The
average of three biological replicates (each performed in triplicate; n $
25 seedlings) is presented. Error bars indicate SEM. Significant differences from the wild type are indicated. *, P , 0.01 (Students t test).
WT, Wild type.

(Fig. 5B). Consistent with an elevated ABA content,

RD29A and COR15A were also induced in msl2 msl3
seedlings (2.3- and 3.6-fold, respectively).
We also found that msl2 msl3 mutants were hypersensitive to ABA-mediated inhibition of seed germination. On standard media, msl2 msl3 mutants exhibited a
modest decrease in germination efciency (Fig. 6A).
However, msl2 and msl2 msl3 seed germination was
signicantly more sensitive than the wild type to the
presence of 1 mM ABA, and the msl2 msl3 seed germinated at a rate similar to the known ABA-hypersensitive
mutant enhanced response to ABA1-2 (era1-2; (Cutler
et al., 1996; Fig. 6B). Notably, the era1-2 mutant did not
accumulate Pro above wild-type levels in the absence
of extracellular water stress (Fig. 3A). Finally, msl2
msl3 pgm-1 triple mutants germinated at a rate similar
to that of the wild type (Fig. 6C), conrming that the
ABA hypersensitivity of msl2 msl3 plants was associated with plastid stress.
Pro Accumulation in msl2 msl3 Seedlings Requires ABA
Biosynthesis and Is More Responsive to Application of
Exogenous ABA

To determine if the elevated level of ABA found in

msl2 msl3 seedlings was responsible for elevated levels
of Pro, 7-d-old wild-type, msl2, and msl2 msl3 seedlings
were grown for 7 d in the presence of 10 mM uridone
(FLU). In the presence of FLU, which inhibits the biosynthesis of the carotenoid precursors of ABA (Gamble
and Mullet, 1986), Pro levels were reduced to wild-type
levels in msl2 mutants and from 7.5-fold to 3.1-fold over
the wild type in msl2 msl3 mutants (Fig. 7A). The Pro
content of seedlings grown on media containing both
FLU and 0.5 mM ABA was restored to the elevated levels
seen in msl2 and msl2 msl3 seedlings in the absence of
FLU. ABA treatment alone produced exceptionally high

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Wilson et al.

To address this same question using a genetic approach, we introduced the abscisic acid-decient2-1
(aba2-1) allele into the msl2 msl3 background (Schwartz
et al., 1997). aba2-1 mutants contain less than 5% (w/w)
of wild-type levels of ABA under osmotic stress (GonzlezGuzmn et al., 2002; Verslues and Bray, 2006). As shown in
Figure 7B, Pro levels in aba2-1 msl2 msl3 seedlings were
signicantly reduced compared with the msl2 msl3 parent
(3.8 and 13.9 mmol g fresh weight21, respectively) and
statistically indistinguishable from the wild type. Thus,
Pro accumulation in msl2 msl3 mutants occurs primarily
through an ABA-dependent pathway, and the presence
of osmotically stressed plastids allows plants to accumulate Pro in response to ABA as if they were under
externally applied osmotic stress.
DISCUSSION

All organisms must respond to differences in CS

across the plasma membrane generated by changing
environmental or metabolic conditions (Yancey et al.,
1982; Wood, 2011). The same is presumably true of CS
across organelle membranes, where metabolism and
transport activities in both the organelle and the cytoplasm can produce substantial differences in solute
content. Organelles have been shown to change shape
and volume in response to alterations in environmental
osmolarity and during metabolic activity (Robinson,
1985; Gupta and Berkowitz, 1987; McCain, 1995; Boustany
et al., 2002; Finan et al., 2009), suggesting that they can
undergo osmoregulation from within the cell cytoplasm.
However, the molecular details of organellar osmoregulation and how it might be integrated into the larger

Figure 6. msl2 msl3 mutants exhibit hypersensitivity to ABA during

germination in an osmotic stress-dependent manner. Germination rates of
mutant and wild-type seed on MS (A) or MS supplemented with 1 mM
ABA (B and C). Germination was scored based on radical emergence.
The average of three biological replicates of $50 seeds each is presented.
Error bars indicate SD. Significant differences from the wild type are indicated. Col-0, Columbia-0. *, P , 0.01 (Students t test).

Pro levels in the msl2 msl3 mutant (14-fold higher than

the wild type compared with 7.5-fold in untreated
seedlings), whereas Pro levels in wild-type seedlings
remained unchanged regardless of treatment. Thus, msl2
msl3 seedlings responded to ABA by accumulating the
high levels of Pro that would be expected if they were
subjected to osmotic stress.
124

Figure 7. Pro accumulation in msl2 msl3 mutants partially requires

ABA biosynthesis and is more responsive to application of exogenous
ABA than the wild type. A, Pro levels in seedlings grown on media
containing 10 mM FLU and/or 0.5 mM ABA. The average of two to three
biological replicates (each performed in triplicate; n $ 25 seedlings)
from three independent experiments is presented. Significant differences from untreated wild type or untreated msl2 msl3 are indicated.
*, P , 0.01 (Students t test). B, Effect of the aba2-1 lesion on Pro
accumulation in the msl2 msl3 mutant background. The average of
two biological replicates (each performed in triplicate; n $ 25 seedlings) is presented. Statistical groups a and b were determined using
one-way ANOVA and Tukeys test (P , 0.01). Error bars indicate SEM.
Col-0, Columbia-0; FW, fresh weight.
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Plastid Stress Activates Cellular Osmoregulation

cellular context remain largely unexplored. In the work

presented here, we used the Arabidopsis msl2 msl3
mutant, which is defective in plastid osmoregulation, as
a tool to interrogate the cellular response to constitutive
organellar hypoosmotic stress. Our results establish that
osmotic imbalance across the plastid envelope can trigger osmotic adjustment as well as other osmotic stress
responses.
msl2 msl3 Mutants Constitutively Activate Cellular
Osmotic Stress Responses

Plastid osmotic stress in the msl2 msl3 mutant activated the following three hallmarks of the cellular environmental osmotic stress response but did so in the
absence of external water stress.
Low CS

The CS of msl2 msl3 seedlings was lower than wild

type (Fig. 2). Furthermore, Pro accumulation can be
used as a proxy for the activation of osmoregulatory
signaling, and msl2 msl3 seedlings contained over 10
times as much Pro as wild-type seedlings (Fig. 3A).
This fold increase is similar to that caused by mild to
moderate low water potentials (1020-fold) and significantly more than that caused by other osmotic stresses,
such as 100 mM mannitol and NaCl (approximately
4-fold; Sharma and Verslues, 2010). P5CS1 and PDH1
are the key regulated enzymes controlling the production and degradation of Pro, respectively (Verbruggen
and Hermans, 2008; Verslues and Sharma, 2010), and in
msl2 msl3 mutants, P5CS1 transcript and protein levels
were up-regulated, whereas expression of PDH1 was
repressed (Fig. 3, B and C). This pattern is similar to
what is seen under osmotic stress, particularly the repression of PDH1 expression, despite high Pro levels.
Furthermore, when plastid osmotic stress was relieved
through media manipulation or the introduction of a
genetic lesion, Pro levels in the msl2 msl3 mutant were
reduced to the levels of the wild-type control (Fig. 4).
Increase in ABA Levels

We also observed a 3.5-fold increase of ABA levels

(Fig. 5A) and a strong 36-fold induction of NCED3
transcripts (Fig. 5B) in msl2 msl3 seedlings. The induction of NCED3 in response to environmental osmotic stress is thought to be primarily responsible for
the enhanced production of ABA resulting from water
stress (Tan et al., 1997; Burbidge et al., 1999; Qin and
Zeevaart, 1999; Chernys and Zeevaart, 2000; Iuchi
et al., 2001). NCED3 itself is induced by ABA as part of
a positive feedback loop (Xiong et al., 2002; Barrero
et al., 2006), and the induction of other ABA metabolism
genes or other mechanisms, such as enhanced ABA
transport or deconjugation, may be responsible for the
up-regulation of NCED3 and the increased synthesis of
ABA in msl2 msl3 mutants (Umezawa et al., 2010). The
early steps of ABA biosynthesis occur in the plastid (Seo

and Koshiba, 2011), suggesting that the transport of ABA

precursors may be disrupted in the mutant. However,
inefcient or decreased ABA production would not explain the observed increase in ABA levels in msl2 msl3
mutants.
Potentiation of ABA Responses

ABA is required for environmental osmotic stressinduced Pro accumulation (Verslues and Bray, 2006;
Sharma and Verslues, 2010), and we found that the
same was true for plastid osmotic stress-induced Pro
accumulation. We used both chemical inhibition (Fig. 7A)
and genetic ablation (Fig. 7B) to disrupt ABA biosynthesis in msl2 msl3 seedlings and observed substantially
reduced Pro levels in both cases, conrming that Pro
accumulation in response to plastid osmotic stress occurred primarily through an ABA-dependent pathway.
In addition, we observed that treatment with exogenous
ABA induced high levels of Pro in the msl2 msl3 mutant
background without additional stress, similar to wildtype plants exposed to low water potential (Fig. 7A;
Verslues and Bray, 2006; Sharma and Verslues, 2010).
In summary, we found that plastid osmotic stress in
the msl2 msl3 mutant was associated with the accumulation of solutes and Pro (Figs. 2 and 3), altered
expression of Pro metabolic enzymes (Fig. 3), elevated
levels of ABA, increased expression of NCED3 (Fig. 5),
and increased sensitivity to ABA in Pro accumulation
and inhibition of germination (Figs. 6 and 7). All of
these phenotypes are hallmarks of the cellular response
to environmental osmotic stress, but in the msl2 msl3
background, they are elicited without any change in
extracellular water potential. These results are consistent with a model where two independent or converging pathways serve to increase cellular osmolytes: one
pathway in response to low cytoplasmic solute concentration relative to the plastid stroma and one pathway in
response to low cytoplasmic solute concentration relative
to the extracellular environment.
The constitutive activation of stress responses in the
msl2 msl3 mutant suggests the existence of a plastidbased retrograde osmotic stress signaling pathway.
Although it is possible that MSL2 and MSL3 are actively involved in repressing osmotic stress pathways
in the wild-type plant, a more straightforward explanation for our observations is that the plastids under
hypoosmotic stress in the msl2 msl3 mutant generate a
signal capable of potentiating ABA accumulation, ABA
responsiveness, and other osmotic stress signals in the
cytoplasm and/or the nucleus. The communication of
information from the plastid to the nucleus has been
extensively documented and is referred to as retrograde
signaling. Multiple retrograde signaling pathways have
been identied, triggered by the accumulation of reactive oxygen species during photosynthesis, the disrupted
redox state of the plastid, defective organellar gene expression, plastid developmental arrest, or the accumulation of metabolic intermediates (Chi et al., 2013; Kleine
and Leister, 2013). Our data suggest the existence of a

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125

Wilson et al.

plastid-based signal triggered by osmotic imbalance

between the plastid and the cytoplasm.

3 d at 4C. After 7 d of vertical growth, seedlings were transferred to plates of

specied treatment and grown for an additional 7 d before tissue harvesting.
All plants were grown at 21C under a 16-h-light regime with light uence
from 150 to 195 mmol m21 s21.

Future Investigation into Plastid Osmoregulation

and Osmosensation

Low Water Potential Treatment and Quantication of CS

These results pose many intriguing questions for

future study. One of these questions is if increased Pro
accumulation in the msl2 msl3 mutant affects plastid
osmoregulation. The answer to this question is difcult
to predict, because it is not yet clear whether Pro accumulates only in the cytoplasm or if it can also do so in
the plastid stroma. The nal step of Pro synthesis is
catalyzed by D 1-pyrroline-5-carboxylate reductase
(P5CR) and P5CR isoforms have been localized to both
the cytoplasm and the plastid in tobacco (Nicotiana tabacum) and spinach (Spinacia oleracea; Szoke et al., 1992;
Murahama et al., 2001). Identifying the subcellular
localization of this enzyme and other Pro biosynthetic
enzymes should help determine if Pro accumulation in
the cytoplasm could help maintain some level of osmotic balance across the plastid envelope in the absence
of MSL2 and MSL3.
Another topic for future investigation is the identication of the proposed plastid-based osmotic stress
signaling pathway and its molecular identity. It is
worth noting that, although it is possible that the increased transport of ABA precursors from the plastid
to the cytoplasm in response to plastid osmotic stress
produces the elevated ABA level observed in msl2 msl3
mutants, a second plastid-based signal would still be
required for the osmotic stress responses documented
here, because the application of ABA has little effect on
Pro accumulation in the absence of plastid or environmental osmotic stress (Fig. 7A; Verslues and Bray,
2006). We anticipate that the answers to these questions
and others will further illuminate plastid osmoregulation, possibly contribute to the eld of retrograde signaling, and may even reveal the nature of the cellular
signal that, in addition to ABA, serves to induce osmotic
responses.

MATERIALS AND METHODS

Arabidopsis (Arabidopsis thaliana) Mutants and Transfer
DNA Lines
pgm-1 (CS210), aba2-1 (CS156), var1-1 (CS271), p5cs1-4 (SALK_063517),
era1-2, and msl2-3 mutant alleles are in the Columbia-0 background. The arc6-1
(CS286) and msl3-1 mutant alleles are in the Wassilewskija background (Haswell
and Meyerowitz, 2006). PCR genotyping of msl mutant alleles was performed as
described by Wilson et al. (2011). The pgm-1 and aba2-1 alleles were genotyped as
described by Veley et al. (2012) and Sharma and Verslues (2010), respectively.

Low water potential treatments were performed as described by Bhaskara

et al. (2012). Seedlings were grown on one-half-strength MS with 2 mM MES
buffer (pH 5.7) without the addition of sugar. Seven-day-old seedlings were
transferred to either fresh control media or PEG-infused agar plates (20.7 MPa),
and samples were collected 96 h after transfer. For CS quantication, seedling
samples were macerated, frozen, thawed, and centrifuged to obtain cell sap, and
CS was quantied using a Psypro system with c52 sample chambers (Wescor).

Quantication of Free Pro

Pro was assayed using the ninhydrin assay by Bates et al. (1973) adapted to
t a 1.7-mL microcentrifuge tube format as described in the work by Abrahm
et al. (2010). To avoid light-dependent Pro oscillation (Hayashi et al., 2000),
tissue samples were collected 8 h into a 16-h-light cycle for all experiments.

Germination Assay
Seeds were surface sterilized and sown on 13 MS with or without 1 mM
ABA. After 3 d of stratication, seeds were exposed to a 16-h-light regime at
21C. Seeds were scored as germinated when the radicle ruptured the testa
and endosperm.

ABA Quantication
Approximately 100 mg of plant aerial tissue was sampled for each of three
replicates, frozen in liquid nitrogen, and stored at 280C before being assayed.
ABA levels were quantied by liquid chromatographymass spectrometry/
mass spectrometry as described previously (Chen et al., 2009).

Quantitative Reverse Transcription-PCR

Quantitative reverse transcription-PCR (qRT-PCR) was performed as previously described (Wilson et al., 2011). Primer mixes (described in Supplemental
Table S1) were added to a cocktail containing 13 SYBR Green PCR Master
Mix (Applied Biosciences) and 0.25 mL of complementary DNA to make
a nal 25-mL reaction. After amplication in the StepOnePlus real-time
PCR system, the data were analyzed using StepOne software (Applied
Biosciences).

Immunoblotting
One hundred milligrams of plant tissue was ground in liquid nitrogen and
resuspended in 200 mL of 23 SDS sample buffer plus protease inhibitors.
Samples were separated on a 10% (w/v) SDS-polyacrylamide gel and electroblotted to polyvinylidene uoride membrane. Membranes were blocked
overnight at 4C with 5% (w/v) nonfat milk in Tris-buffered saline plus Tween
20 and probed with rabbit anti-P5CS1 (1:3,000; Kesari et al., 2012) followed by
anti-rabbit IgG horseradish peroxidase (1:10,000; Sigma-Aldrich). Chemiluminescent detection was performed with the Thermo-Scientic SuperSignal
West Femto detection kit (Pierce). Membranes were stained with Ponceau S
and then stripped and reprobed with mouse anti-a-tubulin (Sigma) as loading
controls.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers MSL2, At5g10490; MSL3, At1g58200; and
PGM, At5g51820.

Plant Growth and NaCl, FLU, and ABA Treatments

Plants were grown on full-strength Murashige and Skoog (MS) medium
(pH 5.7; Caisson Labs) with 0.8% (w/v) agar (Caisson Labs). NaCl was added
before autoclaving, whereas FLU (Sonar; Chem Service) and ABA (Sigma)
were added after sterilization. For FLU and ABA treatments, seed was surface
sterilized, sown on nylon mesh strips overlaid on 13 MS, and stratied for
126

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table S1. Oligos used in qRT-PCR.
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Plastid Stress Activates Cellular Osmoregulation

ACKNOWLEDGMENTS
We thank the Arabidopsis Biological Resource Center (Ohio State University) for aba2-1, var1-1, and arc6-1 mutant seed; Lucia Strader for era1-2 mutant
seed; The Proteomics and Mass Spectrometry Facility of the Donald Danforth
Plant Science Center for performing the ABA quantication; and the Jeanette
Goldfarb Plant Growth Facility staff for assistance.
Received January 24, 2014; accepted March 25, 2014; published March 27,
2014.

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