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4
Plant Science
There have been remarkable advances in the molecular
understanding of a few of the key processes in plants
during the past decade. An important component of these
advancements has been the application of new technologies
for isolating, cloning, and characterizing genes. me
ability to apply these techniques is based on fundamental
knowledge in physiology and biochemistry that has accumu-
lated over the years. Their application has opened new
frontiers in the study of plant growth and development.
Molecular genetic approaches have been applied to
nearly 50 plant genes, primarily those associated with
seed storage proteins, chloroplasts, photosynthesis, and
biological nitrogen fixation. Notably, a number of the
genes that code for important enzymes in photosynthesis
and nitrogen fixation have been identified, cloned, and
sequenced. Several of the genes for storage proteins in
crop plants have been cloned and characterized. In addi-
tion, the ability to regulate the expression of genes
controlling the biosynthesis of the key enzymes in photo-
synthetic carbon dioxide fixation is under active study.
Studies are also being initiated to clone the genes for
phytochrome as well as those for some of the known plant
hormones. Comparable progress is needed in other areas
of plant science research.
A lack of basic information on the biochemistry of
many metabolic and regulatory steps is delaying progress
in using molecular genetics to establish the mechanisms
employed in controlling plant growth. This chapter
suggests ways of strengthening research that emphasizes
integration of traditional biochemical and physiological
54
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55
research with molecular genetic approaches. Three gen-
eral areas of research, all of which are basic to plant
growth, are discussed: (1) the interaction of carbon and
nitrogen metabolism in supporting optimal plant growth;
(2) the role of plant hormones and phytochrome in regu-
lating plant growth and development; (3) and the limita-
tions to growth imposed by physicochemical stresses such
as cold, heat, drought, and salinity.
Carbon and Nitrogen Input for Plant Growth
Both the carbon and nitrogen that are essential com-
ponents for all forms of life ultimately cycle through
the atmosphere. Plants take up these elements, in the
form of carbon dioxide and nitrogen gas, from the atmo-
sphere via photosynthesis and nitrogen fixation. Both
processes involve reduction reactions, which require
energy. Solar energy drives the photosynthetic reduction
of carbon dioxide into sugars. me chemical energy
stored in these products of photosynthesis is then used
for nitrogen fixation. me relative availability of
these two key constituents, reduced carbon and reduced
nitrogen, can greatly regulate plant growth.
Photosynthesis
Photosynthesis encompasses the most important reac-
tions on earth; life depends directly upon solar energy
captured and stored photosynthetically. Not only do
plants, through photosynthesis, reduce carbon dioxide to
the food and fuel products that sustain life, but photo-
synthesis also produces the oxygen required to deoxidize
these products to release their energy.
Light capture, carbon dioxide fixation, and oxygen
evolution were recognized as components of the photosyn-
thetic process more than 200 years ago. These partial
reactions have now been described in considerable detail.
Initial light capture and energy conversion occurs within
a few nanoseconds after a photon is absorbed by chloro-
phyll. Subsequent electron and proton transfers,
occurring within a few milliseconds, generate chemical
energy in the form of adenosine triphosphate (ATP) and
reduced pyridine nucleotide (NADPH). These compounds in
turn power the reduction of carbon dioxide.
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56
Chloroplast Functions
The chloroplast is a remarkable organelle. It con-
tains all the units essential for photosynthesis--the
light-gathering pigments, the membrane components and
cofactors that mediate electron transfer, the enzymes
involved in ATP and NADPH production, the enzymes for
carboxylation and reduction of substrates, and the system
that liberates oxygen from water.
Strong oxidizing and reducing agents are maintained in
close proximity, but their interactions are controlled.
For example, the chlorophylls that capture light and
drive the energy-coupling steps of photophosphorylation
(the process that produces ATP) as well as pyridine
nucleotide reduction are organized within the thylakoid
membranes in the chloroplast. The enzymes that catalyze
the reduction of carbon dioxide for the synthesis of
phosphorylated sugar intermediates occur in the stroma
surrounding these membranes.
Chloroplasts contain DNA, ribosomes, and the other
components needed for protein synthesis. Major advances
in identifying the genes that encode structural compo-
nents of the chloroplast have been made in the last 10
years, largely through integrated studies using genetics,
molecular biology, and biochemical analysis. Chloroplast
DNA accounts for only about 10 percent of the genetic in-
formation needed for chloroplast structure and function.
Most of the structural proteins of the chloroplast are
encoded in the DNA of the cell nucleus, synthesized in
the cytoplasm, and then imported into the chloroplast.
The information now available concerning chloroplast
inheritance is spawning research toward practical appli-
cations. Investigations include manipulating chloroplast
genes that confer selective resistance against specific
herbicides in crop plants and designing genes that pro-
duce the enzymes involved in carbon dioxide fixation to
increase the overall efficiency of the carboxylation
reaction.
Carbon Fixation
The path of carbon in photosynthesis has been clearly
defined. Carbon dioxide is fixed to yield the three-
carbon molecule phosphoglyceric acid, which is then
converted to sugars. This process characterizes the so-
called C3 plants. In other plants, atmospheric carbon
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57
dioxide is initially fixed to yield a four-carbon mole-
cule that is translocated to neighboring cells where it
is decarboxylated to give up the carbon dioxide. These
are known as C4 plants. This released carbon dioxide
is subsequently fixed to yield phosphoglyceric acid via
the C3 pathway.
The formation of phosphoglyceric acid in both C3 and
C4 plants is accomplished by the enzyme ribulose-1,
5-bisphosphate carboxylase-oxygenase, often called
Rubisco. It can react with either carbon dioxide to car-
boxylate ribulose-1,5-bisphosphate to two molecules of
phosphoglyceric acid or with oxygen to oxidize
ribulose-1,5-bisphosphate to one molecule of phospho-
glyceric acid plus the two-carbon molecule, called
phosphoglycolic acid, which is involved in photores-
piration. This oxidation reaction is wasteful since
energy must be consumed to resynthesize the ribulose-l,
5-bisphosphate. It would be advantageous, therefore, to
cause Rubisco to decrease or lose its oxidative reaction
function. Experimental elevation of carbon dioxide in
the air produces major increases in yield of most field
crops by increasing the carboxylation reaction. Although
this technique demonstrates great potential, carbon
dioxide enhancement of large areas is impractical.
Rubisco is inefficient in catalyzing the carboxylation
reaction. Attempts have been made to improve its effi-
ciency, but the changes induced by genetic manipulation
using both mutational selection and site-specific changes
in the DNA sequence have only decreased its activity. In
addition, attempts to specifically inhibit the oxygenate
function without disturbing the carboxylase have been
unsuccessful. If the catalytic sites are different,
theoretically the oxygenase activity of Rubisco could be
blocked. Selective pressures to accelerate the activity
of Rubisco have existed for millions of years; it is
hardly remarkable that a decade of research has not
brought improvement in the carboxylation reaction, which
theoretically might be possible.
The problems in bioengineering an improved Rubisco
focus on the fact that it is a large enzyme consisting of
eight small subunits with molecular weights of 14,000
each and eight large subunits with molecular weights of
56,000 each. me larger subunits are coded for by chlo-
roplast genes, and the small units are coded for by
nuclear genes.
The amino acid sequences of the large and small sub-
units of Rubisco have been completed for some plants.
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58
Research is focusing, in particular, on the large subunit
which contains the catalytic sites for carboxlyation and
oxygenation. Several laboratories are concentrating on
genetically engineering changes in the chloroplast genes
that encode for the large subunits. Their objective is
to locate a form of this enzyme that has an increased
overall activity or a selectively decreased oxygenate
activity. A great technical limitation to this work
exists: mere is no gene transfer system yet available
for inserting recombinant genes into chloroplasts.
Photosynthetic Efficiency
On the average, only one quarter of 1 percent of the
radiant energy reaching the earth is captured by photo-
synthetic organisms. m is largely is a measure of the
density of photosynthetic plants on the earth's surface
and the efficiency with which they can absorb light and
photosynthesize throughout all the seasons. In contrast,
a highly efficient C4 plant, such as corn, may utilize
5 percent of incident radiant energy during its most
rapid period of growth.
What is the potential efficiency of photosynthesis?
One answer is provided by measurements of quantum effi-
ciency. Efficiencies of one carbon dioxide molecule
fixed per 8 to 10 quanta absorbed have been measured
using single-cell algae. m ese efficiencies are high,
in terms of conversion of incident light, and probably
can only be achieved under the optimal experimental
conditions--very low light intensities in comparison to
average sunlight. For this and other reasons, it is
impractical to extrapolate these measurements to field
crops.
Many factors such as the developmental stage of the
plant and the presence of biological and physicochemical
stresses can reduce photosynthetic efficiency. In addi-
tion, each step in the photosynthetic process, from the
absorption of light energy to the conversion and storage
of energy in the synthesis of sugar molecules, can be
affected differently by various limiting factors. Thus,
to improve overall photosynthetic efficiency, researchers
must first understand the steps in photosynthesis and the
factors limiting their efficiency.
Although it is doubtful that quantum efficiency in the
field can approach that achieved with algae in the
laboratory, record yields in field plots are a reasonable
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59
goal. A record corn yield in the United States can be
more than 300 bushels per acre, compared with an average
yield of about 100 bushels per acre. Comparisons
indicate that full photosynthetic growth potential is
seldom realized.
How can photosynthetic efficiency be improved? One
possible approach is suggested by the difference in
photosynthetic efficiency between C3 and C4 plants.
Major crops, such as wheat, rice, and the seed legumes,
are C3 plants. It may be possible to convert them to a
C4-type metabolism.
C4 plants are more efficient, primarily because the
C4 pathway serves as a metabolic carbon dioxide pump
that raises the carbon dioxide concentration, by
ribulose-1,5-bisphosphate carboxylase-oxygenase, at the
site of carbon dioxide -fixation. This increases the rate
of carboxylation and at the same time suppresses the rate
of oxygenation. m is process is carried out at a level
near carbon dioxide saturation, resulting in an enhanced
rate of net photosynthesis.
There are significant anatomical differences between
the leaves of C3 and C4 plants. Specialized bundle
sheath cells in C4 plants contain many chloroplasts;
C3 plants have few if any chloroplasts in their bundle
sheath cells. It is in the bundle sheath cells of C4
plants that the carbon dioxide concentration is elevated
and increases the rate of carboxylation by ribulose-l,
5-bisphosphate carboxylase-oxygenase. C4 plants may
have evolved from C3 plants.
Nature has provided some plants with intermediate ana-
tomical and biochemical properties. m ese plants serve
as encouraging models. They include the composite
Flaveria and the grasses Panicum and Neurachne. These
~-
intermediate plants are tools useful in studying the
inheritance and relative advantages of photosynthetic
efficiency in the C3 and C4 pathways, under different
limiting factors. Some of the leading research on photo-
synthesis in C3 and C4 plants has been conducted in
ARS laboratories.
The photosynthetic potential of a plant cannot be
achieved if its growth is limited by physicochemical
stresses or by nutrient deficiencies. Efforts to improve
photosynthetic efficiency can be enhanced by research
focused on resistance to physicochemical stress and
utilization of nutrients from the soil.
Although outlines of the basic steps of photosynthesis
appear clear, virtually every aspect of this complex
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60
process requires continued investigation. A thorough
understanding of the basic mechanisms of photosynthesis
may reveal new information that will permit researchers
to increase photosynthetic efficiency and productivity
and direct it toward the generation of the most desirable
plant products.
Harvest Index
m e most effective way to improve the harvest index
(the ratio of harvested part of the plant to total plant)
may be to improve the accumulation of photosynthate in
the desired plant part.
Traditional breeding methods have selected for an
improved harvest index in many crops and have led to
substantial gains in crop yields. Such successes through
plant selection have been achieved in the absence of a
clear understanding of the factors under genetic as well
as environmental control that determine crop yield.
Additional improvements in yield and harvest index may
depend on a full understanding of these factors and their
interactions at the molecular and genetic level. With
this information, scientists may now be able to take
advantage of recombinant gene transfer methods to further
improve crop quality and yields.
Plants have often been selected as crops based on
their parts that accumulate the products of photosyn-
thesis. Plants with lush vegetative growth are used for
feed or fodder while they are undergoing rapid photosyn-
thetic growth. Alternatively, plants that deposit their
photosynthate in stems, roots, fruits, or seeds may be
selected for these attributes and harvested after their
storage organs have achieved maximal size. In these
plants the investigator attempts to redirect photo-
synthate to the portion of the plant that will be used
for feed or food. Researchers are currently attempting
to improve the harvest index in soybean, for example.
Before harvest the soybean plant undergoes senescence
and mobilizes a high percentage of its nitrogen from
roots, nodules, stems, and leaves for deposition in the
seeds as protein.
In a sense the plant destroys itself
to produce a viable, energy-rich seed to preserve the
plant line for the next season.
If the soybean plant's delicate control mechanisms can
be manipulated to prolong the period of active photo-
synthesis in the plant without destroying its ability to
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61
go through senescence at the proper time, the harvest of
seed will be greater. Foliar application of plant growth
substances or nutrients can artificially prolong active
vegetative growth. Subsequently, total crop yield may be
increased. Integrated research programs that focus on
both photosynthesis and developmental biology will con-
tribute to a future understanding of the factors that
link photosynthetic productivity and storage mobilization
capacities.
Photosynthesis in the chloroplast produces hexoses and
hexose phosphates that are either converted to and im-
mobilized as chloroplast starch or converted to sucrose.
Sucrose is the major form of carbohydrate transported
from the site of photosynthesis in the leaf to other
parts of the plant. It is readily converted to starch
and other storage products in seeds and storage organs.
Control of the transfer of sucrose to various parts of
the plant and optimization of deposition of carbohydrate,
protein, and fat reserves in seeds and storage organs
determines the harvestable yield of a crop. Using empir-
ical methods in plant breeding and selection, researchers
have successfully increased the harvest index of many
plants. Little is known, however, about the processes
regulating the translocation and metabolism of photo-
synthetically fixed carbon.
Species vary in their rates of accumulation of starch
and sucrose in leaves. Wheat, barley, and spinach
accumulate more sucrose than starch in leaf mesophyll
cells in contrast to species such as peanuts, soybeans,
and tobacco, which accumulate more starch than sucrose.
Studies on the enzymatic steps involved in the biosyn-
thesis of starch and sucrose indicate that inorganic
phosphate and triose-phosphate have profound effects on
regulating the rates of these interconnected biosynthetic
pathways. Research focusing on a full understanding of
the regulation of the storage and transport of carbo-
hydrates has been modest.
The partitioning of photosynthate is a major factor
determining harvest index. Research on the metabolism of
the hexose products of photosynthesis and the regulation
of their conversion to carbohydrate, protein, and lipid
storage products should be increased.
Nitrogen Metabolism
Nitrogen is a key element required by plants, and it
is commonly the limiting element in plant productivity.
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62
me world capacity for commercial fixation of nitrogen is
about 60 million metric tons per year, the bulk of which
is used as fertilizer. Dependence on this source for
agricultural use has created problems. High cost
precludes its use in many areas; natural gas as the feed-
stock for chemical fixation is a limited, nonrenewable
resource that will increase in cost; and the use of too
much fertilizer may be accompanied by excessive losses
through leaching, erosion, and denitrification. Leaching
into water supplies may raise nitrate concentrations to
harmful levels.
When biological fixation is substituted for chemical
fixation of nitrogen, the energy of sunlight is substi-
tuted for the energy of natural gas. Sunlight is
captured through photosynthesis, and its use preserves
fossil fuels. The nitrogen fixed biologically in the
root nodules of leguminous plants is quickly assimilated
into organic nitrogen compounds in the plant and is sub-
ject to very low levels of leaching and denitrification.
Although some major seed and forage legumes such as soy-
beans and alfalfa take advantage of biological nitrogen
fixation, there is potential for improving and extending
the advantages of biological nitrogen fixation to other
crops.
Biological Nitrogen Fixation
The association between leguminous plants and their
root nodule bacteria is the preeminent system for biolog-
ical nitrogen fixation (BNF) in agricultural crop plants.
There are, in addition, certain free-living bacteria and
bacteria in loose association with plants whose nitrogen-
fixing capabilities warrant further investigation.
Substantial advances have been made in studies of the
biochemistry and genetics of nitrogen fixation. The
enzymes and the electron transfer sequence involved in
the steps that reduce nitrogen gas to ammonium can be
described in some detail. Study of the genetics of the
nitrogenase system in the free-living, nitrogen-fixing
bacterium Klebsiella pneumonias has shown that 17 genes
are involved. The function of most of these genes has
been defined. Now it is necessary to establish
comparable detailed information on the genetics of nitro-
genase in other nitrogen-fixing organisms, including the
symbiotic bacteria Rhizobium spp., blue-green algal
species, photosynthetic bacterial species, and the
azotobacter and the clostridia.
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63
In symbiotic biological nitrogen fixation, detailed
information on the contribution of both the bacterium and
the plant must be determined. For example, studies of
the rhizobia-legume association have shown that the
genetic information for production of the globin in
hemoglobin, found in the leguminous nodules, is con-
tributed by the plant. Scientists may eventually manipu-
late genes to improve nitrogen fixation or to introduce
it into other bacteria or higher plants not now capable
of fixing nitrogen. A thorough understanding of the
genetic systems of both the bacterium and the plant will
enhance the chances of success in transferring genetic
elements.
There are marked differences in the effectiveness of
symbiotic nitrogen-fixing systems, but the character-
istics governing good or poor associations have not been
defined. Until these factors are defined, genetic manip-
ulations will remain empirical. In addition, symbiotic
nitrogen-fixing systems require large amounts of
photosynthate--10 to 12 grams of photosynthate are
utilized in fixing 1 gram of nitrogen. Decreasing this
energy requirement is a major research challenge.
Nitrogen-fixing systems dissipate 25 percent or more
of their energy in producing hydrogen rather than in
reducing nitrogen. Hydrogen production is apparently
inherent in nitrogenase action. The only way known
currently to decrease this energy loss is to recycle the
hydrogen to recapture its energy. Oxidation of hydrogen
via a hydrogenate enzyme can be coupled to ATP formation
and to reductant formation. ATP and reductant can then
be used to support nitrogenase activity. The gene for
hydrogenate, Age+, has been transferred to Rhizobium
japonicum. Soybeans inoculated with Age+ rhizobia
produce higher yields of protein than those infected with
comparable t~e~ rhizobia, which lack this enzyme. Sim-
ilar improvements through manipulation of other genes or
modification of the nitrogenase genes for increased
efficiency can be made when other factors limiting
nitrogenase activity are defined.
Improving Symbiotic Nitrogen Fixation
About 85 percent of legume inoculant used in the
United States is applied to soybeans. Indigenous rhi-
zobia are so dominant in most soybean fields, however,
that improved rhizobia strains that are added to the soil
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64
do not compete effectively in the process of Modulation
To improve legume-bacterial symbiosis, the competi-
tiveness of added, improved rhizobia strains must be
increased. A superior nitrogen-fixing strain developed
under controlled conditions in the greenhouse will be of
little use in the field unless it can form nodules on the
soybean plants in competition with indigenous bacteria.
Processes such as primary attraction, binding, and in-
fection may be important aspects of this competition.
Such processes may be influenced by special glycoprotein
molecules, (called lectins) on the root surface, but this
must be established clearly and it must be controlled.
Mutants of rhizobia have been produced that appear to
enhance the early growth of soybeans. m is optimal
growth, however, has not been maintained until harvest.
Improvements under field conditions are of economic
significance; however, few have been verified.
Host Plant Improvement Carbon and nitrogen metabolism
share an intimate relationship. Biological nitrogen
fixation requires great amounts of energy, supplied
primarily by photosynthesis. Progress in the under-
standing of photosynthesis has been impressive, but
further research is needed to define its interactions
with major limiting factors in plant growth. With
advances in experimental techniques and a better under-
standing of the fundamental metabolic steps in both
photosynthesis and biological nitrogen fixation,
researchers are better equipped to study the feedback
relationships between these two processes. An improved
understanding of the interactions among nitrogen metabo-
lism, photosynthetic carbon fixation, and the
distribution of fixed carbon throughout the plant will
contribute to eventual increases in the harvest index.
Recent studies suggest that transformations of carbon
compounds at the site of nitrogen fixation in the plant
may be important in nitrogen fixation. Research con-
ducted by ARS scientists has demonstrated the ability of
root systems of certain leguminous plants to fix carbon
dioxide. These transformations may be a part of the
conversion of photosynthate to compounds especially
useful as acceptors for newly fixed nitrogen.
The transfer of the genes for nitrogen fixation to
nonleguminous plants, such as corn, is appealing and
should be studied on a long-term basis. Genes for
nitrogen fixation have been transferred from the free-
living bacterium Klebsiella pneumonias to the bacterium
, —
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different stages of development. Ultimately, character-
ization of enzymes may lead to the development of genetic
probes that will assist in identifying and cloning genes
that code for and regulate endogenous levels of plant
hormones.
Tools, such as monoclonal antibodies that are specific
for certain enzymes, are needed to identify and localize
hormone biosynthesis within tissues or cells. Plant hor-
mones are low-molecular-weight compounds that do not, by
themselves, have antigenic properties. Monoclonal anti-
body probes for the plant hormones are now being devel-
oped, however, by covalently linking them to the surface
of a carrier protein macromolecule. The carrier protein
serves as the antigen to stimulate antibody production.
Because the attached low-molecular-weight plant hormone
has become a surface characteristic of the carrier pro-
tein, some of the antibodies produced might recognize and
have affinity for free, unlinked hormone molecules.
This approach, using antibodies against plant hor-
mones, is in its early stages. It does offer, however, a
level of sensitivity for both chemical identification and
quantitation that may match the physiologically active
concentrations of the hormones in plant tissues. A dis-
advantage of this method is that tannins and other phen-
olic substances, often found in plant extracts, can
denature proteins, including antibody proteins, and might
obfuscate the sensitivity of the analysis.
Chemical Analysis Sensitive chemical analyses are
greatly aiding studies in plant hormone biosynthesis.
High-resolution analytical instruments are now available
for the chemical identification and quantification of
hormones in the plant. This analytical capability is
based on the use of high-performance liquid chroma-
tography (HPLC) followed by gas chromatography, coupled
with mass spectrometry (GCMS) or nuclear magnetic
resonance or both. m ese methods provide accurate
separation, identification, and quantitation of the
minute amounts of hormones present in plant tissues. The
instrumentation is costly and its operation and
maintenance demand special analytical skills. m e
accuracy and sensitivity provided by these methods,
however, are often required. In addition, the
biosynthetic origin and metabolic fate of these hormones
in plants are being studied using radioactive and atomic
mass labeling techniques.
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71
Genetic Variants Single gene mutants have also been
important in studies on the biosynthetic pathways of hor-
mones in plants. For example, dwarf mutants of corn have
been successfully used to study gibberellin biosynthesis.
Several derivatives of the basic gibberellin chemical
structure are synthesized in plants. For example, only
one gibberellin, gibberellin A1, is active in
~~ ~ ~ ~ Other gibberellins in
controlling shoot growth in corn.
the plant are important intermediates in its
biosynthesis. Dwarf mutants of corn are unable to
synthesize gibberellin A1; they are unable to carry out
one or more of the steps in the interconversion of one
gibberellin to another. Use of those m'~t~nP~ h== h^^n
_ _
. . . . . .
~ ~ ~ _ ~ ~
Critical toot in defining the sequences of conversions of
the many qibberellins to the Final - Act iv" an - ~'l-'
~ — , ~ ~ ~ _ ,
gibberellin A1.
In addition, mutants will be important experimental
models for understanding the regulation of hormone levels
during appropriate stages of development. For example,
in viviparous mutants of corn, the maturing seed does not
become dormant but instead continues to grow and ger-
minate while still on the ear of corn. me dormancy of
normal seed is associated with a relatively high
concentration of abscisic acid. Research indicates that
insufficient levels of abscisic acid are present during
the maturation of the viviparous seed to impose dormancy.
Gene Expression
Scientists have searched for specific
cures that recognize and interact with a hormone or
phytochrome in studies of their mechanisms of action.
Radioisotopically labeled hormone molecules with high
specific activity are used in attempting to locate and
identify the receptor sites that bind the hormone. Thus
far, this method has not succeeded in plants as it has in
the case of identification of the receptor sites of
steroid hormones in animals. Thus far, scientists have
not succeeded in identifying a specific binding site with
characteristics that correlate exactly with the physio-
logical response induced by the plant hormone.
An alternative approach for studying the molecular
mechanisms involved in hormone-related responses is to
study enzymes and other gene products that appear in
response to hormone application. The effect of
receptor mole-
~ ~ ~ & ~ ~ 8~
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72
gibberellins on the synthesis of the starch-hydrolyzing
enzyme alpha-amylase in the aleurone cells of germinating
cereal grains is a classic example. Gibberellin
regulates the expression of these hydrolytic genes in
aleurone cells, as demonstrated by the increased levels
of alpha-amylase messenger RNA s in response to the
gibberellin. Similarly, the level of mRNAs coding for
seed storage proteins in developing seeds has been shown
to be regulated by abscisic acid.
While it is difficult to determine whether the reg-
ulation of gene expression is a primary or secondary
response to the specific hormone, it should be possible
to locate and clone the genes and determine how the
hormone triggers the regulatory sequence.
Photomorphogenesis Light serves an important regula-
tory role in plant growth and development in addition to
providing the energy source for photosynthesis.
Photomorphogenesis, the light-regulated developmental
changes of a plant, is primarily under the control of a
pigment called phytochrome. Phytochrome regulates such
diverse effects as internode elongation, leaf unfurling,
flowering, seed germination, and chloroplast movement.
This high-molecular-weight pigment consists of a
tetrapyrrole chromophore attached to a specific protein.
Phytochrome exists in two molecular configurations that
are reversibly interconver ted by light. One config-
uration, Pfr, is the active form, while the other,
Pr' is inactive. Red light converts Pr to Pfr; far
red light converts Pfr to Pr. Thus, changes in light
quality (the amount of red versus far red light) serve as
a reversible biological switch in plant development.
Research on phytochrome has focused on the chemical
and physical characteristics of phytochrome and on its
location in the cell as well as changes in gene expres-
sion regulated by phytochrome. Specific genes regulated
by phytochrome have now been cloned. Several of these
are for proteins involved in photosynthesis, such as the
small subunit of Rubisco, and the chlorophyll a/b pro-
teins. These phytochrome-regulated genes are useful in
the study of transcriptional regulation of the individual
genes and can be used to study the gene regulatory
sequences that respond to phytochrome.
Chemical approaches that further explore the molecular
structure of the phytochrome protein, immunological
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approaches to localize phytochrome within the cell, and
study of the regulation of gene expression by phytochrome
are contributing to an understanding of photomorphogen-
esis and the relationships that exist between phyto-
chrome and the phenomena it regulates. The recent obser-
vation that phytochrome controls the transcription of its
own gene will have a profound effect on our understanding
of photomorphogenesis.
Cell Culture and Plant Regeneration
Two classes of plant hormones, the auxins and the
cytokinins, must be added to culture media to support
plant cell proliferation in vitro. While it is rela-
tively easy to fulfill the requirements for meristematic
plant cells to continue unorganized cell proliferation in
tissue culture, it is far more difficult to obtain organ-
ized growth and regeneration of plants. Only certain
genotypes of a species will readily regenerate from
tissue culture. The factors necessary for regeneration
of intact plants from tissue culture remain largely
unknown. It appears, however, that plant hormones are
involved, because the relative concentrations of auxin
and cytokinin added to media can promote or inhibit
regeneration.
The ability to regenerate plants from cell cultures at
will is important to progress with gene transfer in
plants. In a useful gene transfer system, DNA is intro-
duced into a cell of the species of interest, and that
cell is regenerated into a functioning plant that has
been altered only by the introduced DNA.
Plant organ and tissue culture is a well-established
technology that originated in the early part of the
twentieth century. In certain horticultural species, use
of tissue culture is a small but important industry.
Progress in manipulating cultures of some major food
crops, including the cereals and legumes, to achieve
plant regeneration has been much slower than with other
crops such as potato, tomato, and tobacco. His major
deficiency in the fundamental knowledge of plant devel-
opment will become an even greater constraint to research
in the future unless it is closed by a major commitment
to the study of plant regeneration from tissue culture.
Understanding the role of plant hormones in organogenesis
and growth is an important aspect of this research.
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Research Status
New and sophisticated techniques, including instru-
mentation for high-resolution chemical analyses,
monoclonal antibodies, and methods for identifying,
cloning, and sequencing genes are rapidly advancing the
understanding of the regulation of plant growth and
development. There is a wealth of descriptive infor-
mation on the roles of plant hormones and the
photomorphogenic pigment phytochrome as regulators in
coordinating the development of form and function in
plants. Increasing evidence points to phytochrome and
plant hormones as major factors in gene expression. As
the molecular understanding of gene expression in plants
increases, so will the opportunities for identifying the
mechanisms of action that plant hormones and phytochrome
use to regulate gene expression. Alternatively, regu-
latory sequences of genes that respond to a plant hormone
or phytochrome can be used as powerful tools in genetic
engineering.
'The original discovery of phytochrome and much of the
outstanding early basic research conducted on this photo-
morphogenic pigment was accomplished by ARS scientists.
The ARS should strive to reestablish its leadership role
in basic research on plant growth and development. The
focus of future research efforts within the ARS should
include:
· Biosynthesis and degradation of plant hormones and
phytochrome, with an emphasis on the regulation of genes
coding for enzymes that synthesize or inactivate these
substances;
· A molecular understanding of the role of
phytochrome and plant hormones in regulating gene
expression, particularly on their effects on the
regulatory sequences of genes; and
o The role of regulatory substances
in major yield-
controlling processes such as flowering, fertilization,
germination, and senescence.
Physicochemical Stress
Physicochemical stresses such as drought, cold, heat,
salt, and toxic ions cause extensive crop losses in the
United States and throughout the world. These stresses
are the main factors limiting expansion of food, feed,
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and fiber production. They are the basis for unrealized
production potential. This is indicated by the fact that
the average yield for eight major U.S. crops including
corn, wheat, soybeans, sorghum, oats, barley, potatoes,
and sugar beets is estimated to be only some 20 percent
of the record yield for the same crops. Of the
unrealized 80 percent of the potential yield, physi-
cochemical stress accounts for about 70 percent with the
remaining 10 percent attributable to insects and
diseases.]
The effects of physicochemical stress may be dramatic,
killing or severely injuring whole crops. Factors
causing such dramatic effects include extremes of temper-
atures and severe drought, such as occurred in the
midwestern United States in 1983 when the average bushel
per acre yield for corn in Illinois dropped 40 percent
compared to the average yield for the state in the
previous year. Less apparent are stress conditions that
cause no visible injury but still retard plant growth and
reduce crop yield. Factors causing these more subtle
effects are limited water supply, unfavorable tempera-
tures, saline soils, and the presence in the soil of
toxic ions such as aluminum. Increasing the tolerance of
major crops to physicochemical stress could produce
enormous benefits by increasing or stabilizing produc-
tivity with little additional cost to the farmer.
The major obstacle to increasing the tolerance of crop
plants to physical and chemical stresses is the lack of
fundamental knowledge of the basic mechanisms of stress
injury and stress tolerance. Past breeding programs for
increased stress tolerance have used a trial-and-error
approach based on the ability of a genotype to survive a
particular physicochemical stress. Determining only
survival, however, gives little indication of the
specific stress effects that contribute to the dramatic
drop in productivity potential noted previously. A
dearth of knowledge of specific mechanisms that confer
increased stress tolerance has precluded combination of
compatible traits into desirable stress-resistant
genotypes. In addition, conventional breeding methods
allow gene transfer between closely related plants only,
which restricts the available gene pool. Recent advances
in plant molecular genetics, however, have opened up the
1J- S. Boyer, 1982. Plant productivity and environment.
Science 281:443-448.
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possibility of transferring specific traits between dif-
ferent, genetically incompatible species. But progress
in conventional breeding and genetic engineering ap-
proaches will remain extremely limited until researchers
have a better understanding of the effects of specific
stress factors on plant growth and the effects of the
genetic traits that mitigate the impact of stress factors
Plant Responses to Stress Factors
The effects of drought, salinity, heat, and cold on
plants are usually multiple and often interrelated.
Studies of the responses of plants to physicochemical
stresses show that many biophysical and biochemical steps
in metabolism can be affected. From this complex of
multiple responses, it is often difficult to identify the
primary site of damage. Thus, many response factors must
be studied to determine the threshold of damage for dif-
ferent responses and to understand the relationships that
might exist between the primary effect and the cascade of
processes that are, in turn, affected.
Drought Water stress reduces or arrests plant growth
because of a variety of effects. The carbon fixation
steps of photosynthesis stop because carbon dioxide
exchange into the leaf is blocked as the stomata! pores
close to halt further water loss from the plant via
transpiration. Heat energy from solar radiation is no
longer effectively dissipated through the evaporation of
water, and leaf temperatures may rise to damaging levels.
Solar energy, absorbed by photosynthetic pigments, is no
longer channeled to carbon fixation, and the photosyn-
thetic system can be inactivated as energy is diverted to
harmful reactions. Without sufficient turgor pressure
the cells at the growing tips of plants cannot expand and
elongate. Growth is also arrested because the products
of photosynthesis needed for cell wall and protein
biosynthesis are inireduced supply. Plant growth
substances involved in regulating the opening and closing
of stomates are also likely mediators of plant growth
changes brought about by water stress.
me number of possible effects in the cascade of
responses to drought will depend on the severity and
duration of the water stress. Some responses, such as
stomata! closure, are quickly reversed when water is
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added. The kinds and degree of damage and the rates of
repair or replacement are unknown. Also, drought may
trigger long-term developmental changes in morphology and
growth pattern that limit flowering, pollination, and
seed development, which can greatly reduce crop yield.
Salinity The osmotic properties of high concentrations
of salt ions in soil water produce the same effects as
those resulting from drought. In addition to creating
this state of so-called physiological drought, the excess
of salt ions can cause ionic imbalances across plant
membranes and in the cytoplasm that lead to impairment of
metabolism.
Low Temperature Chilling temperatures lower the rate
of enzyme activity and retard plant metabolism, including
the processes of photosynthesis. Low temperature can
also cause phase transitions that alter the molecular
configuration of lipid components in membranes. This can
result in leakage of ions and other solutes and
impairment of water uptake. Such phase transitions may
adversely affect the integrity and function of other
important cell membranes such as the vacuolar membrane,
or tonoplast, and mitochondrial and chloroplast
membranes. Freezing temperatures just below O C
disrupt membranes, especially the external plasma
membrane. me formation of ice crystals causes -
mechanical injury to cells and produces severe water
stress and excessive solute concentration.
High Temperature As temperatures in a plant are
raised, water loss increases, thereby causing and
exacerbating the effects of water stress during drought.
Excess heat energy can cause metabolic imbalance by
denaturing enzymes. Photosynthesis and other key
processes in chloroplasts are reduced partly through loss
of the integrity of chloroplast membranes. For example,
increased temperature can result in increased fluidity of
chloroplast membrane lipids. This may be responsible for
decreases in the activity of the photosynthetic
photosystems organized on those membranes within the
chloroplast
.
Stress-Tolerance Mechanisms
Through genetic change and natural selection, many
plants have been able to adapt their physiology and
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morphology to tolerate climatic and environmental
extremes. Mangrove trees grow in sea water, and certain
other wild species can tolerate highly saline soils.
Cacti and other desert plants survive wide temperature
shifts between night and day as well as periods of
prolonged drought. Some plants have adapted to the
frequent freezing and thawing conditions of the tundra.
Others manage to tolerate otherwise toxic levels of ions
found in mine spoils and in serpentine or acid soils.
Many plants survive and grow in poor soils with limited
nutrients. Certain plants also exhibit tolerance to
atmospheric pollutants such as ozone and sulfur dioxide.
Most of the plants that have adapted to survive in
truly extreme conditions are wild and not considered to
be important to U.S. agriculture. Such wild plants,
native to contrasting environments that are extreme in
exhibiting one or a combination of stress factors, how-
ever, can provide invaluable experimental material for
the identification of stress-tolerant mechanisms and
genetic manipulation.
A comparison of physiological responses to stress in
both stress-sensitive and stress-tolerant species is a
valuable experimental approach. For example, tolerance
of water stress by the photosynthetic system may in part
depend on the ability of the plant to minimize the accu-
mulation of excess excitation energy during periods of
high irradiance. Plant-water relationships might also
affect repair processes that may be operating both during
and following exposure of the leaves to high irradiance
levels. The plant's ability to maintain an adequate rate
of repair, even during periods of low water potentials,
may be an important stress-tolerance mechanism.
Tolerance to salinity stress may depend on the ability
to accommodate osmotic changes by concentrating ions and
other solutes in leaves, roots, and specialized cells.
Small molecules such as praline, glycine-betaine, and
polyols accumulate in some species when they are
subjected to water or salinity stress. In addition to
their role in osmotic adjustment, these small molecules
are possible factors in stabilizing supramolecular
complexes in the cytoplasm during water, salinity, and
temperature stress.
Research Status
Research programs on physicochemical stress must be
considered long range. Because the potential impact on
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agriculture is enormous, this research should receive
high priority within the ARS.
Attention must be focused toward research approaches
that characterize basic mechanisms used by plants in
responding and adapting to the stresses of drought,
excessive solar radiation, low and high temperatures,
salinity, and toxic ions. Research approaches should
also be designed to determine interactive relationships
of the effects of major stress factors.
The work must be conducted at varying levels of
organizational complexity to yield an understanding of
the function at the whole-plant level. Research must
include major plant processes such as photosynthesis,
nitrogen metabolism, protein synthesis, and the transport
of water, ions, and other solutes that are either
excluded from or concentrated in intracellular compart-
ments such as the vacuole and other organelles.
Comparative studies on plants that exhibit marked
differences in their tolerance to a given stress factor
provide a powerful approach toward uncovering the basic
mechanisms of tolerance to physicochemical stresses. It
is important, therefore, that the investigator be free to
choose experimental plants best suited for the problem
under investigation.
The three major areas of research that should be
emphasized in ARS programs on physical and chemical
stress are: (1) the primary sites of damage to the plant
caused by a specific stress factor; (2) the mechanisms--
morphological, physiological, biophysical, and
biochemical--employed by stress-resistant plants to avoid
and tolerate stress; and (3) the genetic bases of these
tolerance mechanisms.
More specifically, the ARS must intensify research
efforts in the following areas:
· Mechanisms of water and solute transport, espe-
cially into and within the roots, and the design of
innovative approaches for detecting and measuring changes
in the metabolism and membrane permeability of roots;
The role of small molecules such as praline,
glycine-betaine, and polyols, not only in osmotic
adjustment but also in stabilizing molecular complexes
during stress-induced dehydration;
· The role of excessive light as a destructive agent
when photosynthesis is limited under conditions of water,
salinity, and temperature stress;
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· Identification of genes and gene products
associated with stress tolerance in stress-adapted
genotypes and species; and
O Aspects of membrane properties such as changes in
the biosynthesis of major membrane constituents;
temperature-related changes in lipid fluidity and
membrane protein stability that affect the functional
integrity of the chloroplast, mitochondrial, vacuolar,
and plasma membranes; and related aspects including
dehydration-induced phase transitions, freeze-induced
electrical perturbations, and changes in thermomechanical
properties.
Representative terms from entire chapter:
plant hormones
