of these terrestrial floras enhanced the burial of organic material on land
through the Silurian and Devonian, and thereby gave Earth a huge new source
of oxygen. Oxygen added to the atmosphere by photosynthesis is normally
used up by microbial decomposition, balancing the budget. But if carbon
burial occurs before decomposition, the oxygen instead feeds into the oceans
and atmosphere. Through these processes, the evolutionary assembly of
stomata-bearing terrestrial floras breathed oxygen into the Devonian world.
Chemical analyses of sediment cores drilled from the sea floor reveal the
telltale rise in seawater oxygen levels through the Devonian.20 At the same
time, the Devonian oceans witnessed a dramatic increase in the maximum
size of large predatory fish. Before the Devonian oxygenation event, fossil
fish of Ordovician and Silurian times reached lengths of a few tens of centi-
metres. Afterwards, fish greater than a metre long appear in the fossil record.
Are these changes in the fossil record of fish and ancient atmospheric oxygen
levels merely coincidence, or could they be linked?
The short intense bursts of speed modern fish need to capture prey entail
a high demand for oxygen to fuel their muscles. The same principle also
holds for large Devonian predatory fish, with the implication that the evo-
lutionary arrival of these finned swimmers with energetic lifestyles in the
oceans was only possible after oxygenation of the biosphere.21 It could also
be that this development reflected an expanded food web supported by
nutrient-rich runoff flushed into the oceans from plant-covered continents.
Still, it seems the rise of stomata-bearing plants played a crucial role in
driving a critical transition in Earth history—the development of a fully
oxygenated Devonian Earth system that ushered in the Devonian ‘age of
the fishes’.
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Because as a green, chlorophyllous Eden took hold, plants inadvertently set up
a series of self-reinforcing (positive) feedback processes that generated a climate increasingly agreeable for the further spread of plant life. More plant life on the continents meant the capture of more precipitation by roots and its return to
the atmosphere to seed rainfall (Figure 13). By adding water vapour to the
atmosphere, plants not only cooled the land surface (as sweating cools your
skin) but could also have increased the cloudiness of the sky, casting additional cooling shade. Together, these effects generated convective activity in the
atmosphere, pulling moist air in from the ocean onto the land, further enhancing
precipitation.22
Regional-scale meteorological effects like this partly explain why precipitation
doubled over land in the ‘green world’ versus ‘desert world’ model simulations
mentioned earlier. They also explain how the collective actions of stomata
strengthened over time to air-condition the planet, keeping it a few degrees cooler, and the atmosphere moister, than it would otherwise be without plants.
Of course, there is no grand plan, no teleology about this. The collective
actions of stomata on climate over time are simply a by-product of the crucial
decisions they make on a minute-by-minute basis about when it is safe to open
and close, by how much, and for how long. To do this, they operate as miniature
sensors organized into fantastically complex arrays that integrate multiple cues
from the environment, and chemical and electrical signals communicated from
cells in leaves and roots. Scientists bewitched by investigating details of stomatal function agree that these marvellous cellular structures seem to operate continually in just such a way as to maximize the amount of carbon dioxide captured by
the leaf while simultaneously minimizing water escaping as transpir ation. Yet
they remain baffled as to how stomata achieve this amazing feat.
The late Fred Sack (1947–2015), a giant in the world of stomatal biology, once
likened it to Dr Pangloss’s thoughts in Voltaire’s 1759 French satire Candide. Candide, remember, is the illegitimate nephew of a German baron who grows up in the
baron’s castle under the tutelage of the scholar Pangloss. Sack remarked that ‘life on Earth depends in no small part on stomata’ and noted that stomata offered what
Pangloss called ‘the best of all possible worlds’ for making the planet green and lush.23 Joe Berry, a professor at the Carnegie Institution, Stanford, and another scientist who has also spent his career thinking about stomata, puts it another way,
Gas valves a 103
Plants with
stomata
appear
Wet
Wet
Dry
Dry
Non-vascular plants
Water vapour
Liquid water
Vascular plants
–500
–400
–300
–200
–100
Time (Myr)
Figure 13 The evolutionary progression from small plants lacking stomata to large stomata-bearing trees accelerated the global water cycle, recycling soil water by adding it as transpiration to the atmosphere to later fall as rain.
commenting, ‘You might think of them as analogous to traders on Wall Street
making bets on the cost of water (the currency) for carbon dioxide uptake (a com-
modity)’.24 This useful way of framing the concept extends readily to the global
scale. Just as the activities of Wall Street traders influence the world’s economies, so the collective decisions of stomata influence global water and carbon cycling, and climate.
Today, we understand the basic mechanism of how pressure-operated guard
cells work. In broad terms, molecular signals adjust the pore size by causing them to swell or shrink, although much of the fine detail remains a mystery. Molecular pumps embedded into the cell membranes drive pressure changes in the kidney-shaped guard cells (Figure 14). When environmental conditions are right, usually
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in the morning and when water is plentiful, stomata open by expending energy to
drive molecular pumps that expel positively charged hydrogen ions out of the
cells and create a minute electrical charge across the cell membrane. This charge opens the electronically controlled molecular gates of membrane-spanning channels to allow potassium ions to flood into the cell. In effect, the inside of the guard cells becomes saltier and this causes an influx of water by osmosis, pressurizing and inflating the cell, much like pumping up a bicycle tyre. The inner surfaces of guard cells are thickened, and bend apart in semi-circular fashion as they inflate, creating a pore. This arrangement provides an inherent fail-safe mechanism protecting the leaf against dehydration during severe drought; as the soil dries, wilt-ing due to decreased water pressure automatically closes stomata and conserves
the plant’s remaining water reserves.
As we can start to appreciate, from evolutionary, biophysical, and physiological perspectives—take your pick—stomatal biology seems to have a unique complexity
about it, with far-reaching consequences for all life on Earth, not to mention the global cycling of elements and energy around the planet. A key question follows:
how did stomata originate? The fossil record of plant life suggests they turn up
ready-made, apparently without earlier prototypes. Is it possible plants could
Guard cells (swollen)
Guard cells (shrunken)
Epidermal cells
Cell walls
H O
> 2
H O
2
H O
2
Chloroplast
Potassium ions
H O
2
(K*)
Nucleus
H O
H O
2
H O
2
2
H O
2
Vacuole
Stoma
Stoma opening
Stoma closing
Figure 14 Opening and closing of stomata.
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BEWITCHING STOMATA
-
The sophistication of these microscopic structures often casts a bewitching
spell on the scientists who study them. Nearly two centuries ago, Erasmus
Darwin (1731–1802), Charles Darwin’s grandfather, suffered an obsession
with stomata brought on by suffocating the leaves of Portuguese laurel and
balsams by pouring oil over them; leaves treated in this way died within a day
or two. The same thing happened when he subjected insects to similar treat-
ments. Given insects breathe through tiny pores (spiracles) punctuating their
thoracic segments, Darwin drew the conclusion that there was a ‘similitude
between the lungs of animals and the leaves of vegetables’, both being used
for breathing, without him actually observing stomata. The great botanist Sir
Joseph Banks (1743–1820) had seen them, though, on the leaves of corn and
wheat, noting stems were ‘furnished with rows of pores… shut in dry and
open in wet weather’. Banks was concerned with the cause of mildew on
crops and supposed that infection took place through the stomata, which is
generally correct.
Long before the days of Darwin and Banks these little structures had
caught the attention of the great Italian polymath Marcello Malpighi (1628–
1694).25 Malpighi seems to have been the first person to observe stomata
through a microscope, and later published drawings of them, but Darwin
and Banks were probably unaware of them. I should also mention that the
English anatomist Nehemiah Grew (1641–1712)26 ran him close with what he
described as ‘breathing holes’ on the surfaces of leaves in 1682. Malpighi’s
investigations were not confined to the anatomy of plants and several micro-
scopic anatomical structures of the animal world are named after him. There
are the Malpighi skin layer and two different Malpighian corpuscles in the
kidneys and the spleen, as well as the Malpighian tubules in the excretory
system of insects. It is probably only an accident of history that stomata are
not actually called Malpighian mouths, or some such thing.
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have evolved these sophisticated structures in such a fashion to learn how to
breathe? What sort of genetic toolkits did early land plants like Cooksonia and its cousins deploy to build them? As with questions on the origins of leaves and
roots, we have to delve into the world of evo-devo to see further than the fossil evidence.
Mosses are an important lineage for these sorts of investigations because they
are among the oldest living group of land plants with stomata (Chapter Two).
They diverged from a common ancestral lineage over 400 million years before
flowering plants appeared on the scene. The logic is that if mosses and flowering plants share similar genetic mechanisms for building stomata, then we can infer
that they already existed in the last common ancestor of these plants, and this
takes us a significant step closer to Cooksonia’s stomatal genes.
To develop the evolutionary story, we have to start with what is known about
the business of building stomata in flowering plants. And, in particular, with the breakthrough discoveries made by Dominique Bergmann27 and Keiko Torii28
concerning three ‘master regulator’ genes that sit at the heart of the genetic
machinery for making stomata. Both are world-leading molecular biologists:
Bergmann is a distinguished professor at Stanford University, where she is a col-
league of Joe Berry’s and where a preoccupation with stomata is considered
quite normal; and Torii is a distinguished professor at the University of
Washington, Seattle. For reasons that will become clear shortly, the three major genes they discovered that guide stomatal development are called SPEECHLESS
(SPCH), MUTE, and FAMA, with Bergmann’s lab discovering FAMA 29 and Bergmann and Torii’s labs then separately and yet simultaneously identifying
the other two.30
Together, these genes shape the molecular pathways regulating the processes of
cell division and cell fate by which plants produce guard cells. The cellular process of making stomata begins with the simple act of cell division that produces two
daughter cells of different size. This ability to undergo what is essentially an
asymmetric cell division should not to be underestimated. It sounds like a trivial thing, but it is actually the key to making all sorts of specialized structures in plants and animals because it generates two cells with potentially different fates, leading to the formation of distinct cell types and new organs.31 In stomatal development, the smaller cell gains special, distinctive, stem-cell-like properties and
Gas valves a 107
undergoes several further divisions. Next, the cell at the centre of the rosette
slowly matures, taking on an oval shape, and transitions into a cell that will eventually undergo a symmetric division to form a pair of specialized guard cells.
The role of the newfound genes in regulating these processes of cellular differ-
entiation is well established, and it is worth unpacking a little of the detail to appreciate how it sets the stage for discoveries with the moss genetic toolkit
(Figure 15). SPCH controls the first step in making stomata. In the young leaf, cells that express SPCH look the same as those that do not, but they are doing something unique. They are preparing to undergo the asymmetric cell divisions that
will initiate stomatal development. In Arabidopsis plants lacking SPCH, leaves are unable to develop stomata, stunting growth and ultimately causing them to
die. SPCH does not just act once, but is kept switched on in the smaller daughters of the asymmetric divisions, and this can amplify the number of cells in
the epidermis. These cells are on their way to becoming stomata, but they have
not yet committed themselves to that outcome. Commitment is the role of the
next gene, MUTE. A clue to its role came when geneticists created Arabidopsis lines lacking MUTE and found that the leaf surfaces were covered in rosettes of cells (from asymmetric cell divisions), but no stomata had been made. Arabidopsis
plants with MUTE switched on all the time produces the reverse effect—leaves covered with stomata. FAMA regulates the final stages of stomatal development.
Named after the many ‘false mouths’ of the Roman goddess of rumour (Fama),
this gene is specifically expressed in immature guard cells. Arabidopsis plants lacking FAMA develop leaves with stacks of cells aligned horizontally on the surface that resemble segmented caterpillars. These arise from repeated divisions of
SPCH
MUTE
FAMA
SCRM
SCRM
SCRM
1. Asymmetric
division forms
2. Guard mother cell
3. Mature stomata
meristemoid
Figure 15 Three master genes ( SPCH/MUTE/FAMA) control cell division and cell fate to make stomata on the leaves of flowering plants through
interaction with SCRM.
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proto-guard cells as they attempt but fail to complete the process of stomatal
development. Without FAMA, leaves are unable to close out the final step in building stomata. We can see now that each of the three genes, activated in discrete
places, and at particular times, performs a specialized function to ensure the normal process of stomatal development. Other genes are involved in generating the
diversity of shapes and sizes we see in leaves across the plant kingdom, but this core set of three master switches is all that need concern us here.
Our next task is to see how these findings fit with recently discovered genetic
mechanisms guiding the formation of stomata around the base of the little
spherical capsules (sporophytes) of the moss Physcomitrella. In fact, this moss has two stomatal genes closely related to the SPCH/MUTE/FAMA genes of Arabidopsis,32
called SMF1 and SMF2.33 Until recently, we had little idea what roles either gene played in moss stomatal development, if any, but then, over a period of about a
decade, an international collaborative team of researchers, including researchers from Sheffield, Freiburg, Stanford, and Leeds, began to unpick the mystery.
The long journey to discovery began when we took the obvious step of cancel-
ling millions of years of evolution and manipulating the genetic make-up of moss
by deleting either SMF1 or SMF2 to investigate what happened. To our delight, the resulting lines of moss missing a single gene ( SMF1) developed normal-looking sporophytes in every respect except that they lacked the characteristic ring of stomata around the base of each capsule (Plate 5). It was a ‘eureka’ moment for
Caspar Chater, the dedicated researcher who had started working on the project
as part of his PhD a few years earlier. His time ran out before he obtained definitive results, but he later returned to doggedly pursue it as a postdoctoral researcher with us in Sheffield. On first discovering the ‘missing stomata’, Chater took a
moment to collect himself before anxiously examining other moss sporophytes
under the microscope. In the event, he need not have worried. His observations
confirmed a beautiful anatomic anomaly: the sporophytes of mosses lacking the
SMF1 gene lacked stomata. In contrast, mosses lacking SMF2 developed with stomata as normal. We can conclude, then, that SMF1 but not SMF2 is essential for making stomata in moss, and this fits neatly with earlier complementary findings
Making Eden Page 16