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11
Plant Biology and Agriculture
Plants Constitute the Only Renewable Source of Energy
The enormous quantity of energy from the sun that is captured by the earth
every day becomes available for life processes only through the photosynthetic
activities of plants, algae, and a few kinds of bacteria. These activities have
resulted in the characteristics of the atmosphere that we breathe and have altered
our atmosphere's chemical makeup so that it is hospitable to animal life; pro-
longed over hundreds of millions of years, these activities have given rise to the
fossil fuels that power our civilization. At the same time, photosynthesis consti-
tutes the only renewable source of energy that is available to us for the future: a
source of energy that is clean and potentially inexhaustible. Since plants directly
or indirectly provide for our fuel and fiber needs in addition to being our primary
source of food, they are exceedingly important to us from every point of view.
Understanding their characteristics is of vital importance for the advance of
biological knowledge and for human prosperity as well.
Vegetation plays a major role in maintaining the earth-atmosphere system in
a habitable state. Except for the polar icecaps, snow- and ice-covered mountains,
and certain of the earth's deserts, all land masses are covered with vegetation.
This vegetation contributes to global energy and water budgets through modifica-
tion of the solar energy, water, carbon dioxide, and nitrogen exchanges at the
earth's surface. In short, not the atmosphere, nor the soil, nor any of the other
conspicuous features of the earth's surface would exist in their present condition
if it were not for the existence of photosynthesis, a process that we now believe to
have evolved among the cyanobacteria (blue-green algae) at least 3.5 billion years
ago-at least 2 billion years before the origin of any photosynthetic eukaryotes
and more than 3 billion years before that of plants.
365
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OPPORTUNITIES IN BIOLOGY
The solar energy metabolically fixed through photosynthesis constitutes about
0.3 percent of the total solar radiation that reaches the surface of the earth. In
addition, a substantial fraction of the solar radiation that reaches the earth is
converted into latent heat that leaves the earth's surface through plant transpira-
tion. Some 75 trillion tons of water evaporate each year from the vegetation to the
atmosphere. Agricultural vegetation is responsible for about one-third of this
water flux and also, because of the coupling of these two processes, for one-third
of the total photosynthetic energy fixation. Natural tree ecosystems in the tropical
and subtropical zones constitute the major vegetation mediating global water and
carbon dioxide exchanges.
The greater part of our food is produced by a few species of annual crop
plants, mostly in the temperate and semihumid to semiarid middle latitudes. In
food production, accumulation of dry matter is the process of greatest importance.
Most of the weight in dry matter comes from the 175 billion tons of carbon
dioxide that agricultural plants fix annually through photosynthesis.
In addition to the more obvious activities of plants that occur above ground,
extensive activity in modifying the characteristics of the soil is a role of the roots.
The amount of water passing through a plant in its transpiration stream is many
times the amount required to supply its internal needs. All of this water, together
with the inorganic nutrients that plants require for growth, enters plants by way of
their roots. Plants accomplish this through physical forces and highly specific
transport systems. Interactions between plants and soil microorganisms are of
critical importance for certain assimilatory processes: for example, Rhizobium,
Frankia, and certain free-living bacteria for obtaining nitrogen, and mycorrhizal
fungi, regularly associated with the roots of approximately 80 percent of all plant
species, for phosphorus uptake. In addition, roots produce hormones that are
important in directing the characteristics of shoot growth.
PLANTS AND THEIR ENVIRONMENT
Through Evolution, Plants Have Developed Characteristics to Cope with
Their Environment
One group of unicellular eukaryotes, the green algae (Chlorophyta), consists
of organisms that share a number of biochemical and structural characteristics
with plants. The similarities are so great that it is generally agreed that plants
were derived from green algae, and specifically from organisms that had many of
the features of the multicellular, freshwater alga Coleochaete. The cellulose cell
walls that are such an important feature of the adaptation of plants on land
originated among the green algae, as did the ability to form starch granules within
the chloroplasts rather than free in the cytoplasm, and as did certain unique
features of cell division that are common to all plants. The ancestors of plants
invaded the land at least 430 million years ago, already multicellular and thus
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PLANT BIOLOGY AND AGRICULTURE
367
protected from the environmental extremes that they were to encounter there. The
earliest plants were evidently mycorrhizal, the adaptive features of their symbio-
sis with fungi assisting them in growing on and eventually molding the features of
the raw soils of those ancient times.
With their rigid cellulose cell walls, the bodies of plants are put together as if
from a series of bricks. The sorts of cellular movements characteristic of animal
embryology are impossible among such organisms, as is the ability to move from
place to place in search of more suitable habitats or mates. Consequently, plants
have evolved features that suit them to a sessile existence. Their life processes are
bathed by a continuous stream of water that moves steadily from their root hairs
into their roots, up through specialized conducting cells called xylem through the
stems and into their leaves, and then mostly dissipates through the leaves through
specialized openings called stomata, which also admit the carbon dioxide that
plants require for photosynthesis. A waxy cuticle, similar to the outer covering of
may arthron~xl.s evolved among the earliest Plants and helps to protect them
_ ~ ~ , ~ ~
from drying out.
Rooted in one place, many kinds of plants must tolerate a wide variety of
environmental extremes. The consequent selection pressures led to the evolution
of plant species that can withstand temperatures ranging from that of liquid
nitrogen (-195.8°C) to 90°C and that could grow between temperatures lower
than 0°C and higher than 60°C. Some plants are able to grow in solutions as
concentrated as saturated salt and to withstand desiccation to the air-dry state.
An additional characteristic of plants not found in animals is the ability to
grow endlessly from areas of cell division, or meristems, that occur at the tips of
the roots and shoots. New plants can be propagated from such meristems, and,
depending on their growth form, may grow through the soil into areas of favorable
nutrient status. Each plant can be both embryonic and senescent simultaneously,
and the entire history of a plant's development can often be traced in a single
organ.
Understanding Plant Characteristics Is Important to Agricultural Development
In nature, the ability of plants to reproduce is of fundamental importance.
When plants are grown as crops, it is often their seeds, fruits, or vegetative
reproductive structures, such as tubers or fleshy roots, that people desire. The
characteristics of crops have been modified by selection and hybridization for at
least 11,000 years and are now being modified more precisely by the techniques
of genetic engineering. Cultural practices are also important in promoting crop
yield. In all types of agriculture, opportunities exist to improve yield in different
areas of the world.
The optimal yield of particular strains of pants is usually tested by growing
them in nonlimiting conditions-ones in which they do not encounter stress from
drought, lack of nutrients, pests, or for any other reason. Such conditions are
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OPPORTUNITIES IN BIOLOGY
approximated when crops achieve their highest recorded yields. Record yields
may be some three to seven times as high as average yields obtained under more
usual conditions in the same year. In the United States, for example corn (maize)
had a record yield of 19,300 kilograms per hectare in 1975, but yielded only 4,600
kg/ha on average.
Average agricultural productivity, therefore, falls far short of the genetic
potential present in today's crops. Major environmental pressures must affect
plants in ways that prevent the expression of their full genetic potential. Thus,
improvements in productivity need not rest solely on increases in genetic poten-
tial. For this reason, both the identification of the environmental forces and the
manipulation of crops to express their genetic potential more fully are important
research areas in plant biology.
What are some of the environmental pressures that decrease productivity in
plants? Diseases and insects are important contributors: These pests depress U.S.
crop yields by an estimated 5.1 and 3.0 percent, respectively, below their genetic
potential. In addition, weeds, which compete with the crops, depress yields
another 3.5 percent overall, despite the widespread and relatively efficient appli-
cation of control measures. An additional large depression in yields must be
attributed to the only other factor that can be unfavorable, the physicochemical
environment. An unfavorable physicochemical environment is found in soils and
climates that are ill suited for plants. Adverse physicochemical environments
such as an insufficient supply of water or nutrients-caused yields to be far below
their genetic potential. Sometimes the environments in which crops are grown are
inherently unfavorable, and sometimes farmers choose not to improve these
conditions or cannot afford to do so. At any rate, physiochemical limiting factors
are the most important negative influence on U.S. agriculture. In many other parts
of the world, crop losses caused by diseases, insects, and weeds are considerably
more severe than in the United States, but physiochemical factors usually pre-
dominate everywhere in limiting agricultural yields.
The major physicochemical resources for plants are water, soil type, nutri-
ents, carbon dioxide, oxygen, and solar radiation. Of these, water is generally the
most limiting. Permanently dry and shallow (drought-prone) soils make up about
45 percent of the total U.S. land area. About 40 percent of insurance payments for
crop losses are made to drought-stricken farmers. Cold and wet environments are
also important limiting factors, followed by salinity, hail, and wind. To cope with
water deficits, farmers have for thousands of years irrigated their fields, which has
contributed significantly to higher yields. However, water has become increas-
ingly scarce, and many alternative uses compete with agriculture for it. In
addition, water of poor quality causes progressive soil degradation and a conse-
quent loss of overall productivity. Irrigation, therefore, affords only an incom-
plete solution to the water limitations encountered by plants.
Plant nutrients will probably be less limiting than water in the immediate
future because they are more abundant or can be produced in sufficient quantities
at an acceptable cost. Supplies of nitrogen, phosphorus, and potassium are likely
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PLANT BIOLOGY AND AGRICULTURE
369
to be sufficient to support U.S. agriculture for the next 30 to 40 years. However,
energy must be used in the manufacture of ammonia (the major source of nitro-
gen), and this is the largest energy input to the nonirrigated farm. Thus, the cost of
energy will be a major constraint on the availability of nitrogen. Similarly, the
cost of pesticides will increase with energy costs. Thus, the use of water, certain
nutrients, and pesticides will be increasingly restricted, either because resources
are limited or for economic reasons. These limitations may be overcome, in part,
by the use of plants with lower requirements for these resources, a key aspect of
the potential of Genetic engineering in combination with other methods for crop
~ ~ i, %,
improvement.
The Mechanisms by Which Plants Cope with Adverse Environments Have Only
Begun to Be Understood in Molecular Detail
Unfavorable environmental conditions depress the rate of photosynthesis,
and we are starting to understand the mechanisms by which this process occurs.
Such knowledge will be highly applicable for the improvement of crop perform-
ance. The expansion of cell walls during plant growth is also affected by
unfavorable environmental conditions. These walls contain cellulose as reinforc-
ing microfibrils embedded in a carbohydrate and protein matrix that can flow in a
plastic manner. The large pressures inside the cell, which are generated by
osmotic forces, can cause plastic deformation of the wall and cell enlargement.
The orientation of the cellulose microf~brils determines the direction of growth.
Plant cell enlargement is extremely sensitive to certain environmental conditions
and is retarded by low temperatures and drought. Such adverse conditions, for
example, can cause seeds to fail to germinate or flowers to fail to open. The
changes in molecular architecture of the cell wall that take place under such
circumstances are largely unknown, as are the roles of water transport and plant
hormones. Understanding these factors more completely bears directly on our
ability to improve agricultural yield and quality.
The mechanisms of inorganic ion accumulation by plants also constitute a
critical area for investigation. Plants differ genetically in their ability to accumu-
late nutrients especially nitrogen, phosphorus, and iron from a given kind of
soil, but the molecular bases of these differences are poorly understood. By
manipulating these features, performance would be improved.
Plants vary in their ability to withstand freezing temperatures; for example,
some plants have developed a way to keep water unfrozen in cells at temperatures
as low as 40°C. This ability permits some kinds of trees and shrubs to survive the
extreme freezing temperatures that occur seasonally at high latitudes and high
altitudes, but we do not understand its structural and molecular underpinnings at
all. The reproductive structures of plants are characteristically more susceptible
to low temperatures than their stems and leaves, but, again, we do not understand
the mechanisms involved.
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OPPORTUNITIES IN BIOLOGY
Many tropical and subtropical plants die when temperatures drop below
12°C, but some, such as cotton, can become acclimated to such chilling condi-
tions. Acclimation is accompanied by a change in the phospholipid composition
of the outer membranes of the cells and probably includes similar changes in the
energy-transducing membranes of the mitochondria Energy metabolism seems
to play an essential role in the breakdown of cellular functions in cold-sensitive
plants. Many of the storage problems of fruits and vegetables can be traced to the
breakdown of membranes and the derangement of energy metabolism that occur
at these temperatures. The biochemical basis for chilling resistance and acclima-
tion needs to be established much more firmly to form a basis for improving the
ability of subtropical plants to resist cool temperatures.
A better understanding of water transport in plants can likewise improve crop
performance. The transport of water through the vascular system occurs under
great tension (negative hydrostatic pressures), and the continuity of the water
pathways is sometimes broken an abrupt event that seems to be caused by
cavitation of water under tension. An embolism that forms in the vascular tissue
blocks further transport in that section of the system. Modern methods of
electronic analysis indicate that such events occur frequently and are influenced
by vascular architecture. Knowledge of how to keep the vascular pathways intact
and filled with water is an important need.
Accurate studies of plant biology demand access to controlled environments.
Growth chambers and similar facilities petit the efficient evaluation of factors
affecting growth of plants throughout their entire life cycles. In addition, tissue
culture and seedling culture systems provide convenient ways to study problems
of plant growth. Such systems provide opportunities to explore how limiting
water affects the growth of roots and shoots and allows the use of biophysical
methods, growth regulators, genetic mutants, and molecular genetics to explore
some of the reasons for altered development. Tissue culture systems likewise
permit experimentation under controlled conditions. They have the additional
advantages that metabolites can be supplied in the culture medium and that
selection pressures can be created to identify desired genotypes at the cellular
level.
Taken together, these research areas illustrate some of the ways a better
understanding of plant growth could improve agricultural productivity. In prin-
ciple, most of the features we have discussed should have a genetic basis. Thus,
selection for more efficient water use and nutrient acquisition, as well as for the
ability to avoid toxic ions, should help produce plants able to withstand unfavor-
able environments. The genetic and molecular mechanisms of plant resistance to
disease and insect attack are also becoming known. Pest organisms are not only
responsible for crop loss in the field, but also for a large amount of loss during
storage. In these areas, as in many others, an improved understanding of the ways
plants grow and develop will enhance our ability to produce better crops.
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PLANT BIOLOGY AlID AGRICUL7VRE
PHOTOSYNTHESIS
A Better Understanding of Photosynthesis Is Crucialfor Our Future
371
Plants, like all organisms, depend on the products of photosynthesis for their
growth (Figure 11-1~. The accumulation of plant biomass is a measure of the
plant's total photosynthesis less the respiratory losses Hat have occurred during
its growth. Crop productivity is linked to the seasonal photosynthetic perform-
ance of the crop canopies. For this reason, knowledge of the relation between
productivity and photosynthesis has largely provided the incentive for the broadly
based research effort into this elementary plant process.
Advances in Photosynthesis Research Utilize the Full Range of Modern
Biological Approaches from Biophysics to Molecular Biology
Tremendous slides have been made in gathering information about the
catalytic components of photosynthesis at the level of atomic structure. Wide-
ranging discoveries have created the opportunity to understand photosynthetic
mechanisms at a molecular level. The most significant of these breakthroughs has
Light :~
Starch
CO2 + H2O
Sucrose
F-~-1
Sucrose
~ -1
Sucrose
Vascular
tissue
Starch CO2 + H2O Sucrose
(Stored)
Photosynthetic
plant tissues
(Sources: leaves
stems, etc.)
N on photosynthetic
plant tissues:
(Sinks: roots, seeds,
tubers, etc.)
FIGURE 11-1 Diagrammatic illustration of carbon processing in green plants. [B. B. Buchanan,
University of Califomia, Berkeley]
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OPPORTUNITIES IN BIOLOGY
been the recent crystallization of the photosynthetic reaction center of the purple
bacterium Rhodopseudomonas viridis and the determination of its three-dimen-
sional structure by x-ray diffraction analysis (see Figure 3-2~. The wealth of
existing knowledge concerning the mechanistic features of these complexes,
which lends significance to this structural information, calls for corresponding
structural work on other catalytic components of photosynthesis. The new struc-
tural information contributed immediately to our understanding of the molecular
functioning of photosynthetic bacterial reaction centers. The three-dimensional
structure along with the biochemical and biophysical information about the vari-
ous catalytic and redox-active sites (sites of electron transfer) have focused
attention on specific regions of the amino acid sequence, which seem to have
special significance in light absorption and in charge-transfer processes. In this
prokaryotic photosynthetic organism, designed alterations in the genes coding for
polypeptides that make up the reaction center are possible and becoming routine.
This sort of molecular engineering, coupled with the sophisticated capabilities of
molecular spectroscopy and biochemistry, is certain to contribute much to our
understanding of photosynthesis.
The crystallization of the Rhodopseudomonas reaction center has signifi-
cance beyond the information obtained from its x-ray structure since it represents
a fundamental discovery pertaining to the crystallization of integral membrane
proteins. An intensive effort is under way to crystallize other major polypeptide
complexes of bacterial and plant photosynthetic membranes.
The development and refinement of numerous other physical techniques are
contributing to the revolution in structural information about the catalytic compo-
nents of photosynthesis. In particular, dynamic information about structural
transformations occurring during catalysis, which cannot be obtained from the
static picture provided by x-ray analysis, is now becoming available through the
use of powerful physical methods. The development of high-resolution nuclear
magnetic resonance ~MR) techniques and their application to biology have been
particularly successful. For instance, spin-echo NMR techniques allow the selec-
tive detection of a small subset of highly mobile, charged amino acid side chains
that extend from the protein into the surrounding aqueous environment. The
focus of this technique can be narrowed further to those amino acid residues that
respond during catalysis; in other words, attention can be focused on the catalytic
site as has been done for the chloroplast's coupling-factor enzyme. Even greater
detail about the identity and rearrangements of catalytic site groups can come
from other NMR techniques, such as double resonance and two-dimensional
methods.
NMR is but one example of the array of physical techniques being used to
analyze the structural basis of photosynthetic reaction mechanisms. Neutron
scattering, electron scattering and electron microscopy, linear and circular di-
chroism, resonance Raman spectroscopy, Fourier transform infrared spectros-
copy, extended x-ray absorption fine structure, and electron paramagnetic reso
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PLAINT BIOLOGY AND AGRICULTURE
373
nance spectroscopy are all contributing to the accumulating wealth of information
about the molecular structure of the photosynthetic apparatus.
The amino acid sequence of photosynthetic membrane polypeptides has
recently been determined from the nucleotide sequence of the corresponding
genes. This advance, in tum, has permitted estimation of the two-dimensional
folding patterns of these proteins by hydropathy analysis. This information has
been taken into account in the most recent models of electron transfer through the
complex.
Much Has Been Learned About Regulatory Mechanisms in Photosynthesis, but
Much Remains to Be Done
With our current knowledge about the component processes of photosynthe-
sis, it has become possible to investigate specific questions about their interde-
pendence. The most important mechanism in the regulation of chloroplast pro-
cesses is light activation, a central feature that coordinates the light-driven reac-
tions with the so-called dark reactions of photosynthesis. Light is absorbed by
chlorophyll and is converted to regulatory signals that modulate the activity of
selected enzymes. Such regulation is essential because enzymes that degrade
carbohydrates coexist in chloroplasts with enzymes of carbohydrate synthesis.
Some biosynthetic enzymes are activated by light, whereas degradative enzymes
are deactivated by light. In this way, the concurrent functioning of pathways that
operate in opposing directions (futile cycling) is minimized and the efficiency of
temporally disparate metabolic processes is maximized.
A number of soluble enzymes of photosynthetic carbon dioxide assimilation
and other biosynthetic pathways show a similar activation response to light. Light
regulates specific enzymes through a number of complementary mechanisms that
have been identified during the past few years. These include changes in the
concentration of certain ions, the concentration of regulatory metabolites, and the
oxidation state of thiol groups (-SH) on key regulatory enzymes. Important in
such thiol changes is the ferredoxin-thioredoxin system, a system in which thiore-
doxins small regulatory proteins are reduced in the light by the photosynthetic
apparatus. The reduced thioredoxins, in turn, reduce and thereby activate selected
target enzymes. In this way, the cell can adjust flux through the metabolic
pathways associated with oxygenic photosynthesis in accordance with energy and
metabolite status.
The catalytic activity of ribulose bisphosphate carboxylase/oxygenase (ru-
bisco) is also modulated by light. Rubisco, which is the most abundant enzyme in
the biosphere, performs the carboxylation reaction, the basis for photosynthetic
carbohydrate production. Studies on the mechanism of rubisco activation have
taken an unexpected turn with the recent discovery of the involvement of a
polypeptide dubbed "activase." In certain plants, a newly identified inhibitor, 2'-
carboxyarabinitol-l-phosphate, turns off the enzyme at night. The mechanisms
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OPPORTUNITIES IN BIOLOGY
controlling the formation of this inhibitor and the mode of regulation by the
activase are currently under active investigation.
During the past years, much progress has been made in understanding the
regulation of sucrose production in plants. Sucrose is the mobile form of energy
that most plants form for transport to photosynthetic sinks; for example, to storage
organs such as tubers and seeds that are the source of most of the world's food.
During photosynthesis, chloroplasts convert carbon dioxide, water, and phosphate
to triose phosphates, which migrate to the cytosol and combine to form sucrose.
Photosynthesis requires inorganic phosphate, which is released during sucrose
synthesis; therefore, photosynthesis and sucrose synthesis must be closely coordi-
nated. There is evidence that this coordination is provided in part by phosphate.
Recently, a second compound specifically serving this function has been identi-
fied. Fructose-2,6-bisphosphate coordinates the metabolism of sucrose and starch
and, in so doing, links metabolic processes of the chloroplast with those of the
cytosol. Recent results suggest that fructose-2,~bisphosphate may also coordi-
nate cytosolic and amyloplast (a starch containing plastic) metabolism in sink
tissues.
Our Current Knowledge of the Biochemistry and Physiology of Photosynthesis
Has Made It Possible to Study the Process in Whole Plants or Intact Tissues
Studies on photosynthesis have important applications to the improvement of
agricultural productivity. Low temperatures; drought; photoinhibition; the accu-
mulations of herbicides, pesticides, or fertilizers; and pollution are examples of
frequently encountered conditions that compromise the efficiency of production
in crops. Impaired photosynthesis is a major contributor to these losses, and we
need to understand the mechanisms by which it takes place, something we are
now in a position to do.
One area poised for major advances is the application of recently developed
and adapted physical techniques to investigate component processes of photosyn-
thesis in situ. Techniques such as kinetic absorption spectroscopy, delayed light
imaging (Figure 11-2), Now spectroscopy, flash fluorescence, and photoacoustic
spectroscopy are now applied to diagnostic studies of how particular environ-
mental conditions may influence intersystem electron transfer, adenosine
triphosphate formation and consumption, enzyme activation, light regulation,
photosynthetic reaction center activity, and the transfer of light energy. The use-
fulness of these techniques depends on an underlying experimental basis for inter-
preting the often complex results obtained from in situ measurements. This fact
points to the need for expanding this information base.
The development of "model" plant systems will unquestionably contribute to
the solution of problems in photosynthesis related to agriculture. A notable recent
advance has been the development of vigorous photoautotrophic cultured cell
lines. Because of the difficulty of growing plants to maturity under heterotrophic
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PLANT BIOLOGY AND AGRICULTURE
375
FIGURE 11-2 Delayed light imaging in herbicide-treated bean leaves. Leaves normally emit a tiny
portion of the photosynthetically active radiation that they absorb. Defects in certain chloroplast
processes increase the amount of light that is emitted. This feature of photosynthesis has been
exploited by using the increase in light emission to reveal the photosynthetic performance of different
portions of leaves under stress. The four phytoluminographs depict the spatial distribution of delayed
light emission in a red kidney bean leaf after the lower half of the leaf blade was sprayed with an
herbicide. The herbicide was awlied 5 minutes before illumination; the notations refer to the length
of time the leaf had been illuminated. The herbicide used inhibits the enzyme glutamine synthetase,
and its action results in the accumulation of ammonium ions, which is the most likely metabolic cause
of delayed light emission. [Donald R. on, University of Illinois]
conditions, the screening and selection of photosynthetic mutants is generally
limited to positive selection methods. The advent of these photoautotrophic cell
lines and the promise they hold for expanding the use of mutants in photosynthe-
sis research emphasizes the need for substantial effort aimed at developing reli-
able plant regeneration procedures. It is becoming increasingly evident that
cyanobacteria represent a highly useful model system for chloroplasts. The pro-
gress that has been made in developing a genetic transformation system for
cyanobacteria highlights their potential for the investigation of photosynthetic
processes and events generally. Among plants, Arabidopsis and Petunia are
genetically tractable, and they lend themselves particularly well to being modified
through genetic engineering. They offer many approaches to long-standing agri-
cultural problems with new research strategies having potential far beyond what
could be imagined just a few years ago.
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OPPORTUNITIES IN BIOLOGY
plasmid, the vir region, contains six genes necessary for the events leading to the
integration of the T-DNA into the plant genome. Genes in the vir region are
activated by messenger molecules that are released by Be plant.
In some characteristics, crown gall strikingly resembles a disease on olive
and oleander plants caused by the bacterium Pseudomonas savastanoi. This
bacterium induces tumorous growth by secreting high concentrations of indo-
leacetic acid and cytokinin into the tissues surrounding the point of infection. The
enzymes necessary for the production of these growth regulators in P. savastanoi
are functionally identical to those encoded by A. tumefaciens T-DNA, although
the genes in P. savastanoi are located on separate plasmids and expressed in the
bacterium. No transformation of the plant genome occurs. The nucleotide
sequences in the coding regions of the genes for cytokinin and indoleacetic acid
synthesis show a high degree of homology with those of the corresponding genes
from T-DNA. However, the promoter regions, which control the expression of
these genes, are entirely different in A. tumefaciens and P. savastanoi. This
difference in structure was expected since the Pseudomonas genes are designed
for expression in a bacterial (prokaryotic) cell, whereas the T-DNA genes must
function in a plant (euk~ryotic) cell. The similarities in the structural genes
indicate that the growth-hormone genes in the two tumorigenic systems have a
. .
common origin.
The Ti Plasmid Provides a Vehicle for Gene Transfer in Plants
Once it was known that A. tumefaciens actually transforms its plant hosts,
scientists recognized the potential usefulness of the Ti plasmid as a means of
introducing foreign genes into plants. In a number of laboratories, they designed
so-called disarmed versions of the Ti plasmid. The genes associated with growth-
hormone production were removed from the T-DNA, thereby preventing tumor
formation in transformed plants. An antibiotic resistance gene was introduced as
a selectable marker in an existing T-DNA gene for opine synthesis, and gene-
cloning sites were constructed within the disarmed T-DNA. The genes of the vir
region necessary for the integration process must be retained on the Ti plasmid or
placed into a second helper plasmid. Once the desired gene is spliced into the T-
DNA of the vector plasmid, the recombinant plasmid is reintroduced into A.
tumefaciens. The bacterial cells are incubated with plant protoplasts or with leaf
disks to allow transformation to occur. The protoplasts or leaf disks are then freed
of the bacterium and placed upon a medium favoring plant regeneration. This
transformation procedure has become so standardized that it is now routine in the
hands of trained scientists.
The Ti vector system has been used to introduce DNA sequences that cause
disease and insect resistance into plants. For example, genes responsible for the
production of an insect toxin have been transferred from Bacillus thuringiensis
into plants. These plants became resistant to certain insects as a result of this
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PLANT BIOLOGY AND AGRICULTURE
393
transformation. Disease resistance to some viruses has been introduced by
genetically engineering tobacco and tomato plants to produce the coat protein of
tobacco or alfalfa mosaic virus. These plants show resistance to virulent strains of
these viruses. An alternative method to introduce virus resistance into plants
makes use of small RNA molecules known to act as 'parasites" of some plant
viruses. These entities, called satellite RNA, replicate only in plant tissues that
are infected with a specific virus, frequently reducing the extent of replication of
the virus and ameliorating the symptoms that the virus alone would induce. The
satellite RNA becomes enrobed in the coat protein of the virus and thus may be
co-transmitted from plant to plant with the virus. The protective effect of the
satellite RNA continues even in the subsequently infected plants. Recently DNA
copies of cucumber mosaic and tobacco ringspot virus satellite RNA have been
introduced into tobacco plants. Plants transformed with either one of the satellite
RNAs and then inoculated with the respective virus showed greatly reduced
symptoms in comparison with similarly inoculated, untransformed plants.
Virulence Factors of Certain Plant Pathogens Are Natural Herbicides
Many pathogens produce secondary metabolites that are toxic to plants.
These chemicals are side-products of amino acid, carbohydrate, nucleotide, and
lipid metabolism. One such chemical is tabtoxinine-,B-lactam, which is produced
by a bacterium, Pseudomonas syringae pv. tabaci. If secreted into the cells of its
host, tobacco, tabtoxinine-,8-lactam specifically inhibits the plant's glutamine
syn~etase, an enzyme essential for the production of precursors for protein and
nucleic acid synthesis. The pathogen escapes the inhibitory action of its own
toxin by several mechanisms, among them being the production of a glutamine
synthetase that is less sensitive to the toxin. This phenomenon, called self-
protection, is common among pathogens that produce toxins as a part of their
repertoire of pathogenic determinants.
Because toxins produced by plant pathogens kill or injure plant cells, they
may be viewed as natural herbicides. The activity of such toxins in selective
instances provides a conceptual basis for the use of chemicals for weed control.
Indeed, there is an herbicide that imparts its weed-killing effects by inhibiting the
glutamine synthetase of plants. An alternative to the use of synthetic chemicals
for the control of weeds is seen in fungi that are selectively pathogenic for weedy
plants. Since He basis for this host selectivity is the production of a host-selective
toxin, this phenomenon is being explored as a way to control weeds.
Recognition and Defense Molecules Function in Path~gen-Plant Interactions
Specific molecules function in the maintenance of many kinds of order in
biological systems. Enzymes recognize substrates, cells recognize other cells and
pollen compatibility, and incompatibility determines fertility in plants. Recogni
OCR for page 394
394
OPPORTUNITIES INBIOLOGY
~::~:~:~:~; ::: GENETICALLY EN~GIN~EERED~RESISTANG'E~ ~ ~:~
:~ ~ ~ ~::~AGAIN:ST~:~P~LA:NT~:VI~:RU~SES~ ~ ~:~ ~
~ ~ ~ ~ ~ ~_, . ~, . ~: ~ ~ i, ~ ~., ~, ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~.~ ~ ~
: ::~:
~:~ ~:I/:;~n~e~lraalilonal~;memo~ot~prot~l~ng~crop~plants~aga:Inst~:~speci~:~vl~r~Us~:~:
:~ :::::~: ::: :: ::~ ::::: :::::: ~ ::: i: ::::: ::~ :~ :: :~ : :::::::: ::::::: :~:: i: :::~::~:: ::::: : ~ : ::~::::~ ~ ::: ::: ~ i:: ::~ :: :~ :~: :: :~
~i~d~iseases~has~ :been~1o :~sea~:~ for resistard:~s~ ns~6f~crop~or~its~ clos=e;i~:~ i:
~:~ :atNes in: theft wiW~and~then~td~;~intro~)he~enes~;:~responsi:ble~ f~r~this~ ~:~
~res~lstance~into~ti~t~d~i:~:st~ra~l~ns~with~le~aa~roTn To~mic~Dro~les~:lifDI-~:~
~,~ ~:t~ne~:~:pron~uct~ion~ot ~d~ls~ease-resistan~w~rth~ baa ~ro~no::mic~ ~ l
To JO~years,~a:~r~d.i~Furthe~more,~suitable~
~resi~gen~for~a~tmpor:ant d~ve~not; been~id:e:ntified~:~:rli~:~
~;~ many ~cases. ~ l~e~m~anyr~ crop p;labts~ai~ vu~iln~erable~to~viial~attack.~ AL
: ~;~::situad~lon: that h~as~n~eg~dtJ~e~eco~nom~i~c~conseg~uen¢es c ~:~ ~ ~:~ ~:~ ~:~ ~
:::::::: ~:~::~:::~:~:~::~:~:~:~:~:: ~:::~::~:::~::~:~:~::::~::~:~:::~:~:~:::~:~:~: :~: ~ ::::~:::~:~::~:: ~:~:~:~::~:~ ::: ~ ~:::~::~:
~ ~:~:~ ~ Over~the: past :20 y~ears,~1ant pathos gists: have also :~used:~:thbi~method~ ~ ~
~ ~ ~ ~ :, ~ ~ ~ ~ ~ ~ .: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ ~ ~ ~ :~ ~ ~ ~ ~ ~ i, ~ ~ ~ ~ . ~ ~ ~ ~ . . ~ ~
~;~OJ~ cOoS spr~r~ctio~n~:~to~ gee res~stance~4air~`,l::r!a Amman crop.:
~:~:~ His m~ethod~:~is ~based~on~:~i::a~n~ observation ~mad;e~more~ than:: 6 - act ago
~ ~ ~ .
~ it: ~ ~:th:at~lobacco;~p lants~cou~ld~ be ~p~d~ ~:against~: :virulent~strain:s ~:~toba~w~ :: ~
~ ~ ~ . ~ I. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~rnosa~c~r~us~ ~ l~th~~:werrifirminyulated~w~;th~am~ildi~strain~of~the~virus. ~::~
:~ ~:~ ~ ,:ls~approac ~ ~ has its~::r~s~s~incel~m~ild~ v~iral~sitr~a~ns~ can~Ye~n~se:~:td~viruJ~ent~l~:~:~:~
~ ~ a. ~ . ~ . ~ . :. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ it. ~ ~ ~ ~ ~ ~ . ~ ~
:~:~ones;~ ~ ,= 1~ mews divas :ate~:rat pert ~an~or~em~t Get D ant. ARIA ~so. virus: strains: :: i:
:::: ::~: : :::: :::: ::: :::::::::
:::: i::: :~
::::: ~::~:~:~:~::~:~: ~ ~ ~:~::~:~ ~ ~:~:~
w l t n ~ ~ ~ m ~ ~ ~ e ff e : c t s ~ ~ ~ ~ ~ ~ a n ~ ~ ~ d e ~ e l o p ~ ~ ~ ~ ~ s ~ n e r g ~ i s b c ~ ~ ~ i n t e r ~ i o n ~ s ~ w ~ ~ h ~ o ~ ~ ~ ~ ~ v i ~ u s ~ ~ s ~ .
~Nonethelass~,~the~m~ethod~ has ~bee~n~use~l in~enh~ar~ino~ croD~resistar~ce~in:~ ~1
~ ~ ~ ~ ~ _
~::~:~some~::~instar~ce~s~: :~ ~ ~ ~; ~ ~ ~ : :
~llie phenom~eno~n~;~cross~pro~tion ~prov~id~ the~conceptual~basis~f~r~
~ ~ . . · . . . ~ ~ ~.: ~ ~ ·.. ~ ~ ~ ~
9 e~n~elica y~e~ng~lne~e r Q a r esistance~agal~ns t two~ ~c ~nter e ~nt~t y pes~ 0 f~ p an t YI-
ru~ses,~l ~v ~anc ~ a ta ta~mosalc~v~lrus~(A ~l ~).~Th~e~ A - hdc~n~transior-~
~mation~ ~sys;te~m ~was~ used ~ to~ introduce~the ~ge~nes ~that~encodel~the~ co~at~
~pratein of ~:l MV and AIMV i - ~tomato ;ar d~tobacco~plants.~ ~Coat ;protei~n~'s~
::::: ::: : : :~ : : :~:: :: ~ : :::~ :: ~: ~:~: :: ::~::: ::::~:~ ~:~ ~:: ~ : ::: : :: : ::::: : :~:: ::: : ::::~ : :~ : :::: ~::: :::: : ::: :: :::~:: ::::::
~ :~normal;~; wrapped ~around~the ~viral~n~ucle~c acid~ to form~lthe~ virus~parti^.~
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~
~ants: reg:enerated ~from~the~transfo:rmed:ce}ls~(t~ransg:an:ic~plants)~ producdd~l
~TMV~and~Al~MV~co~protein, but appearedito be~normal~in~l~other~respects.
:~Whe~n~prQg~enyl:::of~:the~transgenic~pldrits~were~:~inocu~:with~::viru~lent~strains~
~:~:: ~f~71~1V~o~r~:Al~MV,:: the~plants:~:~her~esca~d ~:infection:~or~devel~ped~ ~a~ liss
: :: : : : : :: : : ~
~:~sever~form :of :the~d~isease~than~ did:~t~::~nontransformed~p~ts~:~(F~ure 1~1~-~:~:
~::~5)~. ~:~The~mol~ecular mechanism~:~:responsiblelfo:r~ - gineered'~cross-protectidn:~i~
~ . . . . _ ~
:~ 1as ~:not ;~ye '~ ~es~n ~eluadated~. ~ Re£ent~ results::~ ind~icate~:~t~hat~fewer :~:infebtion ::~
~sites~::~are~::established in:~:transpenic~plants,~probably::b~cause~Qf~somei~block ~:
at en: early seage~::~6f:the~infect~ion:~process.::::~::U:~infedtio~n does~occu~r:~:the~::rates: :~::1
:: :: :: : . :: ~ ~ : : ~ : :::: ~ :: ::::::: ~ :::: :: :: ::: : :~ : : :: :: : :: :: :: : : : :: : :: : :: : :: :~ :: ~
: of v~ral~ ~replication:~:and~ ~spread:~:through~:~th:e :~plant~:~:are~: reduced.~: Stud~Ks in
:: prog ress: :~aim:::::at:~::expl ain ing:~:th:e m echari ism of ~ p~rotection, ~ increasing: the:~ level~:~ ~ ::
ot protect~on, and extending~ protection::::: to other:viruses :and other~:plant:::
. ~ . . . .
spec~es.: ~'t ~s ~expectea~:~ t~h at:~genetically~ engin 8 e~red :cross-proteh~n~::will~:::
:relatively: soon beco:me a annQraliv :anal~c~bln mathnd tn intrmH:' '~:~ vira
: res;isttance~:into plants.:: ::: :::
:~:
::
:~:~ :~
:~:~:~::
:: ~:~:~
::~
:: ::~::
:~: ~:~:~
~:::~:~:
~:~
:
~:~
:: :: ::
~:~:~
::
:~
~ :
:~:: ~
~:~:~
::
~:~
:::
~, - ~_ ~. ~.
: :: :: :: ::
:: :~
:
~ ~::
~:~
OCR for page 395
PLANT BIOLOGY AND AGRICULTURE
395
FIGURE 11-5 Genetically engineered cross-proteciion against tobacco mosaic virus (TMV).
(heft) Control tobacco plant (VF36) inocallated with a severe strain of TMV (PV 230). (Right)
Transgenic tobacco plant that expresses the TMV coat protein gene (VF36 +CP) also infected with
TMV strain PV230. [Roger N. Beachy, Washington University]
lion molecules also mediate the interactions between pathogens and their plant
hosts. An example for this is the specific messenger function of small polysac-
charide fragments of fungal cell walls, called elicitors, which induce plants to
produce chemicals called phytoalexins, which in turn might confer disease resis-
tance to plants because they are toxic to the microorganisms that induce phytoalexin
production. A specific molecular configuration is recognized by the plant be-
cause any rearrangement in elicitor structure either abolishes or greatly reduces its
activity.
The biochemical basis for disease resistance and susceptibility is particularly
well investigated in the case of root rot in peas. When attacked by the pathogenic
fungus Nectria haematococca, peas produce a phytoalexin called pisatin. Some
strains of the fungus are sensitive and others tolerant to pisatin. The sensitive
strains cause mild disease in peas, from which the plants recover, whereas pisatin-
tolerant strains are highly virulent and kill the plants. Tolerant strains respond to
pisatin by producing an enzyme, pisatin demethylase, which degrades pisatin to a
nontoxic product. Thus, the fungus has developed a way to circumvent a defense
mechanism of the plant. Pisatin demethylase is a cytochrome P4so monooxyge-
nase, the same type of enzyme that functions in mammalian livers as a detoxifying
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OPPORTUNITIES IN BIOLOGY
agent. The gene encoding pisatin demethylase has been isolated from N. haema-
tococca. Study of the cloned gene will help us to understand how the fungus
recognizes phytoalexins and how it has evolved the capacity to live in their
presence.
In solanaceous plants, such as tomato and potato, small cell-wall fragments
called oligogalacturonides are released when plant tissue is injured by chewing
insects or mechanical rupture. Such oligogalacturonides, or perhaps some other
signal molecules, are transported throughout the plant and systemically induce the
production of a powerful proteinase inhibitor that interferes with the digestion of
proteins. In the initial act of feeding, chewing insects seem to activate a defense
system that renders the plant less digestable and may discourage further feeding.
The systemic production of proteinase inhibitors in response to injury also occurs
in nonsolanaceous plants; it might represent a general plant defense against insect
predation.
In some pathogen-plant interactions, a plant's susceptibility or resistance to a
particular pathogen is determined by a single gene in the host and another in the
pathogen. This gene-for-gene relationship implies a high degree of specificity
and recognition between plant and pathogen. Resistance may arise from a
specific interaction between gene products of the pathogen and the host plant.
Susceptibility would result from a lack of such an interaction. These hypotheses
are being tested by isolating genes from bacteria that confer race specificity for
their hosts. The products of the genes are being identified, and the structures
responsible for specificity will be determined.
With Today's Technology, Fundamental Problems of Plant-Pathogen
Interactions Can Be Investigated at the Molecular Level
The intimate relationship that has evolved between plant and pathogen is now
the focus of attention by plant scientists. These studies have been greatly en-
hanced by new techniques in cell culture, chemical analysis, and molecular
biology. It should be possible now to obtain answers regarding the responses of
plants to challenge by abiotic factors, pathogens, or pests. What is the nature of
disease or insect resistance in plants? How are resistant responses induced? What
controls the expression of these resistance genes? Why are these genes not
expressed in certain host-parasite combinations? Central to much of this research
is our current ability to study mechanisms of communication between organisms
and between cells. Transmembrane signaling, second messenger activity, and
long-distance communication will be areas of active research during the next
decade.
Other research areas with exceptional opportunity include the nature of
pathogen genes that are essential for causing disease. Is specificity determined by
the nature of the plant products that induce the expression of pathogenicity genes
or is it determined by regulatory functions that modify the response of pathogens
OCR for page 397
PLANT BIOLOGY AND AGRICULTURE
397
to these products? The recent success in conferring resistance to plants by
introducing viral coat-protein genes suggests that, as we understand the mecha-
nisms of cross protection, we will also be able to exploit this phenomenon to
control virus disease.
The excitement generated by our ability to transform plants by means of the
Agrobacterium T-DNA should be tempered by our ignorance as to how this plant
pathogen is able to transfer DNA or how this DNA is integrated in the plant
chromosome. The search for other pathogens that can serve as sources of vectors
to introduce useful genes in our major cereal crops will, in Me near future, greatly
expand our ability to improve plants by genetic engineering.
Rapidly expanding computer technology has increased our knowledge of
how plant pathogens are disseminated Cooperation among mathematicians,
computer experts, and plant pathologists will continue to lead to better prediction
of epidemics and, thus, to more rational application of control procedures. There
is now renewed interest in the ability of bacteria to colonize leaf surfaces, for
example. What triggers the change from epiphytic to parasitic habit in certain
bacteria? The recent interest in the possible use of epiphytic, non-ice nucleating
bacteria (which do not act as ice-nucleation centers) to prevent frost damage to
plants is an example of how answers to some fundamental questions in plant-
pathogen interactions can help stimulate the plant biotechnology industry.
GENETIC IMPROVEMENT OF PLANTS
Plant-Breeding Programs Can Now Be Enhanced by Molecular Biology
During this century, plant breeding has led to substantial increases in crop
yield through the production of hybrids with increased vigor, the modification of
plant chemical composition or morphology, and the genetic transfer of disease
resistance, among others. The methods of molecular biology can now be applied
to complement those of conventional genetics, especially where barriers of sexual
incompatibilty or of sterility preclude the introduction of desirable traits through
breeding. Genetic engineering has also made it possible to introduce into plants
genes from other organisms, regardless of their genetic relationship. In addition,
plants such as Arabidopszs thaliana are being used as models for the study of plant
molecular genetics, which will provide basic insights into plant biology.
Tissue Culture
Plant Improvement Through Tissue Culture Is Feasible, but Remains
Technically Difficult
Plant cell and tissue culture is an important tool for improving plant charac-
teristics. Calli originally derived from plant tissue can be subcultured indefinitely
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OPPORTUNITIES INBIOLOGY
:~::~:~:::~:~ARA:B/DOF'SIS - ~TOO~L~FOR~PL:ANT:~MO~R BE - So:
T~:~:;~ ~ 18~: m~tna/l~an~:~an~n~u~aL~:~::~
~ ~ ~ ~ ~ ~: :. ~ ~ ~ ~ ~ ~: I. ~ ~ ~ . ~ ~ ~ ~ .~ ~ ~ ~ ~ ~ ~ ~ ,~ :~ :~ ~
:~m~u:stara owl Yin irass:~cac~ ~stu:oles~n~p ~a~nt~gen~ics~w"
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~:
~:more~tl~8Wyears~:ago bathe German botinist~Eduard:S~r - ~Ariioiig: ~
:~::~:: : :::::::: ::::::::: :::: ::: ::::::: :::: ~:~ ~:~:~: ::::::::::: All::::::: ::: I:: :~:::: ::::::: :::::~:::~::::: ::::::: :~:: ::: I:::: :~ ::::::: :~ :: :: :~: ::: I:::: ::
the advantages are~i~e~sm~all:~size~short gene:r~ioin:~time~ flus set
~:~production~of~this common~pld~^ur9~ 1~6).i Save:
~ ~p~:ce~ntime0~:one~ptant yields:~thousands~of~sedds~, As Lyle:
:~ ~ ~:~ may:: Scoop ~n~sb~6 ~weeks.~l - i: scrams ~DU~Q~ A; a: ~:~:~
I:::
::
: I::
:: ::
::::
:
::: ~::~mam~ as~ 1 u.~uuu seeas~¢an~ c erm~ln~ated:~in~ o~ne~Petrl~d~ ~ ~: ~ ~:~ ~ ~ ~ ~:~
~::~:~:~:~ ~ent~sit~s~::~revealdd:~it:ion~hta m~ Arabi ~:~::
:~: ~:~::~:~:~::: ~:~::~:~:~ ::~:~:~:~:~: :~:~:~:~:~::: ::~::: ~ :::~:~:~:~:::~: ~:~:~: :~:::~:~ :~:~::~ ::~::~:::::::::~:~::::~:~: ::~::~::::::~:::::::~::: :~::~::~
~:~ ~a~ pn~e~:for~studies~in~ plant ~ol~gene~s~:~:
~ ~ ~ ~ ~ ~ ~ ~ ~ · · ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~:
a s t ~ ~ ~ ~ s m a ~ ~ 9 e : n o m e : k n o w n : ~ : i n ~ ~ ~ p l a n t s , ~ ~ i ~ h a b o : l l t ~ ~ 0 ~ k R ~ b ~ ~ s :
~per~h ~c ~ 5~il~k~
~g~no~simpl~yi~::O~e ~:~
~cbni~g of part~u~enes~which~can~:~:~ther ~tor~is~a;ilrig~
: ~ : : : :: ::: : ::::: ::: :~::: :: :: : ::~:::::: : ::: :: :: : ::: ~:: :::: ~: : : : ::: :: : :: :: : ~:: ~ : : :: ::~::~:: :: :: ~ :: :::: : :::: :: ~ ~ :~
~:~oorrespp~dIng~genFes~f~m~otli~sj~r~:n~t~nshr~n:bx~eots.~:~::
~: ~:~:~ Vew:~i~mp~t~:~ intensifi~ed::~ research~with~A~:~arose:~m~:the~:~:~
~ . · ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~ ~rea Iz~on t ~at~nes~d mutations~can ~b~o~ained~with re:l~ e~lbili~is~:~:~
::: ::: ::::::::: ~:: ~:: :::: ::~ : ::~ ::~ :~ :::: ~ :::: :: : ~ :: : :~ : ::
. . . . . ~ .
~ :~process~, ~ : 1~ ~seeas~are~ ltreat~a~ ,~::~a~mutagen~:~ana~fed ~a~ 1e~ ~
,
. . ~ . . , . _
: ::: r esu l~lng~ ~:~p :an :s :::~ are~ ~ a owec~ ~ to:: se t~ertl '~.~ :::~ ~ ~e ~:~p:ro~ny: ~of ~::these ~plar~ts ~:::~ ~ :~
~ ~ ~ ~ ,
4. . ~ .
ca 1 e '~lne~ M2 gen~e~rallon,~:are~::~usea~tor screenlog~tor~:mutants.~:~::~ :Many:~mut~:: ~:~::~
~:~ ::tions~with~ a~::loss:~in~some ~mefabol~ic~h:indtion have~bQan:~f~und~:~a~frequency::~::: ~:
. ~ ~ ~ ~ ~ ~ ~ ~ ~
~:: ot one~:~i~n~2,000:~:MI2:~p):ants.::: ~Specific sereens:~yieW~dd~:~:well-d~ir - ~bioche~m~ical~:~ ~:~
mu T ants,~w~n~Icn~perm~'tted~:spec'hc~m~.tabolic~pathways,~sUth as~thdt:invo~lved~
. , ~ . . . .
~ ~In DnotorQs~DIratic~n~ tn n~ tr~:cQd ~ C3thor m~l~lI::~nt,: t:h:::'t ~: h:~uc~ ~h~"n ~ic~lat^~ :: ~
~:
~:::::
:::: ~
~:~ i nclu:de~some ~thaf ~ ar=e ~ter~d~: in :::~the~ fatty~;~ ac~:co~mplement~ of: :the~ir~ m:em-::: ~::~::
~: ::branes,:~dthers~in ~whtch: btarch sy:nthesis~is~blocked, :arid stillbih:e~rs~ in: wh ch~:~::~ ~1
~:~:~hormone~:::b~sythesTis~:is~:blodkdd~:or:~:holmfone~::~sansitivitir:~altered.~::~S6ch
~ :::~::muta~nts~ar~:: valu~able~tobts:~for:~the~study~6f~plant:~physiology:and~develop-~
:: :::: ~m~ent.~: :~::: ~::~:~ ::::~:::~ ~: ~ ~ ~ ~ ~ :
_ .. . ...
::: ::::: ::::: :: :: ::: ::: ::~ ~:: ::~ ::::: :~ :: ~:: ~: ~::~;~:~:~:::~:::::~:
::: ~;:~:~n~ent~,~^ran'~ops~s~was~used in:~ths:~isolation ~:~heirbicide-res~stance::~:~
:~ ~:~mutants.~ ~:About~o - :~:~o~of:: 100,000~ ~M2~seeds~ :sown::~on:~an~:~:ag~ar~:m~i~um~:~::
~ :~oolita~ning~ ~a~ suHonylurea~ herbicide prov~d:::~to ~:be~:resistant~ :to~:~th:is~compo~und.~::~;:~
_` ., . . . . . . . . .. , ~ ~ ~ ~ ~
~:~:su~on~u~rea~ne~roicides::~'nh~bR::acetola~ate~ synt:hase,~ an~enzyme i:n~:the~: bb-:~:~
:: sYnth:et:ic::~Dathwav of :bra:nch~:~amino acid:s.:~:~ ThQ~m~:~tated~a:Q:nQ~:fnr :~f=t~l:~::
:: :
~:: ~lale symnas~e ~was Clonec:: trom Ar~ aops/s :and :::was~::used: to: transform
: tobacco ce:lls.::::: : The: :~:::regenera ed: t:obacco: plants ::::showed stable: h~erbicide ~
::resistance. ;Expenments~ ~ this:ki:nd un:derscore:the::practical ~and:theoretical:~ :
.
:::importance :of th:e :A:rabi~s~s system :and :::ds potential for future:;:contribu- :::
.
t'^nc, :
,,~, , ~:
:::: :
~::::
OCR for page 399
PLANT BIOLOGY AND AGRICULTURE
Ll\f
~YI
~: L ; - it
~ l
~61
l ;.-\.\
Ad;
/
A: ~ (--
~_~l
FIGURE 11~ Arabidopsis thaluana. [Chris Somerville, Michigan State University]
399
on suitable synthetic media and induced to regenerate roots or shoots by altering
the proportion of auxin to cytokinin in the culture medium. The resulting plants
can be grown to maturity. Altematively, plant cell walls can be digested and calli
regenerated from single protoplasts. The fusion of protoplasts permits the recom-
bination of genetic material, even from unrelated kinds of organisms. Some
plants can also be regenerated by the induction of embryos in cells derived from
tissue that is not normally embryonic. Despite these advances, however, we know
virtually nothing about the principles that allow He regeneration of some kinds of
plants from protoplasts and that seem to preclude such regeneration (at least by
OCR for page 400
400
OPPORTUNITIES IN BIOLOGY
the available techniques) in many others, such as most commercially grown
cereals.
A serious problem that limits the utilization of plant tissue cultures for
various purposes, including the preservation of desirable strains, is the high
frequency of genetic changes in such cultures, a phenomenon called somaclonal
variation. Such changes include alterations of chromosome number, chromoso-
mal breakage, genomic rearrangements, and point mutations. Some of these
changes may be advantageous, conferring such features as disease resistance,
increased sugar yields (sugar cane), tuber uniformity (potatoes), and high levels
of fruit solids (tomatoes). In some widely used cultivars of agronomically
important plants, in which infertility has precluded the introduction of new traits
by breeding, genetic diversity provided by somaclonal variation can be exploited
for the selection of desired phenotypes.
We Need to Learn More About the Principles That Underlie Plant Regeneration
How do nutritional and hormonal factors influence the developmental fate of
plant cells? What biochemical pathways have to be activated for root and shoot
differentiation to occur? How are these pathways regulated? Cultured cells
should be particularly well suited for such investigations because their growth
conditions can be controlled rigorously. Somaclonal variation raises some basic
questions concerning genomic organization and stability in plants. What factors
lead to the observed destabilization of the plant genome, and, conversely, what
factors maintain stability? What precise changes occur in the genome as a result
of culturing? Answers to these questions will have direct consequences for the
application of tissue culture technology to plant improvement and for our basic
understanding of the mechanisms of plant growth and development.
Plant Cell Transformation
Plant Cell Transformation in Agrobacterium Has Become an Important
Research Tool in Plant Molecular Biology
Such fundamental questions as the way gene expression is regulated in plants
have been investigated through the use of Agrobacterium-induced transformation.
Nuclear genes under phytochrome control for example, the gene encoding the
small subunit of the chloroplast enzyme rubisco-have been transferred from one
species into another in such a way that the regulation of gene expression by light
can be studied against a background of precisely defined gene constructions. In
particular, it has been possible to identify the promoter and enhancer sequences
that regulate the expression of these genes. These achievements constitute the
first steps in clarifying the mechanisms by which environmental signals are
transduced in plants.
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PLANT BIOLOGY AND AGRICULTURE
401
The T-DNA of Agrobacterium has also been used successfully to transfer
herbicide resistance into plants. The mode of action of three commonly used
herbicides has recently been elucidated. Glyphosate and sulfonylureas inhibit
specific enzymes in the biosynthetic pathways of aromatic and branched-chain
amino acids, respectively. Triazines block the binding of plastoquinone to an
electron transport protein of the photosynthetic apparatus. Mutant plants that are
insensitive to sulfonylureas and triazines have now been characterized at the
molecular level. In each instance, herbicide resistance was associated with a
single nucleotide change in the genes encoding the two target proteins. Resis-
tance to sulfonylureas and triazines has been conferred on susceptible plants by
transformation, through the use of the respective genes from herbicide-resistant
plants. Plants with increased resistance to glyphosate have also been obtained
through genetic engineering. Creating herbicide-resistant plants is especially
worthwhile in the case of medium- to low-acreage crops, which do not warrant the
development of selective herbicides. The methods of weed control that become
possible in such systems decrease production costs and increase yields.
The use of the Agrobacterium transformation system is limited by the host
range of the bacterium. Although most dicotyledonous plants are susceptible to
crown-gall disease, most monocotyledonous plants, including cereals, are not.
Therefore, only a few monocotyledonous species have been transformed with
Agrobacterium until now. However, striking success has been achieved by
transforming plant cells directly with DNA. With polyethylene glycol used to
perturb the plasma membrane of protoplasts, an antibiotic resistance gene has
been introduced into tobacco cells. Plants regenerated from such transformed
cells and their sexual offspring express the antibiotic-resistance trait in a stable
manner. This same technique was also successful in transforming the cells of a
monocotyledonous plant, the ryegrass (Lolium perenne).
A technique called electroporation offers another method to transform plants
that do not become infected by Agrobacterium. Electrical pulses are used to
temporarily perforate the cell membrane of protoplasts, permitting DNA to enter
the cell. With electroporation, protoplasts of maize have recently been trans-
formed with an antibiotic resistance gene. All that stands in the way of staWy
transforming many species is our inability to regenerate whole plants from proto-
plasts or call). To get around the problem of regeneration from protoplasts or
call), it is possible to shoot DNA-coated microprojectiles directly into intact plant
cells. Potential targets include meristems and pollen, which can be cultured in
vitro or used in sexual crosses, respectively.
The Techniques of Genetic Engineering Hold Enormous Promise for Agriculture
Potential improvements in crop plants through genetic engineering include
increased yield, lowered production costs, improved nutritional qualities, adapta-
tions to unfavorable growing conditions, and new biosynthetic capacities. The
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OPPORTUNITIES IN BIOLOGY
feasibility of obtaining a number of traits of these kinds by genetic engineering
has already been demonstrated in laboratory trials. Other applications of gene
transfer technology, such as the utilization of the plant's biosynthetic machinery
for the production of foreign compounds with high commercial value, have not
yet been realized. "Custom" crop plants capable of synthesizing specific proteins,
valuable oils, or secondary metabolites for medical use could be the bases of new
agricultural industries.
To attain these goals, research in the plant sciences must proceed at all levels.
Discoveries in plant physiology and biochemistry must keep pace with the rapidly
progressing field of plant molecular biology. The metabolic functions of plants
must be explored further. Enzymes involved in the synthesis of plant products
must be characterized and regulatory mechanisms in plant metabolism elucidated.
The mode of action of plant hormones requires intensive study as do the reactions
that mediate compatibility and incompatibility between pollen and stigma, symbi-
onts and roots, and pathogens and plants. A thorough knowledge of the processes
that could be altered for the improvement of plants provides the basis for the
application of recombinant DNA and transformation technologies.
Representative terms from entire chapter:
plant biology
