The Oxygen cycle and Dole Effect--Guy, Berry and Fogel |
Everyone knows about the global carbon
cycle. Humans in the past couple of hundred years have changed it due to the
massive combustion of fossil fuels. Since I was a grad student in 1975 to now
(2020) the concentration of carbon dioxide (CO2) in the atmosphere
has gone from 340 parts per million (ppm) to over 410 ppm and is now increasing
about 10 ppm per year! It may be that we’re not going to be able to reverse
this trend in our lifetimes—maybe not even in the next 100 years. But, the
Earth has gone through many boom and bust periods of high and low concentrations
of carbon dioxide in the atmosphere before, in fact many times. Now, however, we’ve
made substantial changes to carbon dioxide in the air at a much more rapid pace
than ever before.
While increased CO2 in the
air causes global temperatures to rise and sea levels to flood low lying areas,
plants are pretty happy. More CO2 is like more food for plants,
which means more photosynthesis. More photosynthesis means they produce more
oxygen into the atmosphere as well. Currently, oxygen (O2) is about
20% of all the gases in air. If there were much more O2 in the air,
we’d have a significant shift in many of the biogeochemical cycles, not just
with oxygen, but nitrogen and sulfur as well. The interactions between the
carbon and the oxygen cycles—now and in the past—are major drivers of how
organisms manage to grow and reproduce on Earth.
With a PhD in botany, one of the more
intriguing intersections, to me, between botany and biogeochemistry was the
“Dole Effect.” Malcolm Dole, a chemist at Northwestern University, had been doing
experiments with oxygen isotopes in O2 and water in the atmosphere
for over 15 years starting in the 1930s. He and others, including Harold Urey—a
Nobel Prize winner--noted that the O2 in the atmosphere had an
excess of the heavy isotope of oxygen (18O) relative to what it
should be if atmospheric O2 was in equilibrium with Earth’s surface water (Dole, 1935). This
observation was important because the anomaly in the oxygen isotopes in air was
caused by a balancing act in the Earth’s carbon cycle between photosynthesis,
which produces oxygen, and respiration, which consumes it.
In 1956, Dole and his colleagues
published experiments that measured oxygen isotope changes (i.e., fractionation)
during respiration by various organisms. As expected, organisms used the
lighter isotope (16O) faster than they did the heavier isotope (18O).
As a result, the O2 that hadn’t been consumed by respiration had an
isotope composition with an increased 18O concentration. The problem
was that the magnitude of isotope changes Dole and his colleagues measured
didn’t easily explain the isotope values of atmospheric O2.
Something didn’t match up. Studies by Michael Bender, then at the University of
Rhode Island, showed once again that oxygen isotope fractionation during
respiration by marine phytoplankton and microbes in oceans was considerably
less than what had been measured in the oxygen of the atmosphere (Bender and
Grande, 1987). There still was no explanation for the strange isotope values in
the atmosphere. To me it meant that we didn’t understand all the parts and
pieces of Earth’s oxygen cycle.
In 1984, I was looking for additional
challenges to my work as an isotope biogeochemist. I had spent eight years at
the Geophysical Laboratory in the company of geochemists and earth scientists,
far removed from biologists and botanists at the start of my career. I met Joe
Berry at Carnegie’s Plant Biology Department at a Carnegie Institution of
Washington meeting, and we immediately struck up a conversation about how we
could weave together experiments on modern plant biology to tackle the potential
factors that cause the Dole Effect. Berry, Chris Field, and Olle Bjorkman, all
staff members at Carnegie’s Department of Plant Biology, were studying O2
uptake reactions (i.e., photorespiration) in leaves, as well as conducting in
situ work with Rubisco. If conditions are right, Rubisco has the potential
to fix O2, rather than CO2. This can occur when temperatures are high, and the plant has made an
excess of oxygen that might damage the plant via free radical production.
At that time, we could not account for
the factors that caused the Dole effect, which had implications for how the
most important biological processes—photosynthesis and respiration--were
balanced globally. Clearly, the studies to explain the global oxygen cycle
using stable isotopes could not resolve the problem. Thus, I started on a major
discovery endeavor to describe the oxygen isotope changes that occur from living organisms that deal with oxygen. Photosynthesis had been measured.
Earlier experiments had shown that O2 created from the splitting of
water during photosynthesis had the same isotopic composition as that of the
water in the plant (Dole and Jenks, 1944). Nevertheless, we wanted to make sure
we understood it perfectly. Photorespiration, the uptake of photosynthetic O2
by plants in hot, dry environments, had not been measured. Plants also take up
and use oxygen in many other reactions that the geochemists who’d been thinking
about the Dole effect, did not consider. There was plenty of work to do to
figure this out.
Joe Berry and I submitted a proposal to
the Department of Energy to fund our work. We received the following reviews:
“The
investigators underestimate the pitfalls of their experimental technique. The
trapping of oxygen “on a molecular sieve column” (page 8) and the application
of an oxygen electrode (pages 8,9) shift the ratio of the two oxygen
isotopes...The hypothesis that photorespiration can account for part of the
photosynthetic fractionation is interesting. Is there any reason (literature,
preliminary data) to expect it to be so?”
Perhaps we
hadn’t accounted for all of those supposed pitfalls, but both of us were
experimental isotope biological chemists. I was confident that we could figure
this out. Amazingly, the proposal was funded and on we went.
Oxygen is not the only gas in air. The
bulk is nitrogen (about 80%). There are trace amounts of carbon dioxide and
water vapor in air. Another 1% of air is the inert gas argon. To measure the
stable isotopes of O2, we needed to purify it from all of the other
components. We knew how to separate carbon dioxide, water vapor, and nitrogen
from O2, but it was a bigger challenge to remove the argon. To
purify atmospheric oxygen from argon in air samples, Tom Hoering and I designed
and constructed an isotope vacuum line at the Geophysical Laboratory in
Washington, DC. This particular line targeted O2 gas, a procedure
distinct from my first oxygen experiments that did not work out.
We built another homemade gas
chromatograph to separate out the argon. It was a time-consuming process, but
it worked. After oxygen was separated from argon, it was chemically converted
to CO2 in which the oxygen atoms in CO2 came from the oxygen
atoms in air. In late February 1985, we deconstructed the line piece by piece
and shipped it to the Department of Plant Biology in Palo Alto, California. Tom
Hoering and I flew out to California and pieced it back together. Then, working
closely with postdoctoral fellow Robert Guy, we designed and built the
“biological” part of the extraction line where enzyme reactions and cells would
be incubated for experiments. Experiments began in earnest in 1986, when I
spent a year’s sabbatical leave at Plant Biology.
Our vacuum line, 1985-1987 |
Working at another Carnegie department
was an interesting experience for me because I could see there were many things
the same and some things quite different. Both the Geophysical Lab and Plant
Biology had strong leaders. Winslow Briggs, Plant Biology’s Director, made me
feel right at home. I was assigned a comfortable desk space and introduced to
the staff. Winslow had an easy way of checking up on everyone at his
laboratory, including me. The support staff were similar to those at the
Geophysical Lab as well—a building manager, a business officer (Mary Smith), men
in the instrument shop, and grounds keepers. Scientific staff members were
quiet, nose-to-their-work folks who livened up at seminar time. Because Plant
Biology was located on the edge of the Stanford University campus, there were
many more students around than I was used to. In fact, being on a campus give a
larger feel to the small department because you could walk to any number of
seminars, enjoy a big library, and shop at a superb bookstore.
Joe Berry, my host staff member, kept a
certain distance after I arrived in 1986. A tall, lanky man, Berry would come into
the isotope lab maybe every couple of weeks, then we’d regale him about our
successes and failures. He listened carefully, thought over what we’d said, and
provided very measured responses. Sometimes, he even laughed a bit. I was
somewhat disappointed that we didn’t interact more. I was used to seeing my
Geophysical Lab colleagues every day and checking in with a brief “hello, how
ya’ doing?” and “see you later” at the end of the day. Eventually, I didn’t pay
much attention to this and enjoyed working with postdoc Rob Guy and chatting
mostly to Olle Bjorkman, whose lab was next door to ours. Years later, I
learned from Winslow Briggs that Joe really enjoyed having me in residence,
which I couldn’t figure out at the time.
Rob Guy, a Canadian to the hilt, and I
worked hard on the oxygen isotope experiments with long days filled with hard
work operating the vacuum line and preparing samples. Short in stature, but
long in thought and level of detail, Rob was a great colleague for me to work
with. I often had outlandish ideas, whereas Rob was a pragmatist. Sometimes, we
would spend several hours going over an experimental design or an outcome,
followed by a compromise. It was
fortunate for me, looking back, that Rob worked with me on this project,
because his input was critical for its ultimate success.
Our first experiments were with Anacystis
nidulans, an easy-to-culture cyanobacterium. Cells were grown in the lab,
then transferred to a collapsible bag that was bubbled with helium to remove
all traces of atmospheric oxygen. We had an oxygen electrode mounted in the
bottom of the apparatus, which was constantly stirred. Light was turned on, and
oxygen evolved through photosynthesis. We confirmed earlier studies that showed
there was little to no oxygen isotope fractionation during photosynthesis (e.g.,
Stevens et al., 1975). These first photosynthetic O2
measurements allowed us to refine our techniques and understand various
pitfalls. Typically, we would start on Monday to grow cells, get the chamber
and vacuum line in good shape on Tuesday, run the first set of experiments on
Wednesday, then repeat Thursday and Friday. Samples of CO2 were
sealed into Pyrex ampules. Every two weeks or so, I traveled down to NASA’s
Ames Center at Mountain View to work in David DesMarais’s lab, who kindly
allowed me to use his isotope ratio mass spectrometer.
Rubisco, which stands for
ribulose 1,5 bisphosphate (Rubis) carboxylase (c) oxygenase (o),
is an unusual enzyme in that it catalyzes the uptake of both CO2 and O2. Our second
set of experiments was a “revisit” to my PhD dissertation and the
re-measurement of both carbon and oxygen isotope fractionation by Rubisco.
Times had changed. Rubisco from spinach, cyanobacteria, and R. rubrum
could now be expressed in E. coli; no need for growing massive amounts
of cells or purifying enzymes. For these experiments, rather than the
time-consuming purification and crystallization of the enzyme’s product, PGA,
we simply measured the isotopic composition of the remaining CO2. It turns
out that the values I measured for my PhD were slightly different from those I
measured in California, because the carbon isotope ratio of RuBP in my earlier
experiments was influenced by unknown contamination. By measuring only CO2, we
avoided that problem. This time I nailed it. We measured an isotope
fractionation for carbon in CO2 nearly identical to recent experiments
by Roeske and O’Leary (1984).
Oxygen isotope fractionations were
calculated using specialized equations (Rayleigh equations) that compare the
oxygen isotope compositions of the O2 in our reaction chamber at the start of
the experiment to the oxygen isotope compositions of O2 when we
took a sample. Using our oxygen electrode, we could calculate the fraction of O2 that was
consumed (Guy et al., 1993). We determined the isotope fractionation from the
oxygenation part of the Rubisco reaction to be very similar to what was
required to explain the Dole Effect. Approximately 30% of all O2 produced
by photosynthesis is “fixed” by Rubisco prior to its being released. Another
20% is taken up by reactions in which oxygen radicals are converted to peroxide
(H2O2), then converted via another enzyme, catalase, to form
H2O. The isotope fractionation during these reactions yielded further
isotope fractionation.
We followed with measurements of oxygen
isotope fractionations by even more plant enzymes that take up oxygen including
cytochrome oxidase, which is involved in respiration for all plants and animals.
Experiments with cytochrome oxidase were difficult. To perform these
experiments, we needed large quantities of the enzyme cytochrome oxidase, which
we could obtain from Sigma Chemical Company. Cytochrome c, the compound that
the enzyme requires to make it work, was expensive--about $500 per experiment---and
it was destroyed at the end of each experiment. We managed to only have one or
two successful runs, measuring an isotope fractionation that was way too
small--hardly enough to explain the Dole Effect.
We measured oxygen isotope fractionation by
almost all of the enzymes that could possibly take up molecular oxygen. We
concluded that if all of the plant enzymes that took up oxygen were considered,
the Dole effect could be explained. Aerobic respiration is important, but the
other five enzymes we’d measured were also key players in Earth’s carbon and
oxygen cycles. The work remains important—and the only studies to date—for
understanding the biogeochemical cycles of oxygen on Earth.
At the same time I was working at
Plant Biology, two major events in my life and career were unfolding. First,
and most important, Chris proposed in spring of 1986. I returned to the east
coast in July by myself with little dog Sputnik to plan our wedding in
mid-September. We married on September 20th in New Jersey then traveled
across the country on our honeymoon. Unlike many newly weds of that era, we
eschewed going to Florida or Europe and instead went canoeing in Minnesota’s
Boundary Waters Park on the border with Canada. It was absolute heaven! We
stopped off at Yellowstone National Park, where we checked out my former field
areas in the hot springs, then on to California.
Marilyn and Chris, 1986, Geophysical Lab |
The other not-so-pleasant event was
fighting for the sovereignty of the Geophysical Laboratory. President James D.
Ebert discussed with me the possibility of moving fulltime to Plant Biology.
While I would have enjoyed living in California, limiting myself to research
only plants—and not microbes, animals, old rocks, and sediments—wasn’t
appealing to me. After a Visiting Committee assessed the Geophysical Lab’s
science in 1985, they strongly supported keeping the lab the way that it was. The
time I spent at Plant Biology strengthened my resolve to continue in
biogeochemistry and be as interdisciplinary as I wanted to be.