Hi. My name is Eric Hamilton
and I want to tell you today about my research.
My research is all about sex,
the kind painted by the great masters of the Renaissance,
like in this Botticelli.
There's so wonderful going on in this scene,
isn't there?
Like in the corner,
we've got this plant, clearly just about to flower.
It must be so excited for this opportunity
it's been waiting its entirely life for.
And in the background, the bushes and trees
putting all of their resources
into making seeds and fruits,
really the whole purpose of their existence.
Okay, yeah... I study plant sex.
But at least as interesting as all the other kinds,
I think this is really personal to me.
I think it's really personal to you too,
because every calorie that I ate today
or that I'll eat tomorrow,
all the food that all of us will eat
for the rest of our lives,
really comes back to plant sex,
because the product of that are the seeds,
the fruits,
the vegetables that we rely on.
Even if we feed them to animals first,
it really comes back first to plant reproduction.
And as long as humans are going to keep having sex,
and by accounts we are,
because there's supposed to be 2 billion more of us
by the middle of this century,
then we need to make sure that plants
keep having a lot of sex too,
actually about 70% more.
Now, in elementary and middle school,
we all learned that animal reproduction
requires that the swimming sperm
finds its way to the egg cell,
the female partner.
That creates the embryo,
which jumpstarts the whole next generation.
Well, it's actually not so different in the plant world,
but there are a few key differences.
In one, the sperm actually can't move on its own,
it has no tail,
and it's kind of subject to the whims of its environment.
And another, we don't usually call it sperm,
we usually call it pollen.
Now, when I say pollen,
probably the closest association most of us have with that
is seasonal allergies,
because it makes millions of us suffer.
The reason, though, that there's so much pollen out in the world,
dumped by the trees and bushes
out into the wind every spring or summer,
is because plants,
which are literally rooted in the ground,
cannot go out and find a mate,
so they have to play one of the world's biggest numbers games.
Every pollen grain that lands up your nose
is a lost cause.
They're dumping out so much pollen
because they're hoping that just a few grains
find the right female partner.
Now, in order to do this, the pollen...
I like to think about it kind of on this mythological journey.
It faces a lot of obstacles
but it comes equipped with the defenses it needs
in order to overcome them.
One of them is that as it matures
it deliberately loses the vast majority of its water
and ends up looking kind of like a deflated soccer ball, here.
And this is incredibly stressful
for any cell to undergo
and if any of the cells tried to do it,
they just wouldn't be able to recover.
But what's nice about this is that
once you've lost that much water,
frankly, not that much else can hurt you.
It's a strategy that we're familiar
with another common plant tissue
-- dry seeds --
which, under these conditions,
can survive sometimes for thousands of years.
Unfortunately, losing that much water
and going out in this suspended animation kind of state
kind of just kicks the can down the road,
because in order to come back to life
and be a productive cell again
it needs to rehydrate and absorb water.
But it's kind of like taking this deflated soccer ball
and potentially hooking it up to
an overpowered air pump.
It could end disastrously.
Clearly, pollen needs some way
in order to regulate the pressure that's coming in
in order to survive this.
But, like I mentioned,
pollen's really on this epic kind of journey,
because even if it does get lucky enough
to find a female partner
and rehydrate successfully,
it's going to find itself really at the top of what,
to it, is an enormous female flower.
It's going to actually need to grow up to
1,000 times its original size
in order to reach its partner,
and it does this tremendously quickly,
usually in less than a day.
In order to accomplish this feat,
it's driven forward, ever increasing,
by really strong pressure inside the cell,
greater than that inside a car tire.
Now, this is really important
for being able to grow as quickly as it needs to,
but you can easily imagine that
if it outpaces itself,
it's liable to burst open prematurely
-- game over.
So, clearly, whether it's for rehydrating
or for maintaining this level of growth,
but also maintaining the integrity of the cell,
pollen needs some way in order to
regulate the pressure that it naturally experiences.
Now, if we were going to solve this problem,
and we have,
we would invent something like a pressure release valve.
On a radiator or a steam generator, or something.
And it would be able to open when the pressure gets too high
and close again when it's back down to normal.
Well, cells, including pollen,
cannot work through metal and valves.
Their kind of engineering relies on protein
and works over the span of evolution.
So, when we were trying to understand
just what pollen might come equipped with
as a way to control pressure,
we turned to nature's pressure release valves,
which are these mechanically sensitive ion channels.
Now, we actually know a lot about studying this kind of protein
from bacteria,
so let me talk a little bit about what it looks like
from the bacterial point of view
and why we think that it could have a role
similar to that in pollen.
So, if we start up here with a happy little E. coli cell
growing in an environment that it's used to
and we pluck it up and drop it
into a much more dilute solution,
that's going to naturally force a lot of water into the cell,
really increasing the pressure on the membrane
that encloses the cell,
potentially bursting it open.
Except, E. coli comes equipped
with one of these mechanically sensitive ion channels
that opens in response to that pressure,
allowing some ions to flow out,
and that's what balances the pressure inside and outside the cell,
so it can survive this kind of transition.
If we imagine zooming out from this cell
and looking down on top of the membrane
that encloses the cell,
these ion channels form protein pores
studding the membrane at different places.
In response to the pressure, they'll open,
allowing ions to flow through,
but, just as important, they'll then close again,
just like an engineered pressure release valve
that we might use.
So it's this dynamic, cyclical process, even,
that can control the pressure to the amount that the cell needs.
This protein in bacteria is called MscS,
and when we went looking
we found that it has cousins in the plant world,
including one in pollen
that we called MSL8.
Now, outlined in yellow, I highlighted
the family resemblance between these two proteins,
because they're actually related over billions of
years of evolution.
And that region in yellow is the sort of basic component
that allows it to respond to this pressure
and allow ions to flow through.
Well, you can see, in those billions of years,
MSL8 has added a lot onto the protein,
and we just weren't sure if it was still
acting in the same way.
Well, in order to find out, we went ahead
and studied this protein, MSL8,
in this tiny mustard plant that we call Arabidopsis.
Now, 20 or 25 years ago,
this was nothing but a weed,
but in the time since,
hundreds or, really, thousands of plant scientists
have agreed to all study this plant
so that we can learn a lot about,
simply, how plants do plant things.
Now, we knew that if MSL8
was going to be useful for pollen at all,
it has to satisfy at least one very basic criterium
-- it had to be in the right place at the right time.
So, in order to track that,
we hooked it up to the fluorescent protein GFP,
so that wherever MSL8 was
we could see it and we could track it.
And if it was going to be of any use during hydration or germination,
we figured it had to be there
before pollen goes on its long journey,
as it's maturing.
So we dissected a flower, a male flower,
where the pollen was developing,
and took a look.
Each of these little egg-shaped regions
is actually an individual pollen grain,
and the green signal inside
is showing us that MSL8
is being produced, packaged, and prepared
for use later.
Okay... so we checked that off.
It was in the right place at the right time.
But in order to understand whether
it had anything to do with hydration or germination,
this growth of the pollen tube,
we had to go out and study these processes.
Unfortunately, that's really hard to do out in the real world,
so we took it back into the lab
and created an artificial condition that looked
a little bit like it...
what I really mean is that we took pollen
and we dunked it in water.
Now, in order to see whether MSL8
had any effect on that,
we looked at pollen that normally has MSL8
and then we made a mutant
so that it doesn't have this gene,
and then really just tried to see if there was any difference between the two.
We take the pollen and we just put it in water
and we add a dye that shows us
the living cells, in green.
We see the normal pollen that does have this gene
survives fine.
In contrast, pollen that doesn't have MSL8
dies and it dies fast and it dies hard.
You can easily see it under the microscope,
but of course we're scientists,
so we wanted to quantify this
and see just how strong the effect was.
So, here, we took the pollen
and we put it in water for up to 2 hours,
which is basically an eternity for the cell,
but it doesn't matter.
As long as we keep the pollen in the water,
as long as it has MSL8,
it survives fine
-- it doesn't really seem to mind.
On the other hand, as soon as we can look
at pollen without MSL8 under the microscope,
it's dead,
and it really just gets worse from there.
Now, this doesn't really tell us whether or not
MSL8 has any impact on
pollen's function in the real world,
and that function ultimately comes down to
one single thing, which is fertility
-- its ability to make a seed.
And so we wanted to see if
this was true at all,
if it had any impact out, say,
you know, in the real world.
And so in order to do that we did a couple crosses with our plants.
Now, one of them was a control,
where we took regular pollen
-- it has this gene --
and we crossed it to a female partner
where half of the egg cells
did have MSL8 and half didn't.
The reason this is a control is because
we already knew that MSL8 had nothing to do
with the female side of this equation,
so we could expect to find that, in the offspring,
that 50% would carry this mutation through,
and that's exactly what we saw.
But if we flip it around
and use pollen where half of it has this gene
and half doesn't,
well, in that case we recover
less than 50% of this mutation, 44%.
Now, 44% might not seem like it's that different than 50%,
but let me tell you... evolution is brutal.
That slight difference, over just a few generations,
will quickly be selected against and our pollen that doesn't have MSL8
will be no more.
So, MSL8, this gene is definitely
providing an advantage,
and we seem to have some evidence that
one way it could do that is protecting
against excessive pressure during hydration.
But, like I mentioned,
for our poor pollen on its epic journey,
hydration is not sufficient
-- it has to still grow 1,000 times its original size
and it grows at this incredibly fast clip
in order to stay competitive.
And it's driven by pressure,
it needs the pressure,
but that could end dangerously if there's too much.
So instead of just dunking the pollen in water,
we actually provided the nutrients and signals
it needed to germinate and start growing
-- we use the same vocabulary that we do for seeds.
Now, when we look at normal pollen,
after a few hours some of it starts to germinate,
kind of highlighted in the blue color, here.
But the pollen that doesn't have MSL8,
it actually bursts open more often than not.
A zoom in here and you can see that
that grey area coming out of the pollen grain,
that's actually it spilling out its guts into the solution
-- again, game over.
Now, quantifying this,
we see that,
much more frequently than the regular pollen,
pollen without MSL8 bursts open.
And if it does that prematurely,
there is no plant sex and it loses.
Now, this was all evidence that
MSL8 had some kind of adaptive function
-- we knew that it helped pollen survive hydration,
we knew that it maintained the integrity of the cell
as it was trying to grow
at that really fast clip,
and we knew that, ultimately,
there was a real function for it,
because without MSL8
the fertility and its competitiveness was dropped.
Well, we were wondering...
we know it happens without any MSL8,
what would happen with a little bit extra?
Would it provide an extra advantage to the pollen?
Well, we decided to take a look
by producing a whole range of plants
that we forced the pollen to overexpress MSL8,
and again we tagged it with this
fluorescent GFP so we could take a look.
Now, after a couple of generations,
what I was expecting to find was
some plants where 100% of the pollen
was expressing this overexpressed version of the gene,
tagged with GFP.
And so I went looking.
The way I set up my crosses, I was expecting that 1/3,
or 33%, of the plants I looked at,
would be homozygotes,
that's what we call them.
Except when I actually started looking,
I found none... zero...
no homozygotes,
and I just didn't really know what was going on.
Actually, this happens a lot in my job.
Most of my job as a scientist is
dealing with something that doesn't work
and trying to figure out how to fix it.
Now, sometimes it's because the experiment is really hard...
frankly, other times it's because I messed up,
you know?
And I thought that was probably what was going on here,
frankly, that's what happens most of the time.
I thought maybe I was not using the right seeds,
that I wasn't taking care of my plants correctly,
that I wasn't using the microscope right,
and, most importantly,
that I might not have been taking good enough notes,
which is usually the most important thing.
But I decided to take this and find out what was going on.
So, first of all, I made sure to start taking
very good notes, like I should have the whole time,
including the number of plants that I was really looking at.
Once I got up to looking at dozens of plants
and not finding any of them,
let alone a third,
I thought something might be going on.
So I decided to run a couple of experiments
that I knew how to do and see what I could see.
Well, in one case I took the pollen
and I dunked it back in water
-- easy enough.
We know that the normal pollen survives fine
under these conditions,
and when I looked at the pollen that had extra MSL8
it survived fine too.
Well, when I saw this,
it actually made sense to me,
because I figured that the reason MSL8
was helping pollen survive hydration
was relieving excessive pressure.
Well, I couldn't think of a reason
why having too much MSL8 would be bad
under those conditions.
Okay, then I went ahead and
induced the pollen to germinate.
Here, I took regular pollen
and allowed it to germinate for a really long time,
so that basically all of it gets going,
and you can see that there's just pollen tubes everywhere
-- they're just going in every which way,
covering the field of view,
forming this mat where you can barely see what's going on.
Well, in stark contrast,
when I looked at the pollen that had too much MSL8,
it was doing nothing.
Now, it was alive,
we saw that just right before,
and I could see under the microscope right here
that it was trying to do something.
These internal structures
were moving around and kind of bubbling together
-- it was trying to get going, but it just couldn't.
Now, at the same time,
I started looking at more kinds of plants
that had different levels of MSL8.
Now, some of them had a lot more MSL8,
some just had a little bit more than normal,
and when I looked at what effect that
had on germination,
I saw a clear relationship
-- that the more MSL8 there was,
the less it was able to germinate.
And when I took a look at a wider range of plants,
I finally did find the homozygotes
that I thought were just missing before,
not all the time as much as I expected,
but they were at least there.
Now, Martin Chalfie,
who won the Nobel Prize in 2008
for discovering how to use GFP in cells,
the very same fluorescent protein
that I was using in order to track MSL8
in all of these studies,
he gave a talk at our university
a little bit after I was working on this stuff,
and at the end he said that
if you make an observation
and it confirms what you were expecting,
then you've made a measurement,
but if you make an observation and it surprises you
and it contradicts what you were expecting,
then you've made a discovery.
And I realized, after
making sure I took good notes,
growing in my confidence in my data
and in myself as a scientist,
that what I had on my hands was not a mistake,
not a product of failure,
but actually a discovery,
which is that pollen has to balance the amount of MSL8
that it has inside of it
in order to stay competitive.
Because if it has too little,
what we knew early on,
is that the pressure gets too high,
it can burst open.
Sure, it might be able to germinate,
because it has that pressure,
but it doesn't matter because it bursts
and dies too early.
But, what I discovered is that
you can actually have too much of a good thing,
in that if there's too much MSL8 around,
it might be intact,
but it can't build up the pressure
required to burst through
the tough outer coat
and continue with this really rapid pace of growth.
It was alive, but lifeless.
And so pollen really needs to
balance the amount of MSL8 it has
to balance the need for the integrity of the cell on one hand
and the vitality of the cell on the other.
Now, we learned all of this by studying
this tiny little mustard weed Arabidopsis,
but we went looking and tried to see,
is MSL8 in other plants?
Well, we found it in the flowers of corn
and it was there in soybeans too.
We found it in rice
and in poplar trees.
This is pretty good evidence that
MSL8 is likely to be in a whole range of different plants out there.
Now, there are almost 300,000 species of
flowering plants in the world.
I don't know if all of them have MSL8
and I definitely don't know if they all
use it in the same way,
but I think if we're interested in
maintaining this plant sex that we so rely on,
that we should go and find out,
because the more that we understand about the process,
and the more that we understand about
how pollen is equipped with a whole range of different strategies,
not just MSL8,
in order to survive its epic journey,
I think we'll be better off.
So, with that, I'd like to thank you
for joining me with this story
and I'd like to thank all the people listed here,
who have really helped make this a reality.
Thank you.