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					Tree Physiology         14,509-5      19
0 1994 Heron        Publishing-Victoriu,           Canadu




Changes in protein synthesis during drought conditioning in
roots of jack pine seedlings(Pinus banksiana Lamb.)

MICHAEL B. MAYNE,‘,* MOHAN SUBRAMANIAN,‘,2   TERENCE J.
BLAKE,2,3 JOHN R. COLEMAN’%* and EDUARDO BLUMWALD’,*
’ Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario MjS 3B2,
  Cunudu




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2 Center,for Plant Biotechnology, University of Toronto, Ontario M5.Y 3B2, Canada
’ Faculty of Forestry, University of Toronto, 33 Willcocks Street, Toronto, Ontario M.5S 3B3, Canada

Received     June 7. 1993




The impact of drought conditioning                on the ability of eight-week-old            jack pine (Pinus hanks&a Lamb.)
 seedlings      to withstand      drought was assessed. Two progressive                 cycles of drought       conditioning       signifi-
cantly increased           the survival    of seedlings     subjected       to a subsequent       prolonged    drought.      The in vivo
accumulation          of several root membrane           proteins     during drought        conditioning     was correlated       with an
increase in seedling survival.            A group of root proteins,           ranging in molecular         mass from 43 to 47 kDa,
 increased accumulation            during one cycle of drought conditioning              and to a lesser extent during two cycles
of drought         conditioning.       The accumulation         of several      low molecular         mass membrane          and soluble
proteins also increased during drought conditioning,                    suggesting    that these proteins may play an important
role in the enhancement            of drought tolerance.       In vitro translation     studies showed a general increase in the
abundance         of protein products       encoded by mRNAs             from drought-conditioned           seedlings.    Although        the
majority      of the in vitro translation      products     appeared in both control and drought-conditioned                   seedlings,
one mRNA encoding a 15 kDa translated                   protein was more prominent              during the second cycle of drought
conditioning.

Keywords: drought stress, protein uwumulation,                     seedling sunsival, transcriptional and trunslutional
control.


Introduction
The degree of drought resistance exhibited by a particular species is related to one of
four basic responses to drought stress: (1) dehydration tolerance-the      ability of cells
and tissues to tolerate and withstand reduced tissue water potential during water
deficit; (2) dehydration avoidance-the        ability of plants to prevent reduction of
tissue water potentials; (3) drought escape-the ability of plants to complete their
life cycle within the period of water availability thereby not experiencing water
deficit; and (4) drought recovery-the      ability of plants to resume growth and finish
their life cycle after a water-deficit period (O’Toole and Chang 1979, Newton et al.
1991). Of these drought responses, dehydration tolerance and dehydration avoidance
are thought to be important components of drought resistance in forest species
(Newton et al. 1991).
    One procedure for improving forest seedling survival is to condition nursery-
grown plants by exposure to variable environments before transplantation to the
510                                                                    MAYNE   ET AL.



forest site. Drought conditioning to enhance drought resistance is a common proce-
dure. Although the mechanism underlying drought conditioning is not known, it has
been suggested that when a plant is exposed to a moderate drought stress, an
enhanced drought resistance response is triggered (Levitt 1980).
   Modification of gene expression can occur in response to drought (Bray 1988,
Gomez et al. 1988, Guerrero and Mullet 1988, Mundy and Chua 1988). Although
there are reports describing the synthesis of low molecular mass proteins during
drought in various angiosperms, for example, tomato (Bray et al. 1990) and alfalfa
(Luo et al. 1992), there have been few studies on changes in gene expression in




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gymnosperms during drought and, particularly, following drought conditioning. We
have examined the effects of drought conditioning on the ability of jack pine (Pinus
banks&a Lamb.) seedlings to survive prolonged water stress. In addition, we have
described changes in root-localized polypeptides that occur during drought condi-
tioning and have analyzed the impact of drought conditioning treatments on mRNA
abundance by in vitro translation.



Materials   and methods

Plant material
Jack pine seeds were obtained from a northwestern Ontario general seed collection
and sown in a 2/l (v/v) peat/vermiculite mixture. The seedlings were grown as
previously described (Blumwald et al. 1989) in a greenhouse for eight to ten weeks
at 24 “C in an 18-h photoperiod in which natural day length was extended with high
pressure sodium vapor lamps (200 pmol m-2 s-l, minimum light intensity).

Plant drought conditioning
Seedlings were transferred to a Conviron 15 E growth chamber, where conditioning
treatments commenced under the same conditions as in the greenhouse. Three
degrees of drought conditioning of jack pine seedlings were obtained by exposing
the seedlings to cycles of progressive non-lethal drought. Cycle A drought-condi-
tioned seedlings were obtained by withholding water over a 3-day period until the
xylem water potential reached -1.1 MPa as determined with a pressure chamber
(Arinad 2, ARJ, Israel). To generate Cycle B drought-conditioned seedlings, Cycle
A drought-conditioned seedlings were rewatered to full turgor for a period of three
days and then water was withheld for an additional three days at which time xylem
water potential dropped to -1.1 MPa. Cycle C drought-conditioned seedlings were
obtained by subjecting Cycle B drought-conditioned seedlings to an additional cycle
of watering to full turgor (three days) followed by withholding water (three days)
until xylem water potential reached -1.1 MPa. At the end of each conditioning cycle
and before rewatering, some of the seedlings were removed from the soil and
prepared for in vivo protein labeling and RNA extraction. The remaining seedlings
were used for survival experiments (see below).
DROUGHT   CONDITIONING    OF JACK   PINE   SEEDLINGS                                 511


Seedling survival
For seedling-survival experiments, eight-week-old jack pine seedlings at the end of
Cycle A, B or C were rewatered and maintained at full turgor for seven days. At this
time, the seedlings were subjected to prolonged drought by withholding water for
varying periods up to nine days. Over this period, xylem water potential values
declined from -0.2 MPa at Day 0 to -5.5 MPa at Day 9. Following the prolonged
drought, the seedlings were returned to normal greenhouse growing conditions and
allowed to recover for three weeks. Seedling survival was then estimated according
to Koppenaal et al. (1991).




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In vivo labeling of seedlings
 Approximately four hundred intact seedlings (at the end of Cycle A or Cycle B) were
 removed from the soil and their roots submerged in a 500 ml solution containing 0.4
 mol mm3 mannitol (osmotic potential = -1.0 MPa) and 1 g mm3 fertilizer solution
 (20,20,20 N,P,K) containing 1.42 MBq (specific activity of 41.26 TBq mmol13)
L-[35S]-methionine (ICN, Irvine, CA). Incubation of the seedlings in the aerated
 [35S]-methionine solution was allowed to proceed for 16 h under normal greenhouse
growth conditions. Control seedlings were incubated under the same conditions in a
fertilizer solution in the absence of mannitol.

Protein extraction and determination of [35S]-methionine incorporation
Proteins were extracted from excised root segments and purified as previously
described by Blumwald et al. (1989) with the following modifications. The homog-
enization buffer included a final concentration of 10% insoluble PVP and 1 mol mm3
benzamidine. For isolation of membrane-bound proteins, the microsomal pellet was
resuspended and layered onto discontinuous sucrose gradients (20,28 and 34% (w/v)
sucrose) and centrifuged in an SW28 rotor (Beckman, Palo Alto, CA) at 80,000 g for
2.5 h. Enriched tonoplast and plasma membrane fractions (Blumwald et al. 1989)
were collected from the O-20% and 28-34% interfaces, respectively, and im-
mediately frozen in liquid nitrogen. Soluble proteins were purified from the micro-
somal supematant by precipitation with TCA followed by centrifugation at 700 g. To
solubilize lipids, the collected pellet was washed with 50 ml l/l (v/v) cold (4 “C)
100% acetone and 95% ethanol, and stored overnight at -20 “C. Precipitated proteins
were collected by centrifugation, lyophilized and then frozen in liquid nitrogen.
   To determine [“?S]-methionine incorporation, immediately following the drought
conditioning cycles (A, B and C), total protein in the root tissue homogenate was
precipitated on ice with 10% TCA (final concentration) and collected by centrifuga-
tion at 700 g. Incorporation of radioactive label was calculated per g of total protein.
Aliquots containing equal amounts of total protein were spotted onto GFC filter
paper, dried, washed once with 10% TCA, and their radioactivity determined by
liquid scintillation spectrometry.

mRNA Isolation
Root tissue from approximately      200 seedlings was ground in the presence of liquid
512                                                                      MAYNE   ET AL.



nitrogen and stored at -70 “C. Extraction of total RNA from the frozen samples was
carried out according to Schneiderbauer et al. (1991) with some modifications.
Following phenol/chloroform/isoamyl      alcohol extraction (24/24/l, v/v), total RNA
was precipitated from the aqueous phase with 2.5 volumes of ethanol in the presence
of a final concentration of 30 mol me3 sodium acetate (pH 5.2) at -70 “C for 1 h.
Excess polyphenols were removed by extracting the RNA solution twice with 30%
(w/v) PEG 6000. Cesium chloride was added to the RNA solution to a final
concentration of 2.4 kmol m-3 and 3 ml of this solution was layered onto a 0.8 ml
cushion of 5.7 kmol mm3 cesium chloride and centrifuged at 376,000 g (rotor




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TLA100.4, Beckman, Palo Alto, CA) in an Optima TL centrifuge (Beckman, Palo
Alto, CA) for 2.75 h. The total RNA pellet was dissolved in RNase-free water and
purified by precipitation with 2.5 volumes of 4 kmol mm3 sodium acetate (pH 7.0) at
-70 “C for 2 h. Poly (A+) RNA was obtained from the total RNA preparation by the
protocol described for the PolyATtract mRNA Isolation System (Promega Biotech,
Madison, WI).


In vitro translation
In vitro translation of mRNAs isolated from the seedlings was achieved by the
protocol described in the Translation In Vitro Technical Manual (Promega Madison,
WI). One pg of mRNA (optical density ratio 260/280 nm = 1.9) was used for each
in vitro translation. Reactions were carried out in a volume of 100 ~1 containing 70 pl
of rabbit reticulocyte lysate, 600 units ml-’ ribonuclease inhibitor (Promega Madi-
son, WI), 2 pmol mm3 amino acid mixture (minus methionine), 10 mol mm3 magne-
sium acetate and 6.2 TBq mol mm3 of [35S]-methionine (Amersham, Mississauga,
Ontario). Individual reactions were incubated for 90 min at 30 “C.


Polyacrylamide gel electrophoresis and autoradiography
Preparation of protein samples of in vivo and in vitro labeled polypeptides for
two-dimensional SDS-PAGE were carried out as previously described (Barkla and
Blumwald 1991). The accumulation of root-localized proteins synthesized during a
 16-h labeling period in Cycle A and Cycle B drought-conditioned        seedlings was
analyzed by two-dimensional PAGE. Protein profiles obtained from tonoplast,
plasma membrane, and soluble fractions, were also compared with labeled polypep-
tides isolated from control seedlings. Samples were heated at 70 “C for 2 min and
quickly chilled on ice for 2 min before loading onto the gel. To analyze polypeptides
that accumulated in vivo, equal amounts of protein (100 pg) were loaded onto 2-D
PAGE gels (12% acrylamide). To analyze in vitro translation protein products,
500,000 counts per minute of TCA-precipitable protein products from mRNAs from
control and drought-conditioned    seedlings were loaded onto 2-D PAGE gels (12%
acrylamide). Isoelectric points were estimated as described by Barkla and Blumwald
(1991). Autoradiograms were obtained by exposing the dried gels to Hyperfilm-
pmax (Amersham, Mississauga, Ontario) for six (in vivo labeled polypeptides) or
three weeks (in vitro labeled polypeptides) at -70 “C.
DROUGHT       CONDITIONING         OF JACK     PINE   SEEDLINGS                                                      513


Results

Seedling survival
Eight-week-old jack pine seedlings were grown under normal greenhouse growth
conditions and then drought conditioned. The drought conditioning treatments did
not result in seedling mortality. Conditioned seedlings were subjected to prolonged
drought stress over a nine-day period. When control and Cycle A drought-condi-
tioned seedlings had water withheld for eight or more days (xylem water potential
I-4.8 MPa) followed by a recovery period, more than 50% of the seedlings died




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(Figure 1). In contrast, Cycle B drought-conditioned   seedlings exhibited enhanced
survival compared with control plants. Three cycles of drought conditioning did not
further enhance seedling survival (Figure 1).

Radiolabel uptake
The amount of [35S]-methionine uptake was determined by measuring the radioac-
tivity in aliquots of TCA-precipitated, total-seedling, root protein. Incorporation of
[Sj5]-methionine by Cycle A, B and C drought-conditioned      seedlings was increased
twofold compared with control seedlings (Figure 2).

In vivo protein labeling
The 2-D PAGE and autoradiographic analyses revealed an increased accumulation
of tonoplast- and plasma-membrane-associated proteins during both the Cycle A
(Figures 3b and 4b, respectively) and Cycle B drought-conditioning        treatments
(Figures 3c and 4c, respectively). A prominent group of proteins (43 to 47 kDa, p1
ranging from 5.7 to 6.5) associated with the tonoplast (Figure 3b) and plasma
membrane fractions (Figure 4b) from roots of Cycle A drought-conditioned        seed-
lings and to a lesser extent from Cycle B drought-conditioned seedlings (Figures 3c
and 4c) were absent from the autoradiograms of the control roots (Figures 3a and 4a).




                  CONTROL        CYCLE A       CYCLE B      CYCLE C

Figure 1. Seedlings were drought-conditioned,          allowed to recover and then severely drought stressed as
described    in Materials  andmethods.      Percentage  survival of drought-conditioned          seedlings was estimated
according     to Koppenaal   et al. (199 1). Cycle A is one cycle of drought conditioning.           Cycle B and Cycle C
are two and three cycles of drought conditioning,         respectively. Statistical    analysis showed that the means
of the controls    and Cycle A (a) and Cycle B and C (b) were significantly            different   (P = 0.05). Values are
means k SD (n = 50).
514                                                                                                               MAYNE       ET AL.




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                0

                                CONTROL          CYCLE A           CYCLE B          CYCLE C

Figure 2. Incorporation       of [%I-methionine         into root proteins   during the labeling period as outlined     in
the Materials  and methods.      Total root protein was extracted        and precipitated as described in the Materials
and methods.    Incorporation      rates were calculated      based on equal amounts of protein, and radioactivity
was determined     by liquid scintillation      spectrometry.    Values are means * SD (n = 3).




Figure 3. Two-dimensional          PAGE of tonoplast           membrane       polypeptides        synthesized      in roots during
Cycle A and Cycle B drought             conditioning.    Proteins     were radiolabeled         with [35S]-methionine           as de-
scribed in Materials      and methods. The abundance           of proteins enclosed by circles increased               during Cycle
A or Cycle B drought conditioning,             whereas the abundance         of proteins      enclosed by squares decreased
during Cycle A or Cycle B drought conditioning.                 (a) Non-conditioned         seedlings;      (b) Cycle A drought-
conditioned    seedlings;     (c) Cycle     B drought-conditioned          seedlings.      Figure     is representative       of five
independent   experiments.

Two 31 kDa polypeptides were prominent in the tonoplast fractions obtained from
roots of Cycle A (~15.5) and Cycle B (~15.3) drought-conditioned seedlings but were
absent in control tissue (Figure 3). The accumulation of a 40 kDa (~15.2) tonoplast-
associated polypeptide was repressed during Cycle A and Cycle B drought condition-
ing (Figure 3). Several smaller polypeptides (molecular mass less than 25 kDa) were
detected only in the tonoplast and plasma membrane fractions from Cycle B drought-
conditioned seedlings (Figures 3 and 4).
   In contrast with the membrane proteins, soluble proteins isolated from roots of
control seedlings appeared to accumulate at a greater rate during the labeling period
DROUGHT       CONDITIONING           OF JACK     PINE SEEDLINGS                                                            51.5




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Figure 4. Two-dimensional       PAGE of plasma membrane             polypeptides      synthesized     in roots during Cycle
A or Cycle B drought conditioning.           Proteins were radiolabeled         with [3’S]-methionine         as described   in
Materials  and methods. The abundance          of proteins enclosed by circles increased          during Cycle A or Cycle
B drought conditioning.     (a) Non-conditioned         seedlings;  (b) Cycle A drought-conditioned             seedlings; (c)
Cycle B drought-conditioned       seedlings.     Figure is representative      of five independent       experiments.


than soluble root proteins of Cycle B drought-conditioned   seedlings (Figure 5). It
should be noted that, although many differences were not apparent, autoradiographic
analysis following 2-D PAGE of equal amounts of soluble protein showed increased
accumulation of some soluble proteins ranging in molecular mass from 14 to 18 kDa
(p1 ranging from 5.0 to 6.4) and molecular masses of 30 to 32 kDa (~15.8) in roots
of seedlings subjected to the second cycle of drought conditioning (Figure 5b)
compared with the control seedlings.

In vitro translation
In vitro translation studies showed a general increase in the abundance of protein
products encoded by mRNAs from Cycle B drought-conditioned              seedlings com-
pared with the control seedlings. In particular, several low molecular weight transla-
tion products (p1 ranging from 5.0 to 5.6) encoded by mRNAs from
drought-conditioned      seedlings increased in abundance relative to translation prod-
ucts obtained from control seedling mRNAs (Figure 6b). Although the majority of
the in vitro translation products were present in both control and drought-conditioned
seedlings, an mRNA encoding a 15 kDa product (p1 6.5) was more prominent in
seedlings subjected to the second cycle of drought conditioning.


Discussion
Jack pine seedlings exhibited enhanced survival of prolonged drought after two
conditioning cycles, suggesting that the seedlings modified their physiology during
the drought-conditioning   cycles and that these modifications resulted in enhanced
drought resistance.
   It is generally thought that total protein accumulation is decreased by drought
(Hanson and Hitz 1982, Hulbert et al. 1988, Valluri et al. 1988, 1989, Bray 1990).
516                                                                                                           MAYNE       ET AL.




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Figure 5. Two-dimensional              PAGE of soluble polypeptides          synthesized      in roots during the second cycle
of drought      conditioning       (Cycle B). Proteins were radiolabeled              with [35S]-methionine     as described  in
Materials      and methods.      Abundance       of proteins   enclosed    by circles increased        during Cycle B drought
conditioning.       (a) Soluble proteins      from non-conditioned         seedlings;     (b) Soluble proteins    from Cycle B
drought-conditioned          seedlings.   Figure is representative      of five independent        experiments.


          a




Figure 6. Two-dimensional           PAGE of in vitro translation          products    from mRNAs          extracted   from roots of
non-conditioned       or Cycle B drought-conditioned             seedlings.     Products     enclosed     by circles increased   in
abundance       during two cycles of drought            conditioning.        (a) Translation       products      using mRNAs     of
non-conditioned       seedlings;    (b) Translation    products      using mRNAs         from Cycle B drought-conditioned
seedlings.    Figure is representative      of three independent        experiments.

However, we observed that the incorporation of [3sS]-methionine into a TCA-precip-
itable fraction doubled during Cycle A drought conditioning and remained high
during Cycle B drought conditioning (Figure 2). The increase in label incorporation
into the TCA-precipitable fraction during drought conditioning was not due to
changes in [“?S]-methionine uptake because both control and drought-conditioned
seedlings displayed comparable rates of radiolabel uptake (63,000-70,000        cpm
mm-‘).
DROUGHT   CONDITIONING   OF JACK   PINE SEEDLINGS                                  517


    Analysis of labeled proteins indicated that the drought-conditioning   treatments
resulted in the accumulation of several tonoplast and plasma membrane polypep-
tides, and the reduction of many soluble proteins (Figure 5) in both Cycle A and
Cycle B drought-conditioned seedlings (Figures 3 and 4).
    Some polypeptides were expressed solely in response to drought in both Cycle A
and Cycle B drought-conditioned seedlings. The accumulation of membrane-associ-
ated 43 to 47 kDa proteins is of particular interest, because polypeptides of a
comparable molecular mass are expressed in slash pine hypocotyl sections when
exposed to mannitol-induced drought (Valluri et al. 1989). In our study, these




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polypeptides were observed in both tonoplast and plasma membrane fractions.
Although the membrane fractions were enriched in tonoplast and plasma membranes
(Blumwald et al. 1989), these fractions are contaminated by proteins associated with
other endo-membranes (in particular Golgi). The proteins expressed in response to
the drought treatments did not accumulate to concentrations sufficient for detection
by silver or Coomassie stain.
    Several membrane-bound and soluble proteins that increased during Cycle A
drought conditioning also remained high during Cycle B drought conditioning. It is
possible that proteins synthesized in response to a mild drought are also required to
elicit an enhanced drought tolerance response. For example, a group of plasma
membrane proteins within the molecular mass range of 43 to 47 kDa (p1 ranging from
5.7 to 6.5) showed increased accumulation in both Cycle A and Cycle B drought-con-
ditioned seedlings.
    Drought conditioning may enhance drought tolerance through gene regulation
thereby changing mRNA populations. Although few changes in mRNA translation
products were observed during drought conditioning, an abundant 15 kDa in vitro
translation product was obtained with mRNAs isolated from Cycle B drought-con-
ditioned seedlings, but not with mRNAs isolated from control seedlings (Figure 6b).
Stress-induced in vitro translation products of similar molecular weight have also
been identified in tomato (Bray 1988, Cohen and Bray 1990, Ho et al. 1991) and a
protein of similar molecular weight, encoded by abscisic acid-responsive genes (rub
genes), has been reported (Yamaguichi-Shinozaki et al. 1989, Vilardelli et al. 1990,
Plant et al. 1991).
   Joshi et al. (1992) suggested that seedlings store mRNAs when fully watered and
only utilize these transcripts to enhance drought tolerance. A result supporting this
possibility was recently reported by Reviron et al. (1992) who identified a 22 kDa
protein that accumulated in drought-hardened leaves of Brussica naps. The tran-
script for this protein was present in control and drought-hardened leaves. The lack
of many new in vitro translation products from Cycle B drought-conditioned       seed-
lings suggests that the acclimation of drought tolerance could rely partly on post-
translational modifications of proteins; however, the abundant 15 kDa in vitro
translated product obtained with mRNA from Cycle B drought-conditioned           seed-
lings may be indicative of induced transcriptionally regulated mechanisms.
   In summary, progressive cycles of drought conditioning significantly increased the
survival of jack pine seedlings when subjected to a subsequent severe drought, and
518                                                                                                                          MAYNE         ET AL.



the synthesis of several root proteins during drought conditioning                                                  correlated with the
increased survival of the seedlings.


Acknowledgments
Research      was supported         by a Strategic        Grant    from     the Natural       Sciences     and Engineering           Council      of
Canada.


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