Plant Physiol. (1990) 92, 1244-1248
0032-0889/90/92/1244/05/$01.00/0
Received for publication September 29, 1989
and In revised form January 8, 1990
Communication
Differences in Proteins Synthesized in Needles of Unshaded and Shaded Pinus ponderosa var Scopulorum Seedlings during Prolonged Drought 1 Nan C. Vance*, Donald L. Copes, and Joe B. Zaerr
U.S. Department of Agriculture Forest SeNice, Pacific Northwest Research Station, Forestry Sciences Laboratory,
(N.C.V., D. L.C.); and Department of Forest Science, Oregon State University (J.B.Z.), Corvallis, Oregon 97331
patterns in roots of Brassica napus detected on two-dimen­
sional silver stained gels (16). The low mol wt polypeptides
were thought to be associated with drought tolerance (16).
If severe water deficit to which a drought-tolerant plant is
adapted induces the synthesis of specific proteins in dehy­
drating tissue, their function in tolerating progressive cellular
water loss may be inferred. Failure of these proteins to be
synthesized in similarly dehydrated tissue of less drought­
tolerant plants may further implicate their adaptive role.
Severe shading reduces the supply of energy and photoassi­
milates needed to confer resistance to drought stress (14) and
has been reported to lower protein content and the activity of
photosynthetic enzymes (8). In P. ponderosa seedlings, shad­
ing was shown to reduce drought tolerance by altering the
physiology and structural constituents associated with toler­
ance of severe water deficits (14).
In this study we investigated gene expression during increas­
ing plant water deficit and recovery in a drought-adapted
species (P. ponderosa var scopulorum Dougl. ex Laws.). We
ABSTRACT
Proteins were radlolabeled and extracted from needles of Pinus
ponderosa var scopu/orum (Dougl. ex Laws.) seedlings progres­
sively drought-stressed for about 1 month. A set of novel, low
molecular weight proteins was detected in fluorographs of two­
dimensional gels when relative water content of needles fell
below 70%. Their synthesis was undetectable in the fully re­
covered seedlings within 48 hours after rewatering. In similarly
stressed seedlings that were shaded to 10% full light, the low
molecular weight polypeptides were not detected or appeared at
very low levels. The shaded seedlings, In which drought tolerance
was reduced, did not recover upon termination of the drought.
The results suggest that protein synthesis induced by water
deficit in drought-tolerant seedlings may contribute to resisting
the effects of cellular dehydration.
Proteins synthesized during prolonged drought in a plant
of documented tolerance to extreme water deficits may indi­
cate genes that control traits of adaptive value. Ponderosa
pine (Pinus ponderosa Dougl. ex Laws.) is a coniferous gym­
report differences in proteins synthesized in needles of
drought-stressed, unshaded seedlings and seedlings shaded to
10% full light.
nosperm native to semi-arid regions of the western United
States. Its establishment and survival depend on characteris­
tics developed early in the seedling stage which enable it to
tolerate severe water deficits (13, 14). Seedlings have survived
over 30 d of drought and water potentials as negative as -6.0
MPa (L. J. Heidmann, personal communication), and re­
covered rapidly when drought was ended (14). Because of its
tolerance of severe water deficit, seedlings of this species
provide an effective plant system for studying gene expression
as an adaptive response to drought stress.
Water stress-induced changes in gene expression have been
well documented, but few studies use drought to impose water
stress. Specific low mol wt proteins (12-40 M,) have been
induced in a variety of plants water stressed by osmoticum
( l, 6, ll, 12). A family of low mol wt, AHA-inducible proteins
(9) was shown to accumulate in embryonic tissue of maturing
seeds as they naturally desiccated (4). Drought stress was
shown to elicit quantitative and qualitative changes in protein
1
MATERIALS AND METHODS
Plant Material
Seed of(Pinus ponderosa, var scopulorum Dougl. ex Laws.)
were obtained from Dr. L. J. Heidmann, Rocky Mountain
Research Station, Flagstaff, AZ. Germinants were reared in a
greenhouse under day/night temperatures of approximately
25• C/15• C, watered daily, and fertilized twice weekly for 14
weeks. Average photon flux density under full light in the
greenhouse was 700 1-1mol m-2 s-. When seedlings were ap­
proximately 15 weeks old, commercial shade cloth was ap­
plied l week before the drought was initiated. Shading of
approximately 10% full light continued for the duration of
the study. Seedlings were sampled every 2 d during the period
of drought. Increasing plant-water deficit was determined by
measuring predawn xylem vi of each sampled seedling with
2
Abbreviations: 'llx, water potential; RWC, relative water content;
rubisco, ribulose,
1,5-bisphosphate carboxylase/oxygenase; LsU,
SSU, large and small subunit of rubisco, respectively; PYPP, polyvi­
nylpolypyrolidone; DIECA, diethyldithiocarbamate.
Supported in part by U.S. Department of Agriculture Forest
Service grant PNW86-396.
1244
1245
REDUCED SYNTHESIS OF DROUGHT STRESS PROTEINS BY LOW LIGHT
a pressure chamber (3). RWC of needles was determined also.
The approximately month-long drought was discontinued
when 'l'x of four consecutively sampled seedlings was < -5.0
MPa. Survival of remaining seedlings was assessed three
months later.
electrophoretic separation, proteins were transferred onto ni­
trocellulose paper. lmmunoassays were performed with horse­
radish peroxidase detection (Vector Laboratories, Burlin­
game, CA). Antisera raised in rabbits against the Rubisco
holoenzyme were provided by Dr. E. J. Pell, Pennsylvania
State University.
Radioactive Labeling
After predawn 'l'x was measured, radioactivity was incor­
porated into needles of sampled seedlings by introducing L­
[3,4,9H]Ieucine (37 MBqfmL; New England Nuclear, Bos­
ton) into the conducting tissue of the severed stem just above
the cotyledons. The severed epicotyl was placed in the pressure
chamber and pressure was applied until sap appeared on the
surface of the cut stem. After blotting the sap, 6 pL of x 10
concentrated 3H-leucine (2.22 MBq/pL) was applied to the
cut surface. The radioactive leucine infiltrated the conducting
tissue as pressure was slowly released. Following uptake, the
epicotyl was secured in a plastic container in 100% humidity
to stabilize its water status, then placed overnight in the
greenhouse. Approximately 1S h later, predawn 'Itx was re­
measured. If 'llx changed by >0.2 MPa the sample was re­
jected. Otherwise, needles were stripped from the epicotyl,
weighed to the nearest mg, then quickly frozen in liquid N2.
Samples were stored at -so• C until proteins were extracted.
Protein Extraction and Electrophoresis
Approximately 300 mg of frozen leaf tissue were ground in
liquid N2 with mortar and pestle. The powder was rapidly
suspended in 15 mL of an extraction buffer consisting of 50
mM Tris-HCl (pH 6.8), PVPP (125 mg/ml), 2% SDS, 20 mM
sodium metabisulfite, 20 mM DIECA, and 2% P-mercapto­
ethanol. The slurry was expressed through Miracloth and
centrifuged at 12,000g for 20 min at 4• C. Proteins were
precipitated in 4 volumes of cold acetone. The acetone­
washed pellet was suspended in Laemmli (7) sample buffer.
Samples were stored at -so• c. Total proteins were quanti­
tated by the Bradford method (2) and 3H cpm were loaded as
determined by TCA precipitable counts.
Two dimensional SDS-PAGE was performed by the
method of O'Farrell (10). Total ampholytes, (pH 5-7 and pH
3.5-10, Serva), were increased to 4% in sample and gel
solutions. The effective focusing range was determined to be
from pH 4.7 to 7.9. Approximately 30 X 105 cpm were loaded
per sample. Mol wt markers were run in the second dimension
(Gelcode). Fluorography was performed on dried gels that
had been fixed, soaked in Enhance (New England Nuclear),
and exposed to Kodak XAR-5 film at -so• C for 10 to 13
weeks. Replicate gels of each sample were also silver stained
(Bio-Rad). Protein patterns were analyzed visually and with
the aid of a two-dimensional scanner and digitizer (Eikonix
Corp., Bedford, MA). Thirty-two fluorographs and silver­
stained two-dimensional gels were examined with two or more
replicates of different levels of needle water loss: no (98-S5%
RWC), low (84-75% RWC), moderate (74-65% RWC), severe
(<65% RWC).
Immunodetection was used to verify the initial identifica­
tion of the large and small subunit of Rubisco on the gels by
visual comparison to the identified subunits (5, 17). After
RESULTS AND DISCUSSION
Analysis of incorporation of [3H]leucine into TCA precip­
itable protein from needles of 60 seedlings sampled during
the month-long drought indicated protein synthesis gradually
declined as water deficits increased, but persisted even in
severely drought-stressed seedlings. The shaded seedlings gen­
erally showed lower synthesis, but at moderate to severe levels
of water loss, the difference was not significant (data not
shown).
The fluorograph in Figure l A is representative of the usual
protein pattern from needles of well-watered, unshaded seed­
lings. Two major polypeptides synthesized in leaves are the
LSU and SSU of Rubisco. They appear as prominent poly­
peptides in the fluorograph.
Fluorographs of unshaded seedlings that were moderately
(Fig. 1B) and severely (Fig. I C) drought stressed show a
different pattern. Eleven major polypeptides between IS and
30 kD appear on the fluorographs (and corresponding silver­
stained gels, not shown). Polypeptides that were unambigu­
ously identified as increasing or newly synthesized are indi­
cated in the boxed areas. The appearance of the LSU and
SSU suggests rubisco continues to be synthesized in needles
under moderate water deficit (Fig. l B), but at much reduced
levels in needles under severe water deficit (Fig. 1C).
In a fluorograph from an unshaded seedling that recovered
48 h after rewatering the low mol wt polypeptides that were
synthesized in the needles of drought-stressed seedlings are no
longer apparent (Fig. 1D). Synthesis of Rubisco subunits
appears to have strongly recovered in the rewatered seedling
which had been drought-stressed for 31 d (Fig. l D). Seedlings
that recovered 4S h after rewatering continued to survive
three months later. The appearance of the drought stress­
induced proteins apparently does not preclude the plant's
recovery from severe water deficit.
In agreement with others (4, 16), we suggest these polypep­
tides may have a protective role under dehydrating conditions.
Synthesis of the low mol wt 'stress' proteins first occurs at
water deficits (RWC
6S%, 'l'x
-3.0), well within the
normal range encountered in environments to which the
seedlings are adapted. Coupled with the seedlings' high sur­
vival rates from water deficits even greater than those at which
the proteins were first detected, the synthesis of these proteins
suggest an adaptive rather than deleterious response to
drought.
The pattern of proteins synthesized in needles of an un­
stressed, shaded seedling (Fig. 2A) were similar to those of the
unstressed or recovered, unshaded seedlings (Fig. 1, A and
D). However, the 11 low mol wt proteins, highly visible in
the fluorograph from an unshaded seedling that was severely
drought-stressed, are not detectable in the representative fluo­
rograph of a shaded seedling that is severely drought-stressed,
are not detectable in the representative fluorograph of a
=
=
1246
VANCE ET AL.
Plant Physiol. Vol. 92, 1990
REDUCED SYNTHESIS OF DROUGHT STRESS PROTEINS BY LOW LIGHT
1247
Figure 2. Fluorographlc analysis of 3H-Iabeled polypeptides extracted from needles and separated by two-dimensional PAGE of (A) unstressed
(>85% RWC) and (B) severely drought stressed (58% RWC), shaded P. ponderosa seedlings. Polypeptides are marked as stated in Figure 1 .
shaded seedling that is comparably drought stressed (Fig. 2B).
Nor were they detected in six other fluorographs from shaded
seedlings with levels of RWC ranging from 75 to 48%. In one
fluorograph (not shown) several drought stress-induced poly­
peptides were detected, but at such low levels as to make their
identity ambiguous.
The 11 polypeptides that were synthesized in the moder­
ately to severely stressed, unshaded seedlings apparently were
not synthesized in the shaded seedlings, or were synthesized
below the threshold of detectability. Severe shading altered
cellular metabolism (15) which may have influenced synthesis
of the 20 to 30 kD drought-stress induced polypeptides. In
addition, synthesis of LSU and SSU in the needles of shaded
seedlings apparently declined early in the progression of
drought stress and ceased altogether when drought stress
became severe (Fig. 2B). The synthesis of rubisco in general
declined more rapidly with increasing cellular dehydration in
the shaded than in the unshaded, seedlings.
Although proteolytic products were not detected on the
fluorographs and silver-stained gels, inability to detect specific
proteins may have been a consequence of proteolytic enzyme
activity and cannot be ruled out. A breakdown in compart­
mentation of proteolytic enzymes with failure of membrane
integrity may have occurred. Evidence of membrane integrity
loss was reported in the shaded seedlings drought stressed to
RWC < 60% (14).
Severe shading effectively reduced drought tolerance in the
seedlings (14). At the end of the drought period the shaded
seedlings that had the same wx levels as the unshaded seedlings
did not recover when rewatered. Whereas no unshaded seed­
ling died until wx < -4.5 MPa, no shaded seedling survived
at that level of water deficit, and mortality occurred at MPa
as high as -1.85 MPa (14).
We do not know if the induction of the low mol wt stress
proteins represents gene expression at the transcriptional or
translational level. Analysis of mRNA and in vitro translations
of proteins from dehydrated needles, mature embryos, and
pollen is proposed further to characterize these proteins. In
Figure 1. Fluorographlc analysis of 3H-Iabeled polypeptides extracted from needles and separated by two-dimensional PAGE of (A) unstressed
(>85% RWC), (B) moderately drought stressed (68% RWC), (C) severely drought stressed (59% RWC), and (D) recovered (>85% RWC) P.
ponderosa seedlings. The large and small subunits of Rubisco are marked with arrows and the 20- to 30-kD polypeptides induced by drought
stress are marked with darts.
1248
VANCE ET AL.
P. ponderosa normal cellular dehydration (RWC < 30%)
occurs at two stages in the life cycle-in the maturing pollen
grain and seed. If these drought-stress induced proteins are
present in mature embryos of seed, or in the male gameto­
phyte during pollination, this provides further evidence of
their adaptive role in cellular dehydration. The failure to
detect synthesis of stress proteins in the severely shaded seed­
lings under water deficits, whether due to cellular injury,
altered metabolism, or the plant's low energy status, needs to
be determined and warrants further study.
LITERATURE CITED
I. Bewley JD, Larsen KM, Papp JET (1983) Water-stress-induced
changes in the pattern of protein synthesis in maize seedling
mesocotyls: a comparison with the effects of heat shock. J Exp
Bot146: 1126-1133
2. Bradford MM (1976) A rapid and sensitive method for quanti­
fication of microgram quantities of protein utilizing the prin­
ciple of protein-dye binding. Anal Biochem72: 248-254
3. Cleary BB, Zaerr JB (1980) Pressure chamber technique for
monitoring and evaluating seedling water status. NZJ For Sci
10: 133-141
4. Dure L, Crouch M, Harada J, Ho TD, Mundy J, Quatrano R,
Thomas T, Sung ZR (1989) Common amino acid sequence
domains among the LEA proteins of higher plants. Plant Mol
Biol12: 475-486
5. Guy CL, Haskell D (1987) Induction of freezing tolerance in
spinach is associated with the synthesis of cold acclimation
induced proteins. Plant Physiol 84: 872-878
6. King GJ, Hussey CE, Turner VA (1986) A protein induced by
7.
8.
Plant Physlol. Vol. 92, 1990
NaCI in suspension cultures of Nicotiana tabacum accumulates
in whole plant roots. Plant Mol Bioi7: 442-449
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680-
685
Levitt J (1980) Responses of Plants to Environmental Stresses,
Vol 2: Water, Radiation, Salt, and Other Stresses. Academic
Press, New York
9. Mundy J, Chua N (1988) Abscisic acid and water-stress induce
the expression of a novel rice gene. EMBO J 7: 2279-2286
10. O'Farrell PH (1975) High resolution two-dimensional electro­
phoresis of proteins. J Bioi Chern 10:4007-4071
I I. Singh NK, Handa AK, Hasegawa PM, Bressan RA (1985) Pro­
teins associated with adaptation of cultured tobacco cells to
NaCI. Plant Physiol79: 126-137
12. Singh NK, Bracker CA, Hasegawa PM, Handa AK, Buckel S,
Hermodson MA, Pfankoch E, Regnier FE, Bressan RA (1987)
Characterization of osmotin. Plant Physiol 85: 529-536
13. Vance NC, Running SW (1984) Summer climatic influences on
Pinus ponderosa planted on mined lands in eastern Montana.
Reclam Reveg Res4: 129-143
14. Vance NC, Zaerr JB (1990) Influence of drought stress and low
irradiance on plant water relations and structural constituents
in needles of Pinus ponderosa seedlings. Tree Physiol (in press)
15. Vance NC, Zaerr JB (1990) Analysis by high-performance liquid
chromatography of free amino acids extracted from needles of
drought-stressed and shaded Pinus ponderosa seedlings. Phys­
iol Plant (in press)
16. Vartanian NC, Damerval C, de Vienne D (1987) Drought-induced
changes in protein patterns of Brassica napus var . oleifera
roots. Plant Physiol 84: 989-992
17. Zivy M, Thiellement H, de Vienne D, Hofmann JP (1984) Study
on nuclear and cytoplasmic genome expression in wheat by
two-dimensional gel electrophoresis. Theor Appl Genet 68:
335-345
CORRECTIONS
Vol.
Vance N.C., Copes D.L., and Zaerr
92: 1244-1248, 1990
J.B.
Differences in Proteins Synthesized in Needles of
Unshaded and Shaded Pinus ponderosa var Scopulorum Seedlings during Prolonged Drought.
The name of the second author, Donald L. Copes, was spelled incorrectly in the original
article. The online version of the article has been revised.
www.plantphysiol.org /cgi/doi/10.1104/pp.109.900301
Vol. 151:
180-198, 2009
Teotia S. and Lamb R.S. The Paralogous Genes RADICAL-INDUCED CELL DEATH1 and
SIMILAR TO RCD ONE1 Have Partially Redundant Functions during Arabidopsis
Development.
The authors have provided this new version of Figure 6, which includes a revised scale bar
and figure legend for easier understanding of the image.
Figure
6. SEM images of the epidermal cells of the
inflorescence stem of (A) Wild type, (B) rcdl-3 and
(C) rcdl-3;srof·f. The double mutant stems have
less cell elongation. The middle portion of two
independent inflorescence stems is shown tbr each
genotype. The scale bar indicates I OO tm.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.900303
966
Plant Physiology®, October 2009, Vol. 151, p. 966, www.plantphysiol.org © 2009 American Society of Plant Biologists