hi I'm Sharon long from Stanford
University I'm here today to tell you
about some remarkable organisms two
kinds of organisms in fact bacteria and
plants together they can do something
that neither can do alone
they can bring nitrogen in its molecular
form which is usually very inert they
can bring that nitrogen into a
chemically combined form that can be
used for protein nutrition so I'll have
three topics overall first I'd like to
give you an introduction to the nitrogen
fixing symbiosis in particular between
Rhizobium and legumes I'd then like to
focus in my second segment on the
bacterial genes that are used for the
response to the plant and for the
bacteria to be able to provoke the plant
in order to make the home for the
bacteria once they invade and in the
final segment I'd like to tell you some
of our new research on how the plant
responds to the signals that come from
the bacteria so now in the first segment
as I mentioned we have an overview I'm
going to tell you about something called
root nodules and how these root nodules
form and how they work to get us started
I'd like to address a subject near and
dear to all our hearts and that is food
when we think about food we're aware of
the need for balance we need
carbohydrates fats proteins and proteins
it turns out are a particular issue in
nutrition because proteins
unlike carbohydrates and fats for the
most part require a great deal of
nitrogen and nitrogen is scarce that
means that protein malnutrition the
inability to get enough protein can
happen whenever agriculture is deficient
or when yields are low and it's one of
the most devastating problems for human
health worldwide so in Western cultures
when we think about getting protein we
may think well I need to have a protein
rich food something which we're going to
remember it has a lot of nitrogen we're
going to think about for example some
poultry and here they are poultry
protein-rich food now we're thinking
about the theme of nitrogen it's a
critical element where do these animals
get their nitrogen so that they can make
protein they get it from plants and
ultimately of course whether we eat meat
or eat plants all human protein
ultimately comes from plants so that
raises the question where do the plants
get their nitrogen the answer is they
get it from the soil soils include
compounds such as nitrates they will
include organic nitrogen which might be
provided from your manure or a
decomposition of plant matter and the
plants will take up this nitrogen they
will synthesize amino acids from the
soil nitrogen and from those they make
proteins and then we in turn can get our
protein from those plants however if a
field is used over and over again
ultimately it becomes depleted for
nitrogen and that means that this
infertile soil is not able to support
plant yield or plant nitrogen now how do
we overcome that at large scales of
Agriculture when fields are used quite
often then the soil can be restored by
applications of nitrogen fertilizer it's
usually in the form of ammonium now
ammonium remarkably can actually be made
from molecular nitrogen which is in the
air which is very abundant as you may
know it's the vast majority of the
atmosphere is made of nitrogen but it's
incredibly inert it won't combine with
anything these triple bond nitrogen so
in order to coax that nitrogen into a
combined form of ammonium there's a
process called the haber-bosch process
which is able to take molecular nitrogen
can combine it with hydrogen gas at very
high temperature very high pressure with
a metal catalyst and converted to
ammonium that ammonium can then be
applied on a field the plants will take
up the ammonium they'll make protein and
the the yield is therefore supported but
the problem is this how do we get high
temperature and pressure
it's by using fossil fuels it's mostly
natural gas but other fossil fuels can
be used and we're all aware of the
scarcity and the price of fossil fuels
and what lies in the future for their
availability that means that we can't
necessarily depend on unlimited supplies
of this kind of ammonium fertilizer are
there alternatives and it turns out for
millennia farmers in every continent of
the world have known of another way that
is the use of legumes a very special
group of plants it's a plant family the
legume NOC also called fabaceae the
third-largest family in the angiosperms
or flowering plants legumes have this
remarkable property that they can
associate with bacteria from the soil
they can grow a new organ call the root
module the bacteria live inside the root
nodule and once the bacteria are there
the bacteria are converting nitrogen
into ammonia through an enzyme this is
still energy intensive as biochemists we
would see that it uses highly reduced
electrons and a lot of ATP but where is
that coming from it's coming from
sunlight so the advantage of this is
that we're able to nourish plants and
those plants then in turn can nourish
the soil in a process that uses a
biological catalyst at room temperature
ordinary pressure and uses sunlight to
drive it now as I mentioned farmers
throughout the ancient world knew about
this they didn't know why it happened
but they could see that the use of
legumes in agriculture to rotate or to
use as a green manure would assure the
fertility of their soils in the new
world farmers used the common bean which
is a new world plant in the
Mediterranean that you
Lupin's in China the soybean and in
South Asia and Africa the cowpea so
again throughout the world
crop rotation has been used as a key
element of sustainable agriculture so
from that view of real nodules I'd like
to draw back just a moment for a cartoon
so that I can describe this in more
simplified way we know that we're
talking about legumes and the way in
which they form a symbiosis with
nitrogen fixing bacteria and what are
the key elements of this is that it's
able to be used to replace nitrogen
fertilizer how does this happen so in a
schematic form we can imagine that there
are these bacteria the Rhizobium
bacteria that grow in the soil they're
able to grow slowly as sacrifice just
scavenging nutrients for the soil and
here's a plant which again could grow if
there's fertilizer but in the absence
it's going to have a hard time so if
we've got the right bacteria and plant
and I'm going to talk about that
specificity again in a few minutes they
interact to form these what's shown here
in red are root nodules specialized
organs that grow on the root within
which the bacteria are able to fix
nitrogen so this is where it's all
happening I love this these are nodules
they're growing on a soybean plant so
you can see some of the nodules here the
growths that are occurring on the root
the plant is up there and the nodules
once they fix nitrogen and make amino
acid that the plant is able to take
those amino acids put it up in the chute
or it can make chlorophyll where it can
make proteins and it's able and
ultimately to make high protein seeds
now I'd like to make several points
about this first the bacteria in these
nodules are fixing nitrogen but even
that is energy intensive and as I've
mentioned ultimately it's sunlight
through photosynthesis that allows the
plan
support this energy intensive process of
nitrogen fixation it's occurring only in
the root nodules it doesn't occur
anywhere else in the plant this
association is highly specific and I'll
show you that in a moment with a table
it's also established through a complex
developmental process and I'll show you
some microscope it's Mike's microscopic
views of that and remarkably this this
process is restricted with one known
exception only in the world it's
restricted to one family the legumes
family we we know a little bit about why
that occurs but there's a lot left to
find out the legumes I've mentioned are
part of the large family which includes
both agriculturally important and
environmentally important species when
we think of high-protein plants many of
the plants that come to mind are in fact
the legumes in the both in terms of
their seed content and in terms of the
content of their leaves we're familiar
with a soybean cowpea common being all
being high-protein seeds the pea again a
high-protein seeds also alfalfa which is
used as a forage for animals it has very
high protein leaves that's why it's so
nourishing for animals such as cattle
and horses but beyond the the uses for
human nutrition these plants are very
important in many natural ecosystems
throughout the world we might think for
example of this acacia growing up in the
African plain or we could think of this
mesquite tree let me show you this one
over here and here a broom plant a shrub
these legumes shrubs and trees often
grow in relatively harsh environments
where they provide nutrition for the
soil that's around them so coming back
to the developmental process what we
want to know is how is it that those
Incred
diverse groups of plants are able to
establish this association with
Rhizobium and why don't other plants do
it so I'd now like to tell you a little
bit about the specificity the beyond the
specificity for the legumes family
within that very diverse family specific
Legum types such as the soybean our
knowledge elated not by just any old
bacterium but only by certain restricted
groups so here we're looking at the
soybean and here I'm showing you two
different species that are known to form
symbiotic root nodules on soybean here
we have the P it's a leg you also but
it's Nigel ating bacterium is a
different one Rhizobium legume and the
serum
I'll have occasion to mention P and
Rhizobium leg human serum in the second
half of my introduction here we have a
lotus plant called trefoil and it's
nodule ated by a different genus meza
Rhizobium species low t and the system
that i study which is the genus medicago
which includes alfalfa and which is nagy
elated by seiner izo via mela Lodi this
is the species you'll hear about in
terms of the direct experiments that I
described through the rest of my talk
with with the one comparison that I've
mentioned earlier now I've told you that
nodule development is complex but I
haven't really shown you what I mean and
so that's what we want to do next in
looking at this the primary approach
that I'll tell you is through microscopy
but embedded in that microscopy I'll
also tell you a little bit about the
physiology so one of the first changes
that occurs in when Rhizobium eat a
plant is that the root hairs which are
ordinarily straight and here's an
example of a fairly normal looking root
hair right here instead
root hairs grow in a way that's deformed
now don't think of this as root hairs
curling plant cells don't curve think of
a plant cell as a balloon inflated
really hard inside a box
plant cells don't curl but they can grow
so that they form a curl and that's what
happens here if you take a look at this
root hair instead of growing straight
it's grown so that it tops over and
makes what's called a shepherd's crook
or crows ei in that you can see a
bacterial invasion which is called an
infection thread and that's shown here
this highly refract I'll line going down
through the through the root hair now at
the same time that invasion is occurring
on the outside early events are also
occurring inside the plant root so I
want you to imagine that the plant root
is going out this way now here we have
an uninfected plant root it's typical
plant root cells you can see that
they're very long there they look empty
that's because they're mostly filled
with vacuoles a large vacuole plant root
cells in the cortex here have one main
job that is to transport and transport
and transport water ions nutrients and
so forth that's what these cells do they
don't divide but if the correct
Rhizobium and this is an example of
something that happens only if you have
the right species of that tournament
plant if the correct Rhizobium is
infected onto that plant you get this
you can see the mitotic figures
happening here the cells in the inner
cortex of the root have begun to divide
they're going to form what's called a
primordium those cells continue dividing
and the infection that started with an
infection thread continues all the way
through into these newly dividing cells
here you can see a cell that's just
starting to divide and what happens is
that if you can follow this arrow you
can see right here there is a little
membrane bound kind of an empty looking
vacuole and within it a peach-colored
oblong that is a single Rhizobium
bacterium that's being taken up into the
host cell and it's being engulfed by
plant plasma membrane that's going to
differentiate
and perform provide a special
compartment that the Rhizobium will be
in when it starts to fix nitrogen so the
bacteria at this point are released from
the infection thread and they're bound
in plant membrane sacs but that doesn't
do justice to what's going on this
bacterium is part of a more elaborate
compartment and I'm showing that
compartment here this shown in orange is
the bacterium but you see there's an
extra boundary there's not just the
bacterial membrane but there's another
memory that's referred to as The Scindia
stone membrane it's derived from the
plant plasma membrane that was used to
engulf the bacterium when it first
entered the host cell but at this point
it's become a distinctive compartment
with its own protein trafficking signals
I'll just tell you a little bit about
the basics of transport of course this
is the real action we have nitrogen
converted and zu matically into ammonium
this uses ATP and highly reduced
electrons and that ammonium is in turn
transported out to the plant as I've
mentioned before the bacterium gets
energy from the plant male 8 and other
dicarboxylic acids are transported into
the bacterium from there they enter into
the bacterias own metabolism so plant
photosynthate providing energy bacterial
nitrogen fixation providing ammonium
that had been suspected for a long time
but emerging data from a number of labs
shows that there's more going on in fact
genetic data show that a shuttle
involving amino acids that are
transported in and out is also critical
to sustain nitrogen fixation how this
occurs is the subject of a great deal of
ongoing research we've got all of these
cells which are packed with bacteria
which are fixing nitrogen so inside
these cells we've got ammonia converted
of
created from nitrogen of molecular
nitrogen and over here you can see the
vasculature of the plant so we we've got
you can imagine that the stem of the
root is going to go up like this and
this the steel the vascular tissue in
the root is going to transport all of
those amino acids to nourish the rest of
the plant so I've now shown you in this
first part of the overview that
nodulation occurs by a very complex
developmental process
it's species-specific and the events
that I showed you
occur only if the correct Rhizobium and
correct plant come into combination now
in the next segment what I'd like to do
is talk about how the bacteria and the
plant creates signals that stimulate
each other to begin their developmental
process and so in that next slide we're
going to ask this question how do plants
and bacteria use chemical signals for
recognition