Corn, or maize, is an example of human interference with plants. “We mutilated
it,” Asim Esen says. “It can’t survive on its own.” Eight thousand
years ago, maize could both defend itself and drop seeds to grow a new
generation. But as humans selected for such traits as the cob and the
ear, they made it impossible for the seed to get out and disperse — and
therefore sustain itself.
David
Bevan and Asim Esen study the structure of maize beta-glucosidase
and its complex with the natural substrate DIMBOA-glucoside, which
they determined using X-ray crystallography. Young maize uses this
chemistry to protect itself. When insects chew the plant, DIMBOA is
split from the glucoside by the enzyme stored in another part of the
cell. DIMBOA is toxic to insects. The researchers discovered that
in some inbred lines of maize, beta-glucosidase is bound and cannot
be extracted efficiently. Photo by Young Ock Ahn.
Virginia
Tech researchers and collaborators in France determined the 3-D structure
of the enzyme beta-glucosidase and its substrate DIMBOA-glucoside,
which make up the defense mechanism present in maize. This computer
model shows DIMBOA-glucoside bound to the enzyme.
Khidir
Hilu’s computer displays matK sequences. His research on the
sequences. His research on the matK gene to determine gene to determine
relationships among plant species is helping redraw the flowering
plants section of the Tree of Life. Photo by Michelle Barthet
PHOTOS ABOVE
The flowers show a sequence of development: Amborella
(top), the first angiosperm, is a shrub with small flowers. The
waterlily (second) is an aquatic plant with beautiful but simple
flowers. Grasses, such as the sea oats (third), show diversity.
One of the most dominant plants, their small flowers are pollinated
by wind. The passion flower (bottom) is complex to attract pollinators. Photos
above by Khidir Hilu except the Amborella, which is by P.P.
Lowry II of the Missouri Botanical Garden
Plants are everywhere, even in deserts. We eat them, we decorate with
them, we make medicines from them, we add them to or weed them out of
gardens. But some things about plants remain a mystery — things that are
important to their survival and ours.
Virginia Tech biology and biochemistry professors are taking different
approaches in their research to discover plants’ natural defenses and
how they evolved.
Khidir Hilu of biology is examining the way plants are related to each
other and their patterns of differentiation, working with the NSF Tree
of Life Project to reclassify the 270,000 species of flowering plants,
which include all of our food plants. Reclassification is important because
molecular biology and gene sequencing have shown the old classification
system to be wrong, and the system guides researchers who look at plants
for medicinal and other properties.
Asim Esen of biology and David R. Bevan of biochemistry are working under
different National Science Foundation (NSF) grants to discover how certain
enzymes and proteins help plants defend themselves against pests and injuries.
One reason this work is necessary is that humans have cultivated some
plants to serve human needs so that those plants can no longer defend
or propagate themselves without people’s care and attention.
Corn, or maize, is a good example of human interference. “We mutilated
it,” Esen says. “It can’t survive on its own.” Eight thousand
years ago, maize could both defend itself and drop seeds to grow a new
generation. But as humans selected for such traits as the cob and the
ear, they made it impossible for the seed to get out and disperse — and
therefore sustain itself.
However, maize can still defend itself; so Esen and Bevan are looking
at the way its defense mechanism works in hopes that understanding it
will enable them to “;optimize it in such a way that plants can defend
themselves without pesticides,” Esen says. Young maize — the young
seedlings and any growing tissues and organs — has two enzymes that help
protect against insect attacks. Beta-glucosidases are stored in the plastid,
a structure within maize cells, and their substrate DIMBOA-glucoside resides
in the cell’s vacuole cavity. Usually, the two remain separated in an
intact cell. However, when an insect starts gnawing at the young maize,
it breaks the compartments. The enzyme beta-glucosidase breaks the DIMBOA-glucoside
down into glucose and DIMBOA, and the DIMBOA is toxic to insects. However,
14 of 463 inbred lines of maize tested in a study seemed to lack the enzyme.
They are called NULL.
Using spectrophotometric detection and gel assays, Esen and Bevan found
that all the NULL lines actually did have active beta-glucosidase, but
the enzyme became aggregated or bound and could not be extracted efficiently.
“This was a surprise,” Esen says. From this discovery, the scientists
knew the enzyme was there, but something was keeping it in the large aggregate.
Using a procedure called gel filtration that separates proteins according
to size, the researchers then found that the cause of aggregation was
another protein, the beta-glucosidase aggregating factor (BGAF), which
NULL lines produced in excess. They isolated BGAF and proved its aggregating
activity. After further study, they found that BGAF was a hybrid protein
with two distinct regions or domains: a disease-response region and a
carbohydrate binding region, or lectin. In nature, the two occur as separate
proteins, but in all the grass species studied so far, they were fused,
a process that occurred probably millions of years ago in the ancestors
of the grasses. Such things usually happen as accidents, or mutations,
and, if advantageous, they get selected and passed to future generations.
It also can work as a disadvantage. “That’s how we get super bugs,”
Esen says. “They started as deviant types that would have disappeared
some day, but we applied pesticides.” The normal bugs died and the
deviant ones survived, so that the next generation of bugs is resistant
to the pesticide. “The same with antibiotics,” Esen says.
Surfaces of cells have glycoproteins. Lectins recognize their carbohydrate
portion and bind to them. The BGAF’s lectin region is similar to lectins
that recognize mannose and galactose sugars. Esen and Bevan hypothesized
that one of the functions of BGAF is in defense when foreign cells, such
as bacteria, fungus, or viruses, try to enter the cell. BGAF probably
binds foreign cells, marks them, and recruits other components of the
defense system to eventually arrest the development of the foreign elements
and kill them. The defense system acts much the way a football team does
when it surrounds the ball carrier and keeps him from moving.
“Based on data from other plants, anything that stresses plants
will induce this protein. That includes physical wounding as well as insects,”
Esen says. The researchers’ project is to understand the interaction between
beta-glucosidase and BGAF — how they recognize each other and bind so
tightly. Thus far, they have evidence of three genes that make BGAF, but
they need to find out which one, which part of the molecule, is recognized
by the enzyme and binds to it. They will do that through genetic engineering
— changing the gene for BGAF, producing the protein in bacteria and yeast,
and then testing it with the enzyme.
The
ultimate goal is to provide evidence of the biological function of the
binding and aggregation, understand the defense system, and produce plants
that can once again defend themselves without pesticides or with less
pesticide use. The goal is to re-engineer the plants in an artificial
setting to enable them to do what they could originally do: survive on
their own.
That is important for cultivated plants, Esen says. “Cultivated
plants are in an artificial environment that disarms them. There are many
different plants in nature, but under cultivation, you have 100 acres
of corn plants that are identical. In nature, when you have a pest attack,
you have enough diversity there that can withstand it. Some susceptible
ones will die, but enough will live to be able to produce the next generation.”
When you have a great, cultivated crop of identical plants, “they
may all be wiped out when a new selective pressure arrives.” Therefore,
scientists must work to protect or re-engineer plants that will grow in
the artificial setting of cultivation.
Meanwhile, Khidir Hilu’s research to remap the tree of life looks at
which plants came first and which are closely related, revealing the development
of survival characteristics. Hilu’s lab is one of the leaders in the study
of flowering plants, particularly grasses, which provide 80 percent of
the world’s food. In the Tree of Life Project, Hilu is working with colleagues
from six other laboratories across the country. Among them they have research
backgrounds in areas ranging from genes and genomes, morphology, and anatomy
to fossil records and computer modeling of such things as the time of
origin.
“We thought for a while we understood the classification, relationships,
and date of origin and divergence of these plants,” Hilu says of
the flowering plants he studies. Formerly, flowering plants — the most
dominant plant on the surface of the Earth and economically the most important
— were classified primarily by flower and leaf as monocots (such as orchids,
palms, and grasses) and dicots (such as tomatoes, oranges, and cotton).
Now,
based on information from the genes, flowering plants appear as a basal
group that includes both monocots and some dicots (such as magnolias)
and a group called, for now, the true dicots, or eudicots. The new system
has broken up several traditional groups and shuffled and mixed others,
Hilu says. A new pattern of relationship means a new perspective for how,
over time, plants’ physiology, their chemistry (such as the compounds
Esen and Bevin are working with), their reproduction (such as pollination
biology), their anatomy, and their genomes have changed.
Scientists still don’t know where some groups fit and have no formal
classification of others. In the past, most people suggested magnolias
or buttercups were some of the earliest-evolving flowering plants, or
the base of the flowering-plants tree. Now, Hilu says, a small shrub from
New Caledonia called an Amborella forms the base, with water lilies coming
directly after that. “This indicates a major shift from a shrub of
terrestrial habitat to aquatic habitat,” Hilu says. “That's
a major change in structure and ecology.”
The Tree of Life laboratories will depend on results from each other,
including Hilu’s work on the matK gene. He uses material obtained from
botanical gardens or voucher samples in herbaria where plants are preserved
as dried samples. “We like to use leaves because they contain a lot
of the chloroplast, the cell compartments that synthesize food for plants,
and for us,” Hilu says. “The matK gene is located in the genome
of those units.” The leaves are ground with a mortar and a pistil
with the aid of liquid nitrogen, and then the impurities are removed and
the DNA is precipitated with alcohol. Through polymerase chain reaction
in a thermocycler, many copies of the matK gene are created, then sequenced
in an automated machine at the Virginia Bioinformatics Institute to determine
the arrangement of the building units of DNA, or nucleotides. The sequences
are sent to Hilu as a computer file. “We use a computer program to
align the sequences of the various species and then analyze the data by
various computational programs to assess relationships among organisms.”
The complex programs are based on the premise that the more closely related
the species, the more similar their DNA. Some of these calculations may
take more than a month on a computer designated solely for them.
“Time and need for exhaustive analysis of a large number of species
has prompted me to think about the supercomputers we have here at Virginia
Tech, and now I am working with my colleague, Dr. Srinidhi Varadarajan,
director of the Terascale Computing Facility, on an interesting project
for using these computers to speed up the analytical process,” Hilu
says. “This marriage between molecular biology and sophisticated
computing has truly revolutionized our field. It is an exciting time to
be working on biological diversity on a large scale.”
At some point, all the data generated at the seven participating labs
will be combined and computer analyzed. The researchers will then develop
the flowering plants section of the tree of life based on molecular information.
Then they will impose on the tree information from fossils, anatomy, and
morphology to confirm the relationships and come up with reliable groupings
that will be used as the basis for a system of classification. “The
eventual goal of these projects is to have all flowering plants, algae,
mammals, insects, and others put together in a full tree of life,”
Hilu says.
There are several benefits for doing so. For example, chemists and biologists
searching for compounds in medicinal plants need to know their relatives
and ancestors, since families of plants tend to have similar chemistries.
Understanding biodiversity in the frame of patterns of relationships is
important in the assessment of vegetation dynamics and species extinction
due to climatic changes. The new tree would also help in the study of
patterns of gene transfers among plant genomes.
In Esen’s research, for example, the BGAF protein seems to occur only
in grasses, “so the kind of split point between grasses and non-grasses
could be determined by the tree of life,” as could the time of the
fusion of the two proteins into one. Also, Esen says, if plant scientists
do not find the desired trait they need in one plant, they go to the most
closely related plant.
The Tree of Life is their map.
