Biochem. J. (2010) 427, 359–367 (Printed in Great Britain)
359
doi:10.1042/BJ20091762
Proteolytic processing of an Arabidopsis membrane-bound NAC
transcription factor is triggered by cold-induced changes
in membrane fluidity
Pil Joon SEO, Mi Jung KIM, Jin-Su SONG, Youn-Sung KIM, Hie-Joon KIM and Chung-Mo PARK1
Department of Chemistry, Seoul National University, Seoul 151-742, South Korea
Changes in membrane fluidity are the earliest cellular events that
occur in plant cells upon exposure to cold. This subsequently
triggers physiological processes, such as calcium influx and
reorganization of actin cytoskeletons, and induces expression
of cold-responsive genes. The plasma-membrane-anchored NAC
(NAM/ATAF/CUC) transcription factor NTL6 is of particular
interest. Cold triggers proteolytic activation of the dormant NTL6
protein, which in turn elicits pathogen-resistance responses by
inducing a small group of cold-inducible PR (pathogenesisrelated) genes in Arabidopsis. In the present study, we
show that proteolytic processing of NTL6 is regulated by
cold-induced remodelling of membrane fluidity. NTL6 processing was stimulated rapidly by cold. The protein stability
of NTL6 was also enhanced by cold. The effects of cold on
NTL6 processing and protein stability were significantly reduced
in cold-acclimatized plants, supporting the regulation of NTL6
processing by membrane fluidity. Consistent with this, although
NTL6 processing was stimulated by pharmacological agents that
reduce membrane fluidity and thus mimic cold, it was inhibited
when plants were treated with a 18:3 unsaturated fatty acid,
linolenic acid. In addition, the pattern of NTL6 processing was
changed in Arabidopsis mutants with altered membrane lipid
compositions. Assays employing chemicals that inhibit activities
of the proteasome and proteases showed that NTL6 processing
occurs via the regulated intramembrane proteolysis mechanism.
Interestingly, a metalloprotease inhibitor blocked the NTL6
processing. These observations indicate that a metalloprotease
activity is responsible for NTL6 processing in response to coldinduced changes in membrane fluidity.
INTRODUCTION
membranes, it is processed to a nuclear form with a molecular
mass of 65 kDa [5,6]. The transcriptionally active form is
translocated to the nucleus, where it activates genes that
mediate biosynthesis of cholesterol and fatty acids. The SREBP
precursor is integrated into the ER membranes. Upon stimulation,
it is transported to the Golgi complex, where a two-step
processing of SREBP occurs through sequential proteolytic
cleavage events mediated by two membrane-bound proteases, S1P
(site-1 protease) and S2P [1,7].
Several members of the bZIP (basic leucine zipper) and NAC
(NAM/ATAF/CUC) transcription factors have been confirmed
to be membrane-associated in Arabidopsis [8–16]. All of the
reported plant MTFs are type II membrane proteins, having their
N-termini towards the cytoplasm. They have been confirmed or
predicted to be processed by the RIP mechanism, although the
proteases responsible for the MTF activation have not yet been
identified in some cases [4]. Proteolytic activation of the bZIP28
and bZIP60 MTFs is induced by ER stress, and that of the bZIP17
member is triggered by high salinity [11–13]. It has been shown
that a plant S1P mediates the activation of the bZIP MTFs, except
for bZIP60 [11,12].
The NAC MTFs, such as NTM1, NTL6, NTL8 and NTL9,
are involved in plant responses to diverse stress conditions, such
as high salt and cold [15,16], and developmental signals [9,14].
NTM1 regulates cell division by modulating cytokinin signalling
[9]. NTL8 regulates flowering time and seed germination under
Controlled activation of dormant transcription factors is an
essential component of gene regulatory networks in eukaryotic
cells. A pivotal example is proteolytic activation of MTFs
(membrane-bound transcription factors). MTFs are synthesized as
dormant, membrane-bound forms that are anchored to the cellular
membranes, such as plasma membranes, ER (endoplasmic
reticulum) and nuclear membranes. Upon stimulation by
certain environmental and/or developmental cues, they are
processed either via a RIP (regulated intramembrane proteolysis)
mechanism involving specific intramembrane proteases or via
a RUP (regulated ubiquitin/proteasome-dependent processing)
mechanism [1–3]. The processed, transcriptionally active forms
enter the nucleus and regulate expression of target genes.
Numerous MTFs have been identified in animals and plants
[2,4]. Although the functional significance of MTF activation
in regulating gene expression is widely accepted [2,3], molecular
mechanisms underlying MTF activation are poorly understood in
most cases.
One of the best characterized MTFs is the SREBP (sterolregulatory-element-binding protein), which is considered to
be a master regulator for lipid homoeostasis in animals [5].
The SREBP transcription factor is expressed as a membranebound form, having a molecular mass of 125 kDa with two
transmembrane motifs. When cholesterol is depleted in the
Key words: Arabidopsis, cold, fatty acid desaturase, membrane-bound transcription factor, metalloprotease, proteolysis.
Abbreviations used: ALLN, N -acetyl-L-leucyl-L-leucylnorleucinal; BAPTA, 1,2-bis-(o -aminophenoxy)ethane-N,N,N ,N -tetra-acetic acid; bZIP, basic
leucine zipper; CaMV, cauliflower mosaic virus; CD, cytochalacin D; DTT, dithiothreitol; ER, endoplasmic reticulum; FAD, fatty acid desaturase; JK,
jasplakinolide; MS, Murashige and Skoog; MTF, membrane-bound transcription factor; NAC, NAM/ATAF/CUC; qRT-PCR, quantitative real-time reverse
transcription PCR; PR , pathogenesis-related; RIP, regulated intramembrane proteolysis; RT, reverse transcription; S1P, site-1 protease; SREBP, sterolregulatory-element-binding protein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
360
P.J. Seo and others
high salt [14]. NTL9 plays a role in osmotic stress response
and leaf senescence [15]. While the physiological roles of the
NAC MTFs have been fairly well established, very little is known
about the activation mechanisms. Although it has been suggested
that NTM1 is processed by a calpain-like protease activity
[9], it has not been molecularly, genetically and biochemically
confirmed.
The plasma-membrane-anchored NTL6 transcription factor
(At3g49530) plays a role in the cold-induced pathogen-resistance
response [16]. It has been shown that, whereas transgenic plants
overexpressing a truncated NTL6 form devoid of a transmembrane
domain exhibit an enhanced pathogen resistance, NTL6 RNAi
(RNA interference) plants are susceptible to pathogen infection,
particularly under cold conditions. The enhanced resistance to
pathogen infection was correlated with the induction of coldresponsive PR (pathogenesis-related) genes in the transgenic
plants. Interestingly, NTL6 processing is promoted by cold.
On the basis of these observations, it has been proposed that
cold regulation of the NTL6 processing is an adaptive way
of ensuring prompt plant response to psychrophilic pathogens
occuring during cold seasons [16]. However, the effects of cold
on NTL6 processing have not been systematically examined at
the cellular and biochemical levels.
In the present study, we have demonstrated that proteolytic
activation of the plasma-membrane-anchored NTL6 transcription
factor is regulated by cold-induced changes in membrane fluidity.
Accordingly, the processing pattern of NTL6 was greatly altered
in the Arabidopsis mutants with disturbed membrane lipid
compositions. Notably, NTL6 processing was blocked in plants
treated with a metalloprotease inhibitor. We therefore propose that
the metalloprotease-mediated NTL6 processing is a very early
event during cold signalling, which is closely associated with the
cold sensing machinery in plants.
EXPERIMENTAL
Plant materials and growth conditions
Arabidopsis thaliana lines used were in the Columbia (Col-0)
background, unless otherwise specified. Plants were grown in
a controlled culture room set at 23 ◦C with a relative humidity
of 60 % under long day conditions (16 h light/8 h dark) with
white light illumination (120 μmol of photons/m2 per s). The
ssi2 [suppressor of SA (salicylic acid)-insensitivity 2] mutant (no
ecotype) and the fad2 and fad3 mutants were kindly provided by
Dr Pradeep Kachroo (Department of Plant Pathology, University
of Kentucky, Lexington, KY, U.S.A.) and Dr Mi Chung Suh
(Department of Plant Biotechnology and Agricultural Plant
Stress Research Center, Chonnan National University, Gwangju,
South Korea) respectively. The s1p (SALK-020530), ps1 (SALK145544) and ps2 (SALK-013158) mutants were obtained from
a mutant pool of T-DNA (transferred DNA) insertion lines
deposited in the ABRC (Arabidopsis Biological Resource Center,
Ohio State University, Columbus, OH, U.S.A.).
The 35S:6ΔC transgenic plants have been described previously
[16]. The 6C construct contains residues 1–329 of the fullsize NTL6 protein (469 residues). The 6ΔC sequence was subcloned into the pBA002 expression vector [9], which was
transformed into Arabidopsis via a modified floral dip method.
Transcript level analysis
qRT-PCR [quantitative real-time RT (reverse transcription)–PCR]
was employed for measuring transcript levels. Total RNA sample
c The Authors Journal compilation c 2010 Biochemical Society
preparation, reverse transcription and qRT-PCR were carried out
based on the rules that have recently been proposed by Udvardi
et al. [17] to ensure reproducible and accurate measurements.
Extraction of total RNA samples from appropriate plant materials
and qRT-PCR conditions have been described previously [9]. The
RNA samples were extensively pretreated with an RNase-free
DNase to eliminate any contaminating genomic DNA before use.
The qRT-PCR reactions were carried out in 96-well blocks
with an Applied Biosystems 7500 real-time PCR system using the
SYBR Green I master mix in a volume of 25 μl. The PCR primers
were designed using the Primer Express Software installed into
the system. The two-step thermal cycling profile used was 15 s at
94 ◦C and 1 min at 68 ◦C. An eIF4A (eukaryotic initiation factor
4A) gene (At3g13920) was included in the reactions as an internal
control for normalizing the variations in the cDNA amounts used
[18]. The reactions were carried out in biological triplicates using
RNA samples extracted from three independent plant materials
grown under identical growth conditions. The comparative Ct
method was used to evaluate the relative quantities of each
amplified product in the samples. The threshold cycle (Ct ) was
automatically determined for each reaction by the system set
with default parameters. The specificity of the amplifications was
determined by melt curve analysis of the amplified products using
the standard method installed in the system.
NTL6 processing
The 35S:NTL6 transgenic plants, in which a MYC–NTL6
gene fusion was overexpressed under the control of the
CaMV (cauliflower mosaic virus) 35S promoter [16], were
grown for 2 weeks on 0.5 × MS (Murashige and Skoog) agar
plates (hereafter referred to as MS-agar plates) and used
for treatments with cold, MG132, ALLN (N-acetyl-L-leucyl-Lleucylnorleucinal) and plant growth regulators. Harvested plant
materials were ground in liquid nitrogen, and total cellular extracts
were suspended in SDS/PAGE sample loading buffer. The protein
samples were then analysed by SDS/PAGE (10 % gels) and
blotted on to Hybond-P+ membranes (Amersham-Pharmacia).
The NTL6 proteins were immunologically detected using an antiMyc antibody (Santa Cruz Biotechnology).
Effects of pharmacological reagents on NTL6 processing
The assays employing pharmacological reagents were carried
out as described previously [19]. JK (jasplakinolide) was
obtained from Molecular Probes and CD (cytochalacin D) and
BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N -tetra-acetic
acid] were purchased from Sigma–Aldrich. Calcium ionophore
A23187 was obtained from ICN and Bay K8644 was obtained
from Calbiochem. All of the reagents, except for BAPTA, which
was prepared in water, were prepared in 50 % DMSO.
35S:NTL6 transgenic plants (2 weeks old) grown on MS-agar
plates were transferred on to a liquid MS culture and gently
shaken for one additional day before the pharmacological reagents
were applied. DMSO was directly added to the liquid medium at
the final concentrations of 0–12 %, and incubated for up to 6 h.
MG132 (Sigma–Aldrich) and ALLN (Calbiochem) were prepared
in DMSO and used at the final concentrations of 40 μM and 2 μM
respectively. JK, CD, BAPTA, Bay K8644 and A23187 were used
at the final concentrations of 2 μM, 50 μM, 2 mM, 0.1 mM and
0.1 mM respectively. DTT (dithiothreitol) and tunicamycin were
used at the final concentrations of 2 mM and 5 μg/ml respectively.
NTL6 processing and membrane fluidity
361
Histological assays
NTL6 processing is reduced in cold-acclimatized plant cells
The seedlings were incubated in 90 % acetone for 20 min
on ice, washed twice with rinsing solution [50 mM sodium
phosphate (pH 7.2), 0.5 mM K3 Fe(CN)6 and 0.5 mM K4 Fe(CN)6 ]
and subsequently incubated at 37 ◦C for 18–24 h in rinsing
solution containing 2 mM X-Gluc (5-bromo-4-chloro-3-indolylβ-D-glucuronide; Duchefa). The seedlings were then incubated in
a series of ethanol solutions ranging from 15 % to 80 % in order
to remove the chlorophylls from plant tissues. They were then
mounted on to microscope slides and visualized using a Nikon
SMZ 800 microscope.
The earliest cellular event occurring in plant cells exposed to
cold is changes in membrane fluidity [19–21]. The membrane
fluidity is rapidly reduced under cold. However, it is readjusted
after a while when plant growth persists at low temperatures (i.e.
cold acclimation), owing to the activity of fatty acid desaturases
that increase the relative content of unsaturated fatty acids in the
membranes [22]. Indeed, whereas the relative content of the 18:3
fatty acid species is elevated, that of the 18:2 fatty acid species
is reduced in Arabidopsis suspension cells at 16 ◦C compared
with those at 22 ◦C [23,24]. In addition, while the content of
linoleic acid (18:2) is diminished, that of linolenic acid (18:3)
is significantly elevated in rapeseed hypocotyls incubated at 4 ◦C
[23]. Therefore a question was whether the NTL6 processing is
influenced by changes in membrane fluidity.
We first acclimatized the 35S:NTL6 transgenic plants to
16 ◦C for 2 weeks, which were subsequently exposed to lower
temperatures. The processing patterns of the NTL6 protein were
then examined in the plant cells by SDS/PAGE and Western blot
analysis. Although NTL6 processing was still elevated at lower
temperatures in the cold-acclimated plants, the degree of elevation
was markedly reduced compared with that observed in the nonacclimated plants (Figure 2A), supporting that NTL6 processing
is regulated by changes in membrane fluidity. In addition, while
the NTL6 protein stability was promoted in the non-acclimated
plants, the promotive effects were significantly reduced in the
cold-acclimated plants.
We next examined the kinetics of NTL6 processing in plants
treated with cold for up to 14 days. The results showed that the
NTL6 processing was elevated for 1 day after cold treatment, but
slowly reduced during the time course (Figure 2B). Furthermore,
the promotive effects of cold on the NTL6 protein stability
steadily disappeared as well. These are evidently related with the
remodelling of membrane fluidity occurring in cold-acclimatized
plant cells. Taken together, these observations strongly support
the close relationship between NTL6 processing and membrane
fluidity.
Sequence data
Sequence data from the present study can be obtained from the
Arabidopsis Genome Initiative databases under the following
accession numbers: NTL6 (At3g49530), S1P (At5g19660),
S2P (At4g20310), Presenilin-1 (At1g08700), Presenilin-2
(At2g29900), FAD2 (At3g12120), FAD3 (At2g29980), FAD7
(At3g11170) and FAD8 (At5g05580).
RESULTS
Cold regulates NTL6 processing and protein stability
We have recently reported that NTL6 processing occurs to a
high degree in cold-treated plants [16]. However, the mechanisms
underlying the cold effects on NTL6 processing have not been
explored at the molecular level, which is critical for understanding
the role of the NTL6 transcription factor in cold induction of
pathogen-resistance responses.
To more systematically examine the effects of cold on NTL6
processing, the 35S:NTL6 transgenic plants, in which a gene
fusion consisting of six copies of a Myc-encoding sequence fused
in-frame to the 5 end of the NTL6 gene was overexpressed under
the control of the CaMV 35S promoter [16], were exposed to cold
(4 ◦C), and the kinetics of NTL6 processing was analysed for up
to 24 h. The NTL6 processing was initiated within 10 min after
exposure to cold and steadily elevated, reaching a peak at 18 h
after cold treatment (Figures 1A and 1B) (see Discussion). The
processed NTL6 form (indicated by an arrow) increased by more
than 30-fold during the time course. Notably, the full-size form
and the post-translationally modified form were also elevated by
approx. 2-fold after cold treatment, indicating that cold enhances
NTL6 processing, as well as its protein stability.
It seems that the NTL6 activity is also regulated at the
transcriptional level. The NTL6 gene is induced to a moderate
level (∼ 2-fold changes) by cold when the transcript level is
assayed in whole plants [10]. To more precisely examine the
effects of cold on the NTL6 transcription in different plant tissues,
transgenic plants expressing a pNTL6–GUS gene fusion, in which
the NTL6 gene promoter sequence (approx. 2 kbp in length) was
fused transcriptionally to a GUS-coding sequence, were treated
with cold. Under normal growth conditions, the GUS protein was
detected to a relatively higher level in the guard cells and vascular
tissues, including leaf veins and root vasculature (Figure 1C).
The assays revealed that, after cold treatments, the distribution
of the GUS activity in plant tissues was largely unchanged,
but its activity level was visibly elevated in the plant tissues.
Measurements of the GUS gene transcripts in the shoots and
roots by qRT-PCR showed that it is induced to a similar level
(∼ 2–2.5-fold changes) in the plant samples (Figure 1D). These
observations indicate that the NTL6 activity is regulated primarily
at the processing step, and additionally at the transcriptional level.
Heat diminishes NTL6 protein stability
Physico-chemical properties of the membranes, such as lipid
composition and membrane fluidity, are influenced by ambient
temperatures: while lower temperatures reduces membrane
fluidity, higher temperatures increase it [19,25]. Our results
indicate that NTL6 processing and protein stability are closely
related with membrane fluidity, suggesting that NTL6 processing
and protein stability are also affected by heat.
We first analysed the phenotypes of the 35S:6ΔC transgenic
plants, which exhibit severe phenotypic changes, such as dwarfed
growth and altered leaf morphology [16], at different growth
temperatures. When grown at 23 ◦C, more than 70 % of the
35S:6ΔC transgenic plants showed a severe phenotype (type I)
(Figure 3A). Interestingly, the severe phenotype was considerably
rescued when grown at high temperature (28 ◦C), with more plants
exhibiting a mild phenotype (type II). One possible explanation
for the heat effect would be that the NTL6 protein undergoes rapid
turnover at high temperatures.
To examine this hypothesis, the 35S:6ΔC transgenic plants
were treated with high temperature (37 ◦C) for 1–3 h, and the
pattern of NTL6 processing was analysed. As expected, the NTL6
protein stability decreased in the heat-treated plants (Figure 3B).
Although it was unclear whether heat reduces NTL6 processing
or not, since it occurs at a very low level under normal growth
conditions, heat seems to have a negative effect on NTL6
c The Authors Journal compilation c 2010 Biochemical Society
362
Figure 1
P.J. Seo and others
Cold improves NTL6 processing and protein stability
(A) Kinetics of cold effects on NTL6 processing. 35S:NTL6 transgenic plants (2 weeks old) grown on MS-agar plates were used for cold treatments. The NTL6 proteins were detected by Western
blot analysis using an anti-Myc antibody. The full-size (indicated by an asterisk), post-translationally modified (indicated by an arrowhead) and processed (indicated by an arrow) NTL6 forms are
indicated. Part of a Coomassie-Blue-stained gel is shown as a loading control. The molecular mass in kDa is indicated on the left-hand side. The intensities of blots were quantified by densitometry
of images using the Labwork image acquisition and analysis program (Media Cybernetics) (right-hand panel). The ratios of the processed NTL6 forms (arrow) relative to the post-translationally
modified NTL6 forms (arrowhead) at each time point were compared with the ratio at time point 0. Three blots were measured and averaged. Values are means +
− S.E.M. (B) Effect of cold on NTL6
processing and protein stability. Protein sample processing was as described in (A). The ratios of the processed NTL6 forms relative to the post-translationally modified NTL6 forms at 4 ◦C were
◦
compared with the ratios at 23 C (bottom panel). Three blots were measured and averaged. Values are means +
− S.E.M. (C) Cold induction of NTL6 . The pNTL6 –GUS construct, in which a NTL6
gene promoter sequence (approx. 2.0 kbp) was transcriptionally fused to the GUS gene sequence, was transformed into Arabidopsis . Transgenic seedlings (2 weeks old) grown on MS-agar plates
were treated with cold for 6 h and subject to GUS staining. (D) Relative levels of the GUS transcripts in the shoots and roots. The shoots and roots of the seedlings described in (A) were used for
RNA extraction. Transcript levels were determined by qRT-PCR. Biological triplicates were averaged and statistically treated using a Student’s t test (*P < 0.01). Values are means +
− S.E.M.
processing. We were not able to detect the processed NTL6
form in plants exposed to heat. The negative effects of heat
on NTL6 protein stability and processing are consistent with
the positive effects of cold, and further support the potential
correlation between NTL6 processing and membrane fluidity.
arrowhead) NTL6 forms also increased after DMSO treatment.
In contrast, the processed NTL6 form decreased in plants treated
with a 18:3 unsaturated fatty acid, linolenic acid (Figure 4B).
Taken together, these observations strongly support the notion
that changes in membrane fluidity regulate NTL6 processing.
NTL6 processing is regulated by changes in membrane fluidity
Cold-induced NTL6 processing is an early event in cold signalling
We examined more directly the relationship between NTL6
processing and changes in membrane fluidity by employing a
battery of pharmacological agents that affect membrane fluidity
[19].
When the 35S:NTL6 transgenic plants were treated with
DMSO, which is known to reduce membrane fluidity and mimic
cold effects [19], the processed nuclear NTL6 form drastically
increased within 1 h (Figure 4A, indicated by an arrow). Both the
full-size (indicated by an asterisk) and modified (indicated by an
Cold-induced changes in membrane fluidity, which are probably
the earliest direct events that occur during cold acclimation
[19,22], are followed by abrupt calcium influx and restructuring
of actin cytoskeletons.
We therefore examined how the NTL6 processing event
is related to the calcium influx and adjustments of actin
cytoskeletons. NTL6 processing was unaffected by the CD or
JK (Figure 4C), both of which alter the organization of actin
cytoskeletons [19]. Nor was it influenced by the calcium chelator
c The Authors Journal compilation c 2010 Biochemical Society
NTL6 processing and membrane fluidity
Figure 3
Figure 2 Cold effect on NTL6 processing and protein stability is reduced in
cold-acclimated plants
The 35S:NTL6 transgenic plants, cold treatments, SDS/PAGE analysis and detection of the
NTL6 proteins were as described in Figure 1(A). (A) Effects of cold acclimatization on NTL6
processing. The 35S:NTL6 transgenic plants were grown on MS-agar plates either at 23 ◦C or
at 16 ◦C for 2 weeks, and subsequently incubated at 16 ◦C, 8 ◦C or at 4 ◦C for one additional
day before harvesting plant materials. Whole plants were used for preparation of total cellular
extracts. The lower panel displays a graphic presentation. (B) Kinetics of cold effects on NTL6
processing. The 35S:NTL6 transgenic plants grown on MS-agar plates at 23 ◦C for 2 weeks
were exposed to 4 ◦C for the time periods indicated. The molecular mass in kDa is indicated on
the left-hand side. d, days.
BAPTA and by the calcium influx agonist Bay K 8644 or
antagonist A23187 (Figure 4D). These results indicate that coldinduced NTL6 activation occurs at a very early stage of cold signal
transduction. Alternatively, the NTL6-mediated cold signalling
pathway would be independent of the intracellular events occuring
during cold acclimation.
NTL6 processing is altered in fatty acid mutants
To pursue our conclusion further, Arabidopsis mutants with
altered membrane lipid compositions and thus with modified
membrane fluidity were employed in the assays of NTL6
processing. Of particular interest was the ssi2/fab2 (suppressor
of SA-insensitivity 2/fatty acid biosynthesis 2) mutant that was
phenotypically quite similar to the 35S:6ΔC transgenic plants
in that it also exhibits stunted growth with leaf curling [26].
Interestingly, PR1, PR2 and PR5 were up-regulated, but the
expression of PR3 and PR4 was unaltered in the ssi2 mutant
[27], a pattern that is strikingly similar to those patterns observed
in the 35S:6ΔC transgenic plants [16]. The SSI2 gene encodes
363
Heat reduces NTL6 processing and protein stability
(A) Effects of heat on the phenotypes of the 35S:6ΔC transgenic plants. The transgenic plants
were grown on MS-agar plates for 2 weeks either at 23 ◦C or at 28 ◦C (upper panel). The
ratios of the transgenic plants with severe (type I) and mild (type II) phenotypes were calculated
(left-hand lower panel). Three countings, each consisting of approx. 50 seedlings, were averaged
and statistically treated; values are means +
− S.E.M. (Student’s t test, *P < 0.01). (B) Effects of
heat on NTL6 processing. 35S:NTL6 transgenic plants (2 weeks old) grown on MS-agar plates
were treated with heat (37 ◦C) for the time periods indicated, before harvesting plant materials.
Protein sample processing was as described in Figure 1(A). The molecular mass in kDa is
indicated on the left-hand side.
a stearoyl-acyl desaturase governing overall levels of desaturated
fatty acids in plant cells [28]. Similarly, the FAD2 (fatty acid
desaturase 2) and FAD3 desaturases catalyse the 18:1 to 18:2 and
18:2 to 18:3 desaturation steps respectively [29], and function
primarily in the extrachloroplastic membranes [30].
To examine the effects of these mutations on NTL6 processing,
the MYC–NTL6 gene fusion was transformed into these mutants,
and patterns of NTL6 processing were analysed. Although NTL6
processing was unaltered in the fad2 background, it increased
by several fold in the fad3 background, and was further elevated
after cold exposure (Figure 5A; and see the discussion). NTL6
processing was also elevated in the ssi2 background when grown
at 23 ◦C, and even more rapidly after cold exposure (Figure 5B).
We also examined the pattern of NTL6 processing in the
fad3/7/8 triple mutant, in which the level of trienoic fatty acid
is very low and thus membrane fluidity is likely to be greatly
influenced [31]. The MYC–NTL6 gene fusion was transformed
into the triple mutant. The degree of NTL6 processing in the
triple mutant transformed with the gene fusion (fad3/7/8:MYC–
NTL6) was similar to that in the 35S:NTL6 transgenic plants under
normal growth conditions. However, it was higher by approx. 4fold in the triple mutant when exposed to cold (Figure 5C), further
supporting the effects of membrane fluidity on NTL6 processing.
Meanwhile, we observed that DTT and tunicamycin, which
cause ER stress responses [11], did not discernibly affect NTL6
c The Authors Journal compilation c 2010 Biochemical Society
364
Figure 4
P.J. Seo and others
NTL6 processing is elevated by reduced membrane fluidity
Protein sample preparation and processing, quantification of the intensities of blots, and
calculation of the ratios of different NTL6 forms were as described in Figure 1(A). 35S:NTL6
transgenic plants (2 weeks old) grown on MS-agar plates were treated with chemicals for
the time periods indicated before harvesting plant materials. Whole plants were used for
preparation of total cellular extracts. Values are means +
− S.E.M. (A) Effects of DMSO on NTL6
processing. Plants were transferred to liquid MS cultures supplemented with DMSO at different
concentrations for 3 h. (B) Effects of linolenic acid (LA) on NTL6 processing. Plants were
transferred to liquid MS cultures supplemented with 0.4 % LA and incubated for 3 h. (C) and (D)
Effects of pharmacological agents on NTL6 processing. A, A23187; B, BAPTA; K, Bay K8644;
Mo, mock. The molecular mass in kDa is indicated on the left-hand side.
processing (Figure 5D), indicating that NTL6 is not related to the
ER stress response, unlike the bZIP MTFs.
NTL6 is processed via an RIP process
A critical question arose as to how NTL6 is released from the
membranes. Most of the known MTFs were released from
the membranes either by ubiquitin-dependent proteasome
activities or by intramembrane protease activities [2,11,12].
Several membrane-bound proteases, such as S1P, S2P, presenilins
and calpains, have been shown to mediate MTF activation
in higher eukaryotes. S1P and S2P were widely involved in
MTF activation, such as SREBP and ATF6 [1,32]. Presenilins,
membrane proteases (γ -secretase), were involved in APP
(amyloid precursor protein) and Notch activation [3].
c The Authors Journal compilation c 2010 Biochemical Society
Figure 5 NTL6 processing is altered in Arabidopsis mutants with disturbed
membrane lipid compositions
Plants (2 weeks old) grown on MS-agar plates were used in the assays. Protein sample
preparation, processing and detection of the NTL6 proteins, and densitometric measurements
of blots were as described in Figure 1(A). Values are means +
− S.E.M. (A) NTL6 processing
in the fad2 and fad3 backgrounds. A MYC –NTL6 gene fusion was transformed into the fad2
and fad3 mutants. (B) NTL6 processing in the ssi2 background. A MYC –NTL6 gene fusion
was transformed into the ssi2 mutant. (C) NTL6 processing in the fad3 /7 /8 triple mutant. A
MYC –NTL6 gene fusion was transformed into the fad3 /7 /8 triple mutant. (D) Effects of ER
stress-related chemicals. DTT was prepared in water, and tunicamycin (TM) was prepared in
DMSO. The molecular mass in kDa is indicated on the left-hand side. Mo, mock.
Both the full-size and processed NTL6 forms increased in the
35S:NTL6 transgenic plants after treatment with MG132, a potent
26S proteasome inhibitor (Figure 6A), suggesting independence
from ubiquitin/proteasome-dependent proteolysis.
To explore whether any known proteases mediate NTL6
processing, we transformed the MYC–NTL6 gene fusion into
various protease mutants and examined profiles of the NTL6
proteins in the transgenic plants. NTL6 processing was unaltered
in the known mutant backgrounds (Figures 6B and 6C), including
the s1p mutant and the ps1 and ps2 mutants that have mutations
in the presenilin1 and presenilin2 proteases respectively. Clearly,
the processing of NTL6 is distinct from that of other MTFs in
plants, such as AtbZIPs, which are activated by an Arabidopsis
S1P.
Among the MTFs, NTM1, which is structurally similar to
NTL6, is processed by a calpain protease. However, treatments
NTL6 processing and membrane fluidity
Figure 6
365
NTL6 processing is mediated by an RIP mechanism
Plants (2 weeks old) grown on MS-agar plates were used in the assays. Protein sample
preparation and processing and detection of the NTL6 proteins were as described in Figure 1(A).
(A) Effect of MG132. (B) NTL6 processing in s1p . A MYC –NTL6 gene fusion was transformed
into the s1p mutant. (C) NTL6 processing in the ps1 and ps2 mutants. A MYC –NTL6 gene
fusion was transformed into the ps1 and ps2 mutants. (D) Effect of ALLN. The molecular mass
in kDa is indicated on the left-hand side.
with ALLN, which deactivates the calpain proteases [33], also
showed negligible effects (Figure 6D) on NTL6 processing,
indicating that NTL6 processing is not mediated by the calpainlike activity.
NTL6 processing is mediated by a metalloprotease activity
Subsequently, we treated the 35S:NTL6 transgenic plants with
various protease inhibitors. When the plants were treated with a
protease inhibitor cocktail (catalogue number P1599, Sigma–
Aldrich), NTL6 processing was significantly reduced at both 23 ◦C
and 4 ◦C (Figure 7A). To investigate the biochemical identity
of the protease responsible for NTL6 processing, the transgenic
plants were treated with individual protease inhibitors. Among the
protease inhibitors examined, only 1,10-phenanthroline, a zincchelating metalloprotease inhibitor [34], suppressed the NTL6
processing (Figures 7B). In contrast, other protease inhibitors
did not exhibit any discernible effects on NTL6 processing
(Figure 7C), showing that NTL6 is recognized by a plant
metalloprotease activity.
DISCUSSION
Membrane fluidity and NTL6 processing
Accumulating evidence supports the notion that controlled
activation of dormant, membrane-bound transcription factors is
an adaptive way of rapid transcriptional responses, which is critical for plant survival under abrupt environmental changes. In
the present study, we demonstrated that the plasma-membranebound NTL6 transcription factor is proteolytically activated by
a metalloprotease activity under cold conditions. The transcriptionally active NTL6 form is translocated into the nucleus,
where it directly binds to the PR gene promoters, finally
Figure 7
NTL6 processing is mediated by a metalloprotease activity
35S:NTL6 transgenic plants (2 weeks old) grown on MS-agar plates were used in the assays.
Protein sample preparation, processing and detection of the NTL6 proteins, and densitometric
measurements of blots were as described in Figure 1(A). Values are means +
− S.E.M. (A) Effects
of a protease inhibitor cocktail (PI) on NTL6 processing. (B) Effects of a metalloprotease inhibitor
(MI; 1,10-phenanthroline) on NTL6 processing. (C) Effects of individual protease inhibitors on
NTL6 processing. Lane 1, AEBSF; lane 2, antipain; lane 3, aprotinin; lane 4, E-64; lane 5,
leupeptin; and lane 6, phosphoramidon. The molecular mass in kDa is indicated on the left-hand
side.
establishing a cold-induced resistance against psychrophilic
pathogens that frequently propagate during cold seasons [16].
Temperature extremes are critical environmental issues that
profoundly affect plant growth and physiology. Numerous studies
have shown that the physical status of the plasma membranes
is closely related with cold perception and signalling [19–
22]. It has been reported that benzyl alcohol, which increases
membrane fluidity [19], suppresses expression of cold-inducible
genes in Medicago sativa under cold [19]. Inversely, a membrane
rigidifier DMSO induces cold responses even under normal
growth conditions in Brassica napus [35]. Remodelling of
membrane fluidity is one of the earliest cellular events occuring
under cold [19,20]. It has been proposed that cold-induced
membrane rigidification is sensed by certain membrane proteins,
probably acting as cold sensors [19]. Membrane fluidity is
largely determined by the degree to which membrane fatty
acids are unsaturated. It is modulated by a variety of fatty acid
desaturases. Some Arabidopsis mutants with mutations in the fatty
acid desaturase genes exhibit temperature-sensitive phenotypes
[24,36], further supporting the intimate relationship between
membrane fluidity and cold responses. We also found that coldinduced NTL6 processing is elevated by the mutations of the fatty
acid desaturase genes.
c The Authors Journal compilation c 2010 Biochemical Society
366
P.J. Seo and others
Cellular processes adjusting membrane fluidity in response
to low temperatures appear to be somewhat complicated. Coldinduced rigidification of the membranes is established rapidly.
Even a short pulse of cold induces membrane rigidification
[19,35]. However, when plant growth persists at low temperatures,
the membrane fluidity is readjusted, which would be due to
a negative-feedback regulation of the fatty acid desaturase
genes and related cellular mechanisms mediating cold-induced
membrane rigidification [23]. NTL6 processing is induced
by cold, but the inductive effects are evidently reduced in
cold-acclimated plants, unequivocally demonstrating that coldregulated NTL6 processing is affected by changes in membrane
fluidity.
A question arises as to the processing patterns of NTL6 in fatty
acid biosynthetic mutants. While NTL6 processing is unchanged
in the fad2 mutant, which is defective in the conversion step from
18:1 into 18:2 species [37], it is greatly up-regulated in the fad3
and fad3/7/8 mutants, which have a defect in the unsaturation
steps from 16:2 to 16:3 and from 18:2 to 18:3 species [37].
Cold regulation of membrane fluidity is established mainly by
the desaturation of 16:2 to 16:3 and of 18:2 to 18:3 species [23].
It is thus likely that metabolic adjustments of the 16:3 and 18:3
species are not only important for cold-induced adjustment of
membrane fluidity, but also play a role in cold adaptation.
A primary cellular structure for cold perception is considered
to be the plasma membrane, and a protein(s) detecting the coldinduced rigidification of the plasma membrane might serve as
a cold sensor or a primary regulator for cold signalling [19–22].
With this view, it is remarkable that the NTL6 protein is associated
with the plasma membranes and its proteolytic processing is
influenced by changes in membrane fluidity. The transcriptionally
active NTL6 form is translocated from the plasma membranes to
the nucleus. A similar pattern of ligand-induced translocation of
signal receptors from the membranes or cytoplasm to the nucleus
has been demonstrated in many cases [38,39]. For example,
glucocorticoid receptors are cytoplasmic transcription factors.
Upon ligand binding, they are translocated into the nucleus and
regulate glucocorticoid-responsive genes [39]. Epidermal growth
factor receptors are also translocated from the plasma membrane
to the nucleus after ligand binding [38].
It is interesting that a cold-induced transcriptional signalling
is initiated at the plasma membranes, a site that perceives
temperature fluctuations. The subcellular location of the NTL6
transcription factor, cold-induced activation, and its role in
triggering cold signal transduction mediating the pathogenresistance response satisfy, at least in part, the criteria proposed
for as yet unidentified cold sensors [19,24]. However, it is
unlikely that NTL6 processing itself is a constituent of the
cold sensory machinery in plants. Instead, a certain coldregulated fatty acid desaturase(s) or a plasma membraneassociated metalloprotease(s), which is regulated by cold either
at the transcriptional level or at the protein level, may serve
as a primary regulator for the NTL6-mediated cold signalling.
Identification of the protease responsible for NTL6 processing
and elucidation of the mechanisms underlying the regulation
if its activity by changes in membrane fluidity will clarify the
uncertainties.
activity. S2P is a membrane-integrated zinc metalloprotease that
proteolytically activates, in concert with S1P, the ER membranebound SREBP transcription factor in vertebrates [2]. The
Arabidopsis genome has an S2P gene homologue (At4g20310).
We did not examine NTL6 processing in its mutants; they are
currently unavailable in the public databases, possibly because
the mutations may be embryonically lethal [12]. However,
NTL6 processing does not seem to depend on the S2P activity,
since Nicotiana benthamiana leaf cells co-expressing NTL6 and
Arabidopsis S2P did not show altered patterns of NTL6 processing
(P.J. Seo and C.-M. Part, unpublished work). This observation
suggests that the metalloprotease mediating NTL6 processing is
distinct from the S2P protease.
The Arabidopsis genome encodes more than 100 metalloproteases [40]. They play different roles in diverse aspects of
plant developmental processes and stress responses, including
plastid differentiation [41], root and shoot meristem development
[42] and the heat stress response [43]. It is envisioned that
one of the metalloprotease members may be responsible for
NTL6 processing. Large-scale examination of NTL6 processing
in the mutants and transgenic plants overexpressing individual
metalloprotease genes and of their responses to cold and pathogen
infection will help to identify the enzyme that cleaves the NTL6
protein.
Another possibility, although the probability is not high, is
that the NTL6 processing may be autocatalytic. Although the
in-vitro-translated product of the NTL6 gene did not show
any autocatalytic protease activity (results not shown), more
research is needed to further examine whether the NTL6
protein is autocatalytically processed. SDS/PAGE analysis of total
cellular extracts prepared from the 35S:NTL6 transgenic plants
suggested that at least some portion of the NTL6 protein is posttranslationally modified. Post-translational modifications of the
full-size and/or processed NTL6 proteins may be necessary for
the autocatalytic activity to occur. In Synechocystis, a membraneassociated histidine kinase Hik33, which serves as a cold sensor, is
autophosphorylated upon stimulation by cold-induced membrane
rigidification, leading to the induction of cold-responsive genes
[44]. Similarly, Arabidopsis and rice HiK33 homologues, AtHK1
and OsHKs, are also rapidly autophosphorylated upon exposure
to cold, leading to the induction of fatty acid desaturase genes,
such as FAD2 [45]. Similar activation and signalling mechanisms
may be applicable to the NTL6 protein.
AUTHOR CONTRIBUTION
The planning of experiments and writing of the manuscript were carried out by Pil Joon
Seo and Chung-Mo Park. Pil Joon Seo, Mi Jung Kim, Jin-Su Song, Youn-Sung Kim and
Chung-Mo Park contributed to experimental characterization of NTL6 processing. Jin-Su
Song. Hie-Joon Kim and Chung-Mo Park performed work relating to protein structural
analysis of NTL6.
ACKNOWLEDGEMENTS
We would like to thank Dr Pradeep Kachroo for ssi2 and Dr Mi Chung Suh for fad2 and
fad3 , and Dr John Browse (Institute of Biological Chemistry, Washigton State University,
Pullman, WA, U.S.A.) for fad3/7/8 .
FUNDING
Proteolytic activation of NTL6
NTL6 activation depends not on the proteasome activity and
on the known membrane-associated proteases, such as S1P (a
subtilisin-related serine protease), calpains (serine proteases)
and presenilins (γ -secretases), but, rather, on a metalloprotease
c The Authors Journal compilation c 2010 Biochemical Society
This work was supported by the Brain Korea 21, Biogreen 21 [grant number
20080401034001]; National Research Laboratory Programs; the Plant Signaling Network
Research Center [grant number 2009-0079297]; the Korea Science and Engineering
Foundation [grant number 2007-03415]; the National Research Foundation of Korea [grant
number 2009-0087317], the Agricultural R&D Promotion Center [grant number3090175]; and the Korea Ministry for Food, Agriculture, Forestry and Fisheries.
NTL6 processing and membrane fluidity
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Received 16 November 2009/12 February 2010; accepted 16 February 2010
Published as BJ Immediate Publication 16 February 2010, doi:10.1042/BJ20091762
c The Authors Journal compilation c 2010 Biochemical Society