THE plastic chemical model is falling apart in Sharon Long’s hands.
“Oops—glycosidase,” she mutters, as a section of the red, yellow, black
and blue structure snaps in two, and “Uh—boy, we have a deacylation,” as
the decomposition worsens. Long deftly mends the molecule and continues with her
demonstration of how a soil-borne chemical informs a plant, down in the dark
underground, that some very special bacteria are waiting patiently to come
inside and take up residence in its roots.
As a rule, plants will do all they can to keep microorganisms out of their
tissues. They have a panoply of chemical defences to ward off disease by keeping
the interlopers at bay. But the alfalfa plant that Long studies is only too
eager to usher this particular microorganism inside. In return for a cosy home
and plenty of energy-yielding sugars, Rhizobium meliloti will do the
plant a mighty service: it will convert nitrogen gas in the atmosphere (which is
superabundant and tantalisingly inaccessible to other plants) into ammonia that
the plant can use to make proteins and grow. Fields of alfalfa—unlike rice
and wheat, for example—do not need nitrogen fertiliser.
Long’s office at Stanford University in Palo Alto, California, gives plenty
of clues to her academic obsession. The walls are decorated with pictures of
legumes, the family that includes peas, beans and clover. Legumes are the envy
of the rest of the plant kingdom because of their ability to form bulbous
nodules on their roots that are stuffed with nitrogen-fixing bacteria. Long is
not alone in her obsession. A goodly array of scientists around the world are
busy, like her, teasing apart the molecular dance between a bacterium and a root
which ensures that they set up house together.
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They study the dance because they’re curious. But the payoff could be much
more than mere satiated inquisitiveness. Today, there’s mounting evidence that
one of the key chemical signals that prompts nodulation may help to govern
growth and development in all plants, not just legumes, and perhaps even in
animals, including humans. And, of course, nobody studying nodulation is
ignoring the possibility that one day—just maybe—by tracing the
blueprints that allow a nodule to be built, scientists might figure out a way to
make important staple crops, like rice and wheat, fix nitrogen too, boosting
yields to help to feed the world’s burgeoning population.
Even though the vast majority of legumes form nodules, they are picky about
just which Rhizobium species they let into their roots. Nothing much
happens when a bacterium that normally nodulates a pea comes into contact with
an alfalfa root, for instance. Only when the alfalfa bacterium happens along, is
the required cascade of events set in train. Within hours, tiny hairs on the
roots, each made from a single cell, begin to wrap themselves around the
bacteria. Next, the bacteria slowly move into the root hair and are shunted
along an elaborate tunnel plastered with freshly-synthesised cell wall
material.
Chemical decorations
Towards the centre of the root, meanwhile, cells that were quiescent are busy
dividing and reorganising. Nine days later, the nodule is complete, a brand new
organ encasing thousands of bacteria housed in specialised plant cells, all
fixing nitrogen like crazy. Other plant cells in the nodule are busy too,
converting the ammonia made by the bacteria into organic, nitrogen-containing
chemicals for delivery to the rest of the plant.
How on earth does this elaborate transformation come about? Unlike the
nodules, the nodulation researchers are not entirely in the dark. They know that
the dance is initiated by the plant. From its roots seep molecules known as
flavonoids that enter the bacterium and interact with a pivotal protein inside
it.
This protein, a gene regulator, normally sits on the microbe’s DNA, not doing
anything much. But when the flavonoid comes along, it turns on certain key genes
in the bacterium, called nod (as in “nodulation”) genes. Long was the
first to identify some of these. The enzymes encoded by the nod genes,
in turn, make their own special “hello, got your message, here we are” signal
that seeps back through the soil to the plant. It is this signal—a
chemical known as the “Nod factor”—that tells the plant to make a
nodule.
It was only as recently as 1990 that the structure of the first Nod factor,
the one that triggers nodule growth on the roots of alfalfa, was identified.
Today, scientists know the structure of a whole battery of Nod factors for a
wide range of legumes, from peas to alfalfa to soya.
At their core, the molecules all consist of a short and specially modified
fragment of chitin, the chemical that makes crab shells tough. But this is just
the basic structure. Just as a decorator crab adorns its body with bits of
seaweed and shell, the Nod factor is similarly adorned. And the decorations get
very individual, courtesy of the particular nod genes that each
bacterium possesses. For example, the Nod factor from the pea Rhizobium
sports an acetate group. While the Nod factor from R. meliloti, which
nodulates alfalfa, has both a sulphate and an acetate group attached.
Somehow, the plants can distinguish between them and respond accordingly. In
fact, the bacterium doesn’t even have to be present. All you have to do is
squirt the tiniest quantity of Nod factor onto a compatible root, and the nodule
grows.
Sexy genes
“It’s really mind-blowing when you think about it,” says Gary Stacey, a
microbial geneticist at the University of Tennessee in Knoxville, who works on
soya bean nodulation. “It’s as if you were to take a single molecule and drop it
on your shoulder and have a new hand or finger grow. It’s like something from
The X-Files.”
From an evolutionary point of view, the fact that plants can recognise the
bacterial Nod factors is intriguing. Unlike crabs and fungi, plants had never
been shown to make chitin; their tough outer walls are made of cellulose. So why
do they possess the biological hardware to respond to it? Perhaps it is because
they have evolved to recognise pathogenic or symbiotic fungi that do contain
chitin. Another tantalising possibility is that the plants do, after all, make
chitin molecules. The evidence for this is mounting.
In 1993, molecular biologist Sacco De Vries and his colleagues at Wageningen
Agricultural University in the Netherlands discovered that a certain carrot
mutant responds to minute quantities of chitin-like Nod factor. Under the right
conditions, normal carrot cells will develop into little carrot plants, but
mutant cells don’t—except in the presence of a Nod factor. This suggests
that bacterial Nod factor replaces something that is lacking in the mutant. And
if a molecule akin to Nod factor is crucial for normal development in a
carrot—which is not a legume—why not for plants in general?
Herman Spaink’s group at Leiden University in the Netherlands has since
extracted short chitin-like molecules from another non-legume, thale
cress—as well as the leguminous sweet pea. Now his group is trying to find
out just how similar these molecules are to bacterial Nod factors, and what, if
anything, they are doing in the plant. Meanwhile, Jeff Schell’s group at the Max
Planck Institute for Plant Breeding in Cologne has shown that synthetic chitin
chains that resemble bacterial Nod factors can coax tobacco cells into dividing.
According to Jean Denarie, a molecular geneticist at the INRA/CNRS Institute in
Toulouse, France, who was the first to unpick the structure of a Nod factor,
this finding could lead to the discovery of a new class of plant growth
regulators. Such chemicals would join the toolkit of plant hormones such as
auxins and cytokinins, that are currently used in the lab to generate
fully-fledged plants from plant cells.
Other bridges between nodulation by Rhizobium and more workaday
plant growth have been discovered. In the push to understand how plants detect
Nod factor, and what happens next, researchers in various labs have been working
fast and furious. Long, for instance, discovered last year that the plant root
cells, just like many animal cells, may use successive bursts of calcium to send
a signal around their interiors that a Nod factor has been detected. Other labs,
meanwhile, have come up with dozens of genes that blink on during
nodulation—and almost all of them are present in non-leguminous plants,
too.
One of the sexiest of these genes, going by the decidedly unsexy name
Enod40, was identified in 1993 by molecular biologist Ton Bisseling’s group
at Wageningen. Enod40 is switched on in the first hours after the root
hairs get their first sniff of the Nod factor drizzling towards them, which
suggests that the gene helps to govern the early stages of nodulation. Indeed,
it seems to play a key role in cell division inside the root. If you take the
Enod40 gene and attach DNA sequences that make sure it is switched on
all the time, then insert it into the cells of a leguminous plant, they will
divide as they do during nodule formation—all without any Nod factor. This
finding was reported in July at the 8th International Congress on Molecular
Plant-Microbe Interactions in Knoxville, Tennessee, by Adam Kondorosi, a plant
molecular biologist at the CNRS Institute of Vegetable Science in Gif-sur-Yvette
near Paris.
Enod40, it turns out, also encodes a very unusual molecule: a short
string of just 12 to 14 amino acids. Unusual, that is, for a plant. There are
plenty of small “peptides” in animals, where they often function as hormones,
but only once before has a peptide hormone been found in a plant—and in
that case it was important for keeping dangerous microbes out of the plant, not
inviting them in. “Now here’s another one—and if there are two, there are
probably going to be forty,” says Stacey.
Once again, the effect is not confined to legumes. Schell and Bisseling found
that the Enod40 peptide also stimulates the growth of cultured tobacco cells,
when it would normally be inhibited by one of the well-known plant hormones, an
auxin. Once again, says Bisseling, this tells us that “most of the machinery
that is used in nodulation is probably present in all plants”.
Chitin catastrophe
If all plants use Nod-like factors for some everyday housekeeping function,
then the hypothetical “receptor” proteins that bind bacterial Nod factors may
also be surprisingly widespread. Finding such a receptor—one that allows a
species from one kingdom, a bacterium, to shake hands with species from an
entirely separate kingdom, plants, is the Holy Grail of nodulation research.
Spaink, meanwhile, plans to seek out a Nod receptor in animals—and no,
he’s not out of his mind. He has already shown, with Phil Robbins at the
Massachusetts Institute of Technology in Cambridge that the short chitin chain
that forms the backbone of all bacterial Nod factors is also present in the
embryonic zebra fish and African clawed toad, two animals that biologists use to
study development. What is more, when Spaink and Robbins treated developing
mouse embryos with enzymes that mess up the structure of the chitin chains,
catastrophic things happen to their development. Spaink declined to describe the
catastrophes in detail until the findings are published, but he will go so far
as to speculate that the chitin molecule is vital for normal organ development
in both plants and animals.
Animal links aside, for those who want to feed the world, the fact that the
basic nodulation machinery seems to be in place in plants other than legumes is
good news. This brings a little closer the dream that plants that don’t
nodulate— important crops such as rice and wheat—might one day be
made to fix their own nitrogen and lose their dependence on chemical
fertilisers. There would have been little chance of doing that if one had to
insert scores of genes into a plant before it could set up shop with bacteria,
and start churning out ammonia from its own nodules. Instead, plant biologists
may well face the simpler—although, still daunting—task of tinkering
with a few genes in a non-leguminous plant.
But it’s still a very long shot. Long and other nodulation experts are much
more optimistic about less complicated ways to exploit the new-found
understanding of nodulation. For instance, she says, “I’m pretty confident that
over time we’ll be able to make Rhizobium symbioses in their native
plants more efficient”. This could make legumes like alfalfa and soya even
better nitrogen-fixers, bumping up their yields.
For Long, however, the driving force behind her research remains the mystery
of how a bacterium and root chemically seduce one another. “At times, it makes
your insides quiver,” she says. “I just want to know how it works.”
- Further reading: “The molecular basis of infection and
nodulation by rhizobia: the ins and outs of sympathogenesis”, H. P. Spaink,
Annual Review of Plant Pathology, 1995. - “Rhizobium Symbiosis: Nod Factors in perspective”, S. Long, The Plant Cell,
1996, vol 8, p 1885.