Influence of inadequate water supply on metabolism in biological systems

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Influence of inadequate water supply on metabolism in biological systems
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West, S. H.
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Abstract:
Data have been obtained that show the effect of drought on growth itself and how this reduction in growth may be a result of specific changes in total protein production, nucleic acid metabolism and on functional activity of a fraction of nucleic acids. While the drought treatments decreased total protein by only 40 percent, growth was reduced 80 percent. These data suggested that the synthesis of growth-dependent proteins was being hindered. Although total nucleic acid production was not reduced by the lack of water, the function of the nucleic acid fraction responsible for delivering the genetic information to the process of protein synthesis was altered. This fraction of nucleic acid was not, under water-stress conditions, getting attached to ribosomes. This malfunction prevented the synthesis of a proline-rich protein which is probably required for cell wall production during growth. This information provides a specific selection criterion and should aid in the development of plants and perhaps other organisms that can withstand stress from drought.

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THE INFLUENCE OF INADEQUATE WATER SUPPLY
ON METABOLISM IN BIOLOGICAL SYSTEMS


by


S. H. WEST
Professor of Agronomy
University of Florida


PUBLICATION NO. 9

of the

FLORIDA WATER RESOURCES RESEARCH CENTER


RESEARCH PROJECT TECHNICAL COMPLETION REPORT

OWRR Project Number A-008-FLA

Annual Allotment Agreement Numbers
14-01-0001-1077 (1968)
14-01-0001-1628 (1969)
14-31-0001-3009 (1970)
Report Submitted: August 26, 1970


The work upon which this report is based was supported in
part by funds provided by the United States Department
of the Interior, Office of Water Resources Research,
as authorized under the
Water Resources Research Act of 1964.














TABLE OF CONTENTS


Page
ABSTRACT .................... ...... .... .. ...... .

PROJECT SUMMARY ..... .. ... ................. ...... 2

INTRODUCTION .,. ............ ...... ... ,.. . 3

INITIAL RESEARCH PLAN AND RESULTS .......,.............. 4

CHARACTERIZATION OF RNA ACCUMULATED IN DROUGHT ......... 5

PROTEIN SYNTHESIS ALTERED BY DROUGHT ....... ...,...... 7

LITERATURE CITED ............. ................ 11















ABSTRACT


THE INFLUENCE OF INADEQUATE WATER SUPPLY ON
METABOLISM IN BIOLOGICAL SYSTEMS WITH EMPHASIS ON
PROTEIN SYNTHESIS AND NUCLEIC ACID METABOLISM

Data have been obtained that show the effect of
drought on growth itself and how this reduction in growth
may be a result of specific changes in total protein produc-
tion, nucleic acid metabolism and on functional activity of
a fraction of nucleic acids. While the drought treatments
decreased total protein by only 40 percent, growth was re-
duced 80 percent. These data suggested that the synthesis
of growth-dependent proteins was being hindered. Although
total nucleic acid production was not reduced by the lack
of water, the function of the nucleic acid fraction respon-
sible for delivering the genetic information to the process
of protein synthesis was altered. This fraction of nucleic
acid was not, under water-stress conditions, getting attach-
ed to ribosomes. This malfunction prevented the synthesis
of a proline-rich protein which is probably required for
cell wall production during growth. This information pro-
vides a specific selection criterion and should aid in the
development of plants and perhaps other organisms that can
withstand stress from drought.







West, S. H.
THE INFLUENCE OF INADEQUATE WATER SUPPLY ON METABOLISM IN
BIOLOGICAL SYSTEMS WITH EMPHASIS ON PROTEIN SYNTHESIS AND
NUCLEIC ACID METABOLISM
Completion Report to the Office of Water Resources Research,
Department of the Interior, August, 1970, Washington, D.C.
20240
KEYWORDS: water supply*/ water shortage*/ water utiliza-
tion/ plant growth*/ metabolism/ droughts*/ water consump-
tion (plants)*/ soil moisture/ drainage.















PROJECT SUMMARY


The goal of these studies was to determine what
basic processes or biochemical reactions are susceptible
to stress imposed by reducing the supply of water. This
information would be used in the development of drought-
resistant strains or management practices to alleviate
water stress. The proposed research organism was plants
but extrapolation of the findings to other biological ma-
terials would be meaningful at this subcellular level.
The objectives of the research proposal included an adap-
tation of the research results to screening procedures.
All of these goals and objectives have been obtained.

The initial experiments were designed to survey
the effects of various degrees of water stress on certain
chemical identities that may reflect the response of meta-
bolic processes. These studies revealed that when stress,
as a result of reduced water supply, decreased protein
content by 40 percent, growth as measured by dry weight
was reduced by 80 percent. The effect of water stress on
other constituents, such as total amino acids and nucle-
otides, was similar to that on dry weight and therefore
reflected no specific response to drought. However, total
ribonucleic acid (RNA) increased in seedlings subjected to
drought conditions.

The RNA that accumulated in water stressed
plants was characterized chemically to determine why it
was not functional in protein synthesis. Extensive studies
showed that the nucleotide composition of this RNA had
not been altered by water stress. However, the physical
characteristics had been affected. The individual com-
ponents of RNA were markedly smaller in drought treated
than in control plants. The reduced size resulted because
of the failure of ribosomal RNA to become attached to
messenger RNA. Therefore, information required from the
genetic material to direct the synthesis of specific
proteins was not being delivered to the ribosomes. The
lack of attachment can be used as a selection criterion
to screen for plants that are resistant to drought.

The amino acid proline accumulated in plants sub-
jected to water stress treatments. Isotope incorporation
studies showed that this accumulation was a result of the









lack of synthesis of a proline-rich protein. Together
these results support the conclusion that drought altered
the function of messenger RNA in plants and this malfunc-
tion prevents the synthesis of a cell-wall-type protein.

Publications that have resulted from this project
thus far are:

West, S. H., 1966, How Water Affects Plant Life.
Weeds, Trees and Turf Magazine, p. 12-14.

West, S. H., 1966, Sub-cellular Physiology as
Affected by Drought. Proc. X International Grassland
Congress, Helsinki, Finland, p. 91-94.

Shiralipour, Aziz and S. H. West, 1968, Effects
of Water Stress on Amino Acids, Protein and DNA Content of
Corn Shoot at Different Ages. Soil and Crop Sci. Soc. of
Fla. Proc. Vol. 28, p. 115-122.

Shiralipour, Aziz and S. H. West, 1970, Specific
Protein Synthesis Altered by Water Stress. In preparation.


INTRODUCTION

Throughout the worlddrought conditions reduce the
growth of plants and often contribute significantly to
stand failure. Slight improvements in adaptation to these
stress conditions have been made, but progress is slow.
The time required for the development of drought resistant
plants is prohibitive because of the selection criteria
available. Selection is usually based upon survival. Often
failure to survive cannot be attributed to one factor of
the environment. Furthermore, the environment is variable
from year to year and selections that are adapted to an
environmental factor that predominated in one season may
be lost in another season. A response to the environment
at the molecular level is needed if a specific effect is
to be resolved.

The purpose of these studies was to determine
what basic processes are affected by the stress imposed by
reducing the supply of water. This information would be
used in the development of drought-resistant strains or
management practices to alleviate water stress.










INITIAL RESEARCH PLAN AND RESULTS


The data reported here are from experiments de-
signed to survey the effect of various degrees of water
stress on certain chemical identities that may reflect the
response of metabolic processes. The quantitative changes
of endosperm-scutellum and seedling protein, nucleotides,
and RNA during germination and growth under water stress
have been measured. The qualitative changes in seedling
nucleotides and RNA were determined by ion-exchange chro-
matography and spectral analyses.

The design of these experiments was such that the
effect of a sub-optimal supply of water to plant roots
could be observed without the usual complicating factors
of loss of turgor and reduced ion absorption. The atmos-
phere of the growth chamber was maintained near 100 percent
relative humidity so that wilting did not occur, and only
nutritive ions from the seed were available to all treat-
ments. The quantity of mannitol that moved into the seed-
lings was not measured. The effect of mannitol on growth
is assumed to be only a result of a decreased supply of
water.

Reducing available water by increasing the osmotic
pressure of the medium surrounding the seeds resulted in a
marked reduction in growth rate of the seedlings as shown
by the dry- and fresh-weight. The dry-weight increases of
the seedlings grown without water stress were similar to
those reported elsewhere and after 6 days of incubation
were approximately three times as great as the increase of
the seedlings subjected to the greatest water deficiency
(14.7 atm of osmotic pressure) for the same period. The
increase in dry weight of the seedlings occurred at the
expense of the endosperm-scutellum dry weight. Treatments
that limited water entry and plant growth also reduced the
dry-weight loss in the endosperm-scutellum.

Protein accumulated in the seedling during 1 to
6 days of germination and growth at a rate similar to the
dry-weight increases. Also like the dry-weight changes, a
decrease in endosperm-scutellum protein accompanied the
increase in seedling protein. Restricting the water supply
slowed protein degradation in the endosperm-scutellum and
the formation of protein in the seedling.

Nucleotides increased in seedlings and endosperm-
scutella during incubation. The magnitude of increase was
greater in the seedlings. The quantity of nucleotides per
plant was reduced by water stress, and the level per gram
of dry weight was reduced initially. However, the initial










decrease was followed by an accumulation to a level higher
than the check treatment. With increasing water stress,
more time was required for the accumulation. Throughout
the 6 days of growth the nucleotides in the check seedlings
remained constant. A disruption in normal nucleotide meta-
bolism as indicated by lower nucleotide content would be
expected to adversely affect RNA formation.

The RNA in the seedlings increased during the 6
days of growth and the endosperm-scutellum RNA decreased.
The rapid degradation of endosperm-scutellum RNA that oc-
curred during the first 3 days of germination in the check
plants was slowed by the water stress. Unlike seedling
protein and nucleotides, seedling RNA increased when water
was limited.


CHARACTERIZATION OF RNA ACCUMULATED IN DROUGHT

The initial or survey studies showed that growth,
amino acids, protein, and nucleotide contents decreased
with imposed water-stress treatments, but RNA increased.
The following studies were designed to determine what
fractions of RNA were accumulating and to characterize that
RNA. Protein is an important constituent of the enzymes
which catalyze the thousands of chemical reactions required
for the growth of a plant. Protein synthesis is dependent
upon ribonucleic acid in ribosomes, messenger (mRNA) and
transfer RNAs. Ribosomes form the site upon which amino
acids are joined. Transfer RNA brings the amino acids to
the ribosomes. Finally, mRNA is the vehicle by which the
genetic information in the nucleus is conveyed to the ribo-
somes where this information directs the polymerization of
the various proteins.

Zea mays L. seedlings were grown in darkness in
a constant temperature of 800F and 95 percent relative
humidity. The test material consisted of one-centimeter
sections harvested from the stem immediately above and below
the first node of 6-day-old plants. The treatments were:
(1) plants grown for 6 days with a continuous supply of
water to the roots, (2) similar to treatment (1), except
that the roots were supplied with a solution containing
sufficient mannitol to provide 9.8 atmospheres of osmotic
pressure, (3) plants grown in the mannitol treatment for
4 days and then transferred to the water supply for 2 days.

These experiments showed that drought conditions
reduced fresh weight to approximately one half that of the
control plants, but when the stress was released, growth










was resumed at a rapid rate. Total cytoplasmic RNA in-
creased per section and per cell. A large part of the
increase was attributed to an increase in ribosomes. The
level of RNA was not significantly decreased in the stress-
release plants. These data should be compared to those that
have shown a positive correlation between RNA content and
the capacity to make protein. Furthermore, the level or
content of ribosomes have been shown in other studies to
regulate the rate of protein synthesis. Our data show
that drought-stressed plants synthesized more ribosomes
upon which protein could be made, yet growth was reduced.

The author surveyed some of the characteristics
of the ribosomes that accumulated in plants subjected to
drought conditions to determine why they were not functional
in the period of slow growth. Conceivably the composition
of nucleotide bases in the RNA may reflect changes suffi-
cient to render the ribosomal units non-functional in pro-
tein synthesis. To check this feature, ribosomes were
isolated from the control and water-stress plants, the RNA
was hydrolyzed, and chromatographed on a Dowex-1 column.
These experiments showed that the quantities of each of the
4 nucleotides were similar in plants from each treatment,
In other tests, P32 was added to the incubation solution
and the amounts of the isotope occurring in each nucleotide
showed also that the base composition was not altered by
the water-stress treatment. These data show: (1) that the
ribosomes which accumulated in the water-stress condition
were not abnormal in base composition, (2) that there was
no preferential synthesis of nucleotides, and (3) that
there was no preferential incorporation of nucleotides into
RNA.

The above studies indicated that the single ribo-
some unit was not altered by the drought condition and
therefore, would not account for the effect on growth. In
previous studies the number of single 70s ribosomes that
clustered together into aggregates appear to be related to
the rate of protein formation. The ratios of the amounts
of RNA occurring in fractions containing particle sizes
70s, 70s x 2, 70s x 3, and those greater than 70s x 3 show
a positive correlation between ribosome cluster size and
growth rate. Slow growth of plants under drought stress
was associated with a high level of single 70s particles.
The plants in the control treatments contained a higher
level of clusters larger than single 70s particles than
that of the stress plants. The rapid rate of growth in the
drought-released plants resulted in levels of clusters mark-
edly higher than that of the control plants. Electron-
microscope studies using permanganate fixing and platinum
shadowing revealed more large clusters in the grid fields
in fast growing plants than in those growing under stress.










Clusters of ribosomes may be formed by the attach-
ment of messenger RNA (mRNA) to individual ribosomes as
visualized in various reports. If this is a necessary step
in the process of protein synthesis, measurements of
clusters are some measure of amounts and participation of
the mRNA in groqth. In addition, mRNA has been described
as a rapidly P" labelled fraction of RNA, in contrast to
ribosomes. Therefore, providing test plants with the iso-
tope for a brief period and then isolating the RNA fractions
with their associated counts is still another way of cor-
relating mRNA with growth and with polysome formation. Re-
presentative data from these experiments are shown in Table
5. These data show that the RNA fractions that contain more
than one ribosome contain high levels of P32 and suggest
that growth is indeed associated with polysome formation.

These data further indicate that stress from
drought adversely affects mRNA. This information provides
a specific selection criterion and should aid in the de-
velopment of strains of herbage plants that can withstand
stress from drought. Plant materials can now be screened
and selections made on the basis of susceptibility of mRNA
to water-stress. In addition, methods of inducing resis-
tance to drought can now be accurately followed.


PROTEIN SYNTHESIS ALTERED BY DROUGHT

The regulation of growth in biological systems
is considered an expression of highly organized and ordered
protein synthesis. Drought, in most plants, invariably
reduces growth. This reduction in growth appears to be
partially associated with an alteration of protein meta-
bolism (Barnett and Naylor 1966, Ben-Zione et al 1967,
Henckel 1965, Shah and Loomis 1965, and West 1962). The
development of drought resistance in plants might be fa-
cilitated if the mechanism of growth regulation were by a
specific protein and if this mechanism were elucidated.
The previous work on this project showed that the appa-
ratus for polymerizing proteins was altered by water
stress. Since the altered portion was that involved with
the direction of various proteins, our research was aimed
at determining if water-stress affected specific proteins.

Research reported earlier by our laboratory and
by others has shown that drought stressed plants have a
high level of the amino acid proline. The research re-
ported here was designed to determine if the accumulation
of the amino acid proline during drought is a result of
protein degradation or of the reduced synthesis of a
specific proline-rich protein.










Drought treatments resulted in a 58 percent re-
duction in fresh weight per shoot and 40 percent in dry
weight as compared with the control treatment. The re-
duction for fresh and dry weight of the root and shoot was
58 percent and 30 percent respectively. The plants grown
under control conditions for 1 day after 4 days of drought
made a marked recovery in both fresh and dry weights. All
compounds were compared on a per cell (mg DNA) (Ingle
et al 1964) basis. Drought affected weights and protein
content of roots and shoots similarly. Protein content
per cell was not changed by the treatments; however,
drought reduced the total protein per seedling.

Total free amino acids in the shoots and roots of
the seedlings grown under drought conditions were not
significantly increased as compared with controls. In the
24 hours after release from drought treatment, the change
in total amino acids was negligible. While proline,
phenylalanine, glutamine and asparagine increased in the
shoots of the drought treated seedlings, the increase in
proline was greater than that for any other amino acid.
Many workers (Barnett and Naylor 1966, Chen et al 1964,
Kemble and Macpherson 1954, Mathes 1956, Saunier 1968,
Steward et al 1966) have reported increases in free amino
acids under drought conditions. In one case free proline
increased 10 to 100 fold (Barnett and Naylor 1966). The
increase in amino acids has been attributed to a degrada-
tion of protein (Kemble and Macpherson 1954, Mathes 1956).
However, our results which show no significant decrease
in protein, or increase in total amino acids tend to refute
this suggestion. The lack of decrease in protein in
drought treated plants does not preclude the possibility
that some protein was being degraded while others were being
formed.

Instead of degradation of protein we propose that
the accumulation of certain amino acids, especially proline,
in the drought treated seedlings was partially, if not com-
pletely caused by their continued synthesis or transfer
from seed to shoot and root with a reduction in the rate
of incorporation nto protein. This is supported by our
observation that C labeled proline, an amino acid which
consistently accumulated in drought conditions was incor-
porated into cytoplasmic protein of shoot at only about
one-third the rate in drought treated plants as in the con-
trols. In a similar comparison of treatments in roots, the
proline-14C incorporated into the cytoplasmic proteins of
drought treated plants was about one-half that in control
plants. Furthermore, plants that had been in dr ght
conditions for 4 days incorporated more proline- -C when
released from drought for 1 day than did the plants in
continuous control treatments. These data further demon-
strate that some protein synthesis was inhibited during









drought conditions and was rapidly reinitiated when drought
stress was released, and that these proteins are especially
rich in proline. The reduction in the rate of incorporation
of 14C-proline in stressed seedlings can not be attributed
to isotopic dilution, since maximum specific activities of
proline pools in control and stressed shoots and roots are
very similar.

In contrast to proline, serine did not accumulate
under drought conditions and its incorporation into protein
was neither reduced by drought nor appreciably increased
after the plants were released from drought.

From the data it is possible to propose that plant
cells under drought stress actively accumulate proline not
only because of lack of incorporation into protein, but also
for other, as yet undetermined, requirements of the stressed
plants. The total uptake of proline- 1C by the cells of
shoots from plants under drought conditions was about 3
times greater than in the control treated plants and the up-
take in the drought treated roots was about 2 times that in
the control roots. The level of proline in the shoots was
reduced when water was supplied to the drought treated
plants. Again, in contrast to proline, the total uptake of
serine-14C by the roots and shoots of drought stressed
plants was less than by control plants (comparison to the
control treatment). Plants under drought conditions may
preferentially synthesize proline and this may also contri-
bute to the accumulation of that amino acid.

Results from studies on the effect of drought on
nucleic acid metabolism are consistent with findings concern-
ing accumulation of certain amino acids and lack of incor-
poration into protein discussed above. Marcus and Feeley
(1965) showed a low level of amino acid incorporation into
protein in the cotyledon of the unimbibed peanut seed.
After water was added, imbibed seeds incorporated the amino
acids at a much higher rate. Furthermore, they showed that
the unimbibed seed possessed the entire apparatus for pro-
tein synthesis except messenger RNA was not adequate or was
in an inactive form. Polysomes were formed and protein
synthesis was resumed upon addition of water in the cotyle-
dons and embry (Marcus and Feeley 1965). West (1966) attri-
buted a reduced polysome formation under water stress con-
ditions to a possible messenger RNA malfunction. In our
experiments the addition of water caused rapid incorporation
of proline- C into protein, possibly due to the formation
of activation of specific messenger RNA and eventually
polysome formation.









The partial inhibition of proline incorporation
into protein by water stress appears to be completely re-
versible through the addition of water and the treatment
does not affect the genetic apparatus since the plant re-
covers. The amounts of proline- 4C and serline- 4C incor-
porated into protein are inverse to their quantities as
free amino acids.


Acknowledgement. This research was supported in
part by the Office of Water Resources Research, U. S. De-
partment of the Interior, as authorized under the Water
Resources Research Act of 1964, public law 88-379.















LITERATURE CITED


Barnett, N. M. and Naylor A. W.: Amino acid and protein
metabolism in Bermudagrass during water stress. Plant
Physiol. 41: 122-30. 1966.

Ben-Zione, Aliza, Itai, C., and Vaadia, Y.: Water and salt
stresses, kinetin and protein synthesis in tobacco
leaves. Plant Physiol. 42: 361-65. 1967.

Chen, D., Kessler, B., and Monselise, S. P.: Studies on
water regime and nitrogen metabolism of citrus
seedlings grown under water stress. Plant Physiol.
39: 379-86. 1964.

Henckel, P. A.: Physiology of plants under drought. Annual
Rev. Plant Physiol. 15: 363-86. 1965.

Ingle, John and Hageman, R. H.: Studies on the relationship
between ribonucleic acid content and the rate of growth
of corn roots. Plant Physiol. 39: 730-34. 1964.

Kemble, A. R. and Macpherson, H. T.: Liberation of amino
acids in perennial rye grass during wilting. Biochem.
J. 58: 46-50. 1954.

Marcus, A., and Feeley, J.: Activation of protein synthesis
in the inhibition phase of seed germination. Proc.
Natl. Acad. Sci. (U.S.) 51: 1075-79. 1964.

Marcus, A., and Feeley, J.: Protein synthesis in imbibed
seeds. J. Biol. Chem. 240: 1675-80. 1965.

Mathes, K.: De einfluss des wasserzustandes auf ferment-
prozesse und staffumsatz. In: Ency. of Plant Physio.,
v.e., W. Ruhland, ed., p. 656-64. 1956.

Saunier, R. E.: Aspects of the drought tolerance in crea-
sotebush (Larrea divaricate). Plant Physiol. 43:
401-04. 1968.

Shah, C. B. and Loomis, R. S.: Ribonucleic acid and pro-
tein metabolism in sugar beet during drought. Plant
Physiol. 18: 240-54. 1965.









Steward, Cecil, R., Clayton, Morris, J., and Thompson, J.
R.: Changes in amino acid content of excised leaves
during incubation. 11. Role of sugar in the accumu-
lation of proline in wilted leaves. Plant Physiol.
41: 1585-90. 1966.

West, S. H.: Protein, nucleotide, and ribonucleic acid
metabolism in corn during germination under water
stress. Plant Physiol. 37: 565-71. 1962.

West, S. H.: Sub-cellular physiology as affected by drought,
Proc. of the X International Grassland Congress
(printed in Finland) page 91-94. 1966.




Full Text

PAGE 1

TijE or INADEQU.4\TE WA,TE;It SUPPLY ON ME'l'ABOLISM IN B;I:OLOGICAL SYSTEMS by S. H. WEST Qf Agronomy University of Floriqq PUBLICATION NO.9 of the FLORID.4\ WATER RESOURCES RESEARCH CENTER PROJECT TECHNICAL COMPLETION REPORT OWRR Projeot Number Annual Agreement Numbers 14-01 1"0001 .. 1077 (1968) 14 .. 01 or-POQ1.,.1628 (l9q9) 14 ... 31 .. 0001-3009 (1910) SUblll'l .. August 26, 1970 The wQrk upon whic::n thiq 'is based was in part by funds provideclby t;:he l)pitedStats$ pepartment of the Interior, Office ot Water ResourCes Research, as under the Water Research Act of 1964,

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TABLE OF CONTENTS ABSTRACT ,. II 'f II ,. III ". Page 1 P"R.O.)ECT RY III II ,. ,. II II II III II 2 INTRODUCTlON II '1/ ... ........... I II II I II II II II II 3 INITIAL RESEARCH PLAN AND RESULTS ....... ",.... ....... 4 CHARACTER,IZATION OF RNA ACCUMULATED IN DROUGHT 5 :PROTEIN SYN1HESIS ALTERED BY DROUGHT 7 LITERA TURE C I TED ., ..... .. .... "....... 1 1

PAGE 3

ABSTRACT THE INFLUENCE OF INADEQUATE WATER SUPPLY ON METABOLISM IN BIOLOGICAL SYSTEMS WITH EMPHASIS ON PROTEIN SYNTHESIS AND NUCLEIC ACID ME':rABOLISM Data have been obtained that show the effect of drought on growth itself and how this reduction in growth may be a result of specific changes in total protein production, nucleic acid metabolism and on functional activity of a fraction of nucleic acids. While the drcught treatments decreased total protein by only 40 percent, growth was re duced 80 percent. The$e data suggested that the synthesis of growth-dependent proteins was being hindered. Although total nucleic acid production was not reduced by the lack of water, the function of the nucleic acid fraction responsible for delivering the genetic information to the process of protein synthesis was altered. This fraction of nucleic acid was not, under water-stress conditions, getting attached to ribosomes. This malfunction prevented the synthesis of a proline-rich protein which is probablY required for cell wall production during growth. This information provides a specific selection criterion and should aid in the development of plants and perhaps other organisms that can withstand stress from drought. West, S. H. THE INFLUENCE OF INADEQUATE WATER SUPPLY ON METABOLISM IN BIOLOGICAL SYSTEMS EMPHASIS ON PROTEIN SYNTHESIS AND NUCLEIC ACID METABOLISM Completion Report to the Offi6e of Water Resources Research, Department of the Interior, August, 1970, Washington, D.C. 20240 KEYWORDS: water water shortage'''"! water utilization/ plant metabolism/ droughts,'-/ water consumption (plants),'-/ soil moisture/ drainage. 1

PAGE 4

PROJECT SUMMARY The goal of these studies was to determine what basic processes or biochemical reactions are susceptible to stress imposed by reducing the supply of water. This information would be used in the development of droughtresistant strains or management practices to alleviate water stress. The proposed research organism was plants but extrapolation of the findings to other biological materials would be meaningful at this subcellular level. The objectives of the research proposal included an adaptation of the research results to screening procedures. All of these goals and objectives have been obtained. The initial experiments were designed to Survey the effects of various degrees of water stress on certain chemical identities that may reflect the response of metabolic processes. These studies revealed that when stress, as a result of reduced water supply, decreased protein content by 40 percent, growth as measured by dry weight was reduced by 80 percent. The effect of water stress on other constituents, such as total amino acids and nucleotides, was similar to that on dry weight and therefore reflected no specific response to drought. However, total ribonucleic acid (RNA) increased in seedlings subjected to drought conditions. The RNA that accumulated in water stressed plants was characterized chemically to determine why it was not functional in protein synthesis. Extensive studies showed that the nucleotide composition of this RNA had not been altered by water stress. However, the physical characteristics had been affected. The individual com-: ponents of RNA were markedly smaller in drought treated than in control plants. The reduced size resultecl because of the failure of ribosomal RNA to become attached to messenger RNA. Therefore, information required from the genetic material to direct the synthesis of specific p:r:'oteins was not being oelivered to the ribosomes. The laCk of attachment can be used as a selection criterion to screen for plants that are resistant to drought. The amino acid proline accumulated in plants subjected "to water stress treatments. Isotope incorporation studies showed that this accumulation was a result of the 2

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lack of synthesis of a proline-rich protein. Together these results support the conclusion that drought altered the function of messenger RNA in plants and this malfunction prevents the synthesis of a cell-waIl-type protein. Publications that have resulted from this project thus far are: West, S. H., 1966, How Water Affects Plant Life. Weeds, Trees and Turf Magazine, p. 12-14. West, S. H., 1966, Sub-cellular Physiology as Affected by Drought. Proc. X International Grassland Congress, Helsinki, Finland, p. 91-94. Shiralipour, Aziz and S. H. West, 1968, Effects of Water Stress on Amino Acids, Protein and DNA Content of Corn Shoot at Different Ages. Soil and Crop Sci. Soc. of Fla. Proc. Vol. 28, p. 115-122. Shiralipour, Aziz and S. H. West, 1970, Specific Protein Synthesis Altered by Water Stress. In preparation. INTRODUCTION Throughout the world drought conditions reduce the growth of plants and often contribute significantly to stand failure. Slight improvements in adaptation to these stress conditions have been made, but progress is slow. The time required for the development of 'drought resistant plants is prohibitive because of the selection criteria available. Selection is usually based upon survival. Often failure to survive cannot be attributed to one factor of the environment. Furthermore, the environment is variable from year to year and selections that are adapted to an environmental factor that predominated in one season may be lost in another season. A response to the environment at the molecular level is needed if a specific effect is to be resolved. The purpose of these studies was to determine what basic processes are affected by the stress imposed by reducing the supply of water. This information would be used in the development of drought-resistant strains or management practices to alleviate water stress. 3

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INITIAL RESEARCH PLAN AND RESULTS The data reported here are from experiments designed to survey the effect of various degrees of water stress on certain chemical identities that may reflect the response of metabolic processes. The quantitative changes of endosperm-scutellum and seedling protein, nucleotides, and RNA during germination and growth under water stress have been measured. The qualitative changes in seedling nucleotides and RNA were determined by ion-exchange chromatography and spectral analyses. The design of these experiments was such that the effect of a sub-optimal supply of water to plant roots could be observed without the usual complicating factors of loss of turgor and reduced ion absorption. The atmosphere of the growth chamber was maintained near 100 percent relative humidity so that wilting did not occur, and only nutritive ions from the seed were available to all treatments. The quantity of mannitol that moved into the seedlings was not measured. The effect of mannitol on growth is assumed to be only a result of a decreased supply of water. Reducing available water by increasing the osmotic pressure of the medium surrounding the seeds resulted in a marked reduction in growth rate of the seedlings as shown by the dry-and fresh-weight. The dry-weight increases of the seedlings grown without water stress were similar to those reported elsewhere and after 6 days of incubation were approximately three times as great as the increase of the seedlings subjected to the greatest water deficiency (14.7 atm of osmotic pressure) for the same period. The increase in dry weight of the seedlings occurred at the expense of the endosperm-scutellum dry weight. Treatments that limited water entry and plant growth also reduced the dry-weight loss in the endosperm-scutellum. Protein accumulated in the seedling during 1 to 6 days of germination and growth at a rate similar to the dry-weight increases. Also like the dry-weight changes, a decrease in endosperm-scutellum protein accompanied the increase in seedling protein. Restricting the water supply slowed protein degradation in the endosperm-scutellum and the formation of protein in the seedling. Nucleotides increased in seedlings and endospermscutella during incubation. The magnitude of increase was greater in the seedlings. The quantity of nucleotides per plant was reduced by water stress, and the level per gram of dry weight was reduced initially. However, the initial 4

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decrease was followed by an accumulation to a level higher than the check treatment. With increasing water stress, more time was required for the accumulation. Throughout the 6 days of growth the nucleotides in the check seedlings remained constant. A disruption in normal nucleotide metabolism as indicated by lower nucleotide content would be expected to adversely affect RNA formation. The RNA in the seedl ings increased during the 6 days of growth and the endosperm-scutellum RNA decreased. The rapid degradation of endosperm-scutellum RNA that occurred during the first 3 days of germination in the check plants was slowed by the water stress. Unlike seedling protein and nucleotides, seedling RNA increased when water was limited. CHARACTERIZATION OF RNA ACCUMULATED IN DROUGBI The initial or survey studies showed that growth, amino acids, protein, and nucleotide contents decreased with imposed water-stress treatments, but RNA increased. The following studies were designed to determine what fractions of RNA were accumulating and to characterize that RNA. Protein is an important constituent of the enzymes which catalyze the thousands of chemical reactions required for the growth of a plant. Protein synthesis is de:pendent upon ribonucleic acid in ribosomes, messenger (mRNA) and transfer RNAs. Ribosomes form the site upon which amino acids are joined. Transfer RNA brings the amino acids to the ribosomes. Finally, mRNA is the vehicle by which the information in the nucleus is conveyed to the ribosomes where this information directs the polymerization of the various proteins. Zea mays L. seedlings were grown in darkness in a constant temperature of SOoF and 95 percent relative h1,lmidity. The test material consisted of one-centimeter sections harvested from the stem immediately above and below the first node of 6-day-old plants. The treatments were: (1) plants grown for 6 days with a continuous supply of water to the roots, (2) similar to treatment (l), except that the roots were supplied with a solution containing sufficient mannitol to provide 9.S atmospheres of osmotic pressure, (3) plants grown in the mannitol treatment for 4 days and then transferred to the water supply for 2 days. These experiments showed that drought conditions reduced fresh weight to approximately one half that of the control plants, but when the stress was released, growth 5

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was resumed at a rapid rate. Total cytoplasmic RNA creased per section and per cell. A large part of the increase was attributed to an increase in ribosomes. The level of RNA was not significantly decreased in the stressrelease plants. These data should be compared to those that have shown a positive correlation between RNA content and the capacity to make protein. Furthermore, the level or content of ribosomes have been shown in other studies to regulate the rate of protein synthesis. Our data show that drought-stressed plants synthesized more ribosomes upon which protein could be made, yet growth was reduced. The author surveyed some of the characteristics of the ribosomes that accumulated in plants subjected to drought copditions to determine why they were not functional in the period of slow growth. ConceivablY the composition of nucleotide bases in the RNA may reflect changes sufficient to render the ribosomal units non-functional in pro tein synthesis. To check this feature, ribosomes were isolated from the control and water-stress the RNA was hydrolyzed, and chromatographed on a Dowex-l column. These experiments showed that the quantities of each of the 4 nucleotides in plants from each treatment, In other tests, P was added to the incubation solution and the amounts of the isotope occurring in each nucleotide showed also that the base composition was not altered by the water-stress treatment. These data show: (1) that the ribosomes which accumulated in the water-stress condition were not abnormal in base composition, (2) that there was no preferential synthesis of nucleotides, and (3) that there was no preferential incorporation of nucleotides into RNA. The above studies indicated that the single ribosome unit was not altered by the drought condition and therefore, would not account for the effect on growth. In previous studies the number of single 70s ribosomes that clustered together into aggregates appear to be related to the rate of protein formation. The ratios of the amounts of RNA occurring in fractions containing particle sizes' 70s, 70s x 2, 70s x 3, and those greater than 70s x 3 show a positive correlation between ribosome cluster size and growth rate. Slow growth of plants under drought stress was associated with a high level of single 70s particles. The plants in the control treatments contained a higher level of clusters larger than single 70s particles than that of the stress plants. The rapid rate of growth in the drought-released plants resulted in levels of clusters markedly higher than that of the control plants. Electronmicroscope studies using permanganate fixing and platinum shadowing revealed more large clusters in the grid fields in fast growing plants than in those growing under stress. 6

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Clusters of ribosomes may be formed by the attachment of messenger RNA (mRNA) to individual ribosomes as visualized in various reports. If this is a necessary step in the process of protein synthesis, measurements of clusters are some measure of amounts and participation of the mRNA in In addition, mRNA has been described as a rapidly P labelled fraction of RNA, in contrast to ribosomes. Therefore, providing test plants with the isotope for a brief period and then isolating the RNA fractions with their associated counts is still another way of correlating mRNA with growth and with polysome formation. Representative data from these experiments are shown in Table 5. These data show that the RNA fractions that contain more than one ribosome contain high levels of p32 and suggest that growth is indeed associated with polysome formation. These data further indicate that stress from drought adversely affects mRNA. This information provides a specific selection criterion and should aid in the development of strains of herbage plants that can withstand stress from drought. Plant materials Can now be screened and selections made on the basis of susceptibility of mRNA to water-stress. In addition, methods of inducing resistance to drought can now be accurately followed. PROTEIN SYNTHESIS ALTERED BY DROUGHT The regulation of growth in biological systems is considered an expression of highly organized and ordered protein synthesis. Drought, in most plants, invariably reduces growth. This reduction in growth appears to be partially associated with an alteration of protein metabolism (Barnett and Naylor 1966, Ben-Zione et al 1967, Henckel 1965, Shah and Loomis 1965, and West 1962). The development of drought resistance in plants might be facilitated if the mechanism of growth regulation were by a specific protein and if this mechanism were elucidated. The previous work on this project showed that the apparatus for polymerizing proteins was altered by water stress. Since the altered portion was that involved with the direction of various proteins, our research was aimed at determining if water-stress affected specific proteins, Research reported earlier by our laboratory and by others has shown that drought stressed plants have a high level of the amino acid proline. The research reported here was designed to determine if the accumulation of the amino acid proline during drought is a result of protein degradation or of the reduced synthesis of a specific proline-rich protein. 7

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Drought treatments resulted in a 58 percent reduction in fresh weight per shoot and 40 percent in dry weight as compared with the control treatment. The reduction for fresh and dry weight of the root and shoot was 58 percent and 30 percent respectively. The plants grown under control conditions for 1 day after 4 days of drought made a marked recovery in both fresh and dry weights. All compounds were compared on a per cell (mg DNA) (Ingle et al 1964) basis. Drought affected weights and protein content of roots and shoots similarly. Protein content per cell was not changed by the treatments; however, drought reduced the total protein per seedling. Total free amino acids in the shoots and roots of the seedlings grown under drought conditions were not significantly increased as compared with controls. In the 24 hours after release from drought treatment, the change in total amino acids was negligible. While proline, phenylalanine, glutamine and asparagine increased in the shoots of the drought treated seedlings, the increase in proline was greater than that for any other amino acid, Many workers (Barnett and Naylor 1966, Chen et al 1964, Kemble and Macpherson 1954, Mathes 1956, Saunier 1968, Steward et al 1966) have reported increases in free amino acids under drought conditions. In one case free proline increased 10 to 100 fold (Barnett and Naylor 1966). The increase in amino acids has been attributed to adegradation of protein (Kemble and Macpherson 1954, Mathes 1956). However, our results which show no significant decrease in protein, or increase in total amino acids tend to refute this suggestion. The lack of decrease in protein in drought treated plants does not preclude the possibility that some protein was being degraded while others were being formed. Instead of degradation of protein we propose that the accumulation of certain amino acids, especially proline, in the drought treated seedlings was partially, if not completely caused by their continued synthesis or transfer from seed to shoot and root with a reduction in the rate of incorporation protein. This is supported by our observation thatC labeled proline, an amino acid which consistently accumulated in drought conditions was incorporated into cytoplasmic protein of at only about one-third the rate in drought treated plants as in the controls. In a similar comparison of treatments in roots, the proline-14C incorporated into the cytoplasmic proteins of drought treated plants was about one-half that in control plants. F1,lrthermore, plants that had been in conditions for 4 days incorporated more proline-'C when released from drought for 1 day than did the plants in continuous control treatments. These data further demonstrate that some protein synthesis was inhibited during 8

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drought conditions and was rapidly reinitiated when drought stress was released, and that these proteins are especially rich in proline. The reduction in the rate of incorporation of 14C-proline in stressed seedlings can not be attributed to isotopic dilution, since maximum specific activities of proline pools in control and stressed shoots and roots are very similar. In contrast to proline, serine did not accumulate under drought conditions anct its incorporation into protein was neither reduced by drought nor appreciably increased after the plants were released from drought. From the data it is possible to propose that plant cells under drought stress actively accumulate proline not only because of lack of incorporation into protein, but also for other, as yet undetermined, requif:z;ments of the stressed plants. The total uptake of proline-C by the cells of shoots from plants under drought conditions was about 3 times greater than in the control treated plants and the uptake in the drought treated roots was about 2 times that in the control roots. The level of proline in the shoots was reduced when water was supplied to the drought treated plants. Again, in contrast to proline, the total uptake serine-14C by the roots and shoots of drought stressed plants was less than by control plants (comparison to the control treatment). Plants under drought conditions may preferentially synthesize proline and this may also contribute to the accumulation of that amino acid. Results from studies on the effect of drought on nucleic acid metabolism are consistent with findings concerning accumulation of certain amino acids and lack of incorporation into protein discussed above. Marcus and Feeley (1965) showed a low level of amino acid incorporation into protein in the cotyledon of the unimbibed peanut seed. After water was added, imbibed seeds incorporated the amino acids at a much higher rate. Furthermore, they showed that the unimbibed seed possessed the entire apparatus for protein synthesis except messenger RNA was not adequate or was in an inactive form. Polysomes were formed and protein synthesis WaS resumed upon addition of water in the cotyledons and embry (Marcus and Feeley 1965). West (1966) attributed a reduced polysome formation under water stress conditions to a possible messenger RNA malfunction. In our ?f water. caused rapid of due to the of activation of specific messenger RNA and eventually polysome formation. 9

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The partial inhibition of proline incorporation into protein by water stress appears to be completely reversible through the addition of water and the treatment does not affect the genetic <;tppafatus since re: covers. The amounts of 4C and C porated into protein are inverse to their quantities as free amino acids. This research was supported in part by the Office of Water Resources Research, U, S. Department of the Interior, as authorized under the Water Resources Research Act of 1964, public law 88-379. 10

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LITERATURE CITED Barnett, N. M. and Naylor A. W.: Amino acid and protein metabolism in Bermudagrass during water stress. Plant Physio1. 41: 122-30. 1966. Ben-Zione, Aliza, Itai, C., and Vaadia, Y.: Water and salt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physiol. 42: 361-65. 1967. Chen, D., Kessler, B., and Monselise, S. P.: Studies on water regime and nitrogen metabolism of citrus seedlings grown under water stress. Plant Physiol. 39: 379-86. 1964. Henckel, P. A.: Physiology of plants under drought. Annual Rev. Plant Physiol. 15: 363-86. 1965. Ingle, John and Hageman, R. H.: Studies on the relationship between ribonucleic acid content and the rate of growth of corn roots. Plant Physiol. 39: 730-34. 1964. Kemble, A. R. and Macpherson, H. T.: Liberation of amino acids in perennial rye grass during wilting. Biochem. J. 58: 46-50. 1954. Marcus, A., and Feeley, J.: Activation of protein synthesis in the inhibition phase of seed germination. Proc. Nat1. Acad. Sci. (U.S.) 51: 1075-79. 1964. Marcus, A., and Feeley, J.: Protein synthesis in imbibed seeds. J. BioI. Chern. 240: 1675-80. 1965. Mathes, K.: De einfluss des wasserzustandes auf fermentprozesse und staffumsatz. In: Ency. of Plant Physio., v.e., W. Ruhland, ed., p. 656-64. 1956. Saunier, R. E.: Aspects of the drought tolerance in creasotebush (Larrea divaricate). Plant Physio1. 43: 401 0 4 1 968 Shah, C. B. and Loomis, R. S.: Ribonucleic acid and protein metabolism in sugar beet during drought, Plant Ph y s i 01. 1 8 : 240 54 1 965 11

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Steward, Cecil, R., Clayton, Morris I J., and Thompson, J. R.: Changes in amino acid content of excised leaves during incubation. 11. Role of sugar in the accumulation of proline in wilted leaves. Plant Physiol. 41: 1585-90. 1966. West, S. H.: Protein, nucleotide, and ribonucleic acid metabolism in corn during germinqtion under water stress. Plant Physiol. 37: 565-71. 1962. West, S. H.: Sub-cellular physiology as affected by drought, Proc. of the X International Grassland Congress (printed in Finland) page 91-94. 1966. 1,2