Spring.wmf (18300 bytes) Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Gas Exchange/Transpiration 

I. Definitions

II. Photosynthesis/Transpiration Paradox (or perhaps more accurately, a "Compromise" or "Dilemma")
    Recall the equation for photosynthesis where:

CO2 + H2O (CH2O) n + O2

This equation suggests that:

  1. Gases, such as carbon dioxide and oxygen, are important in the overall energy metabolism of plants;
  2. Plants must exchange gases with the environment; and
  3. In order to obtain carbon dioxide plants will necessarily loose water (transpire).  In other words, transpiration is a necessary evil of photosynthesis.

III. Theoretical considerations

  1. A large surface area is required for efficient gas exchange.
        Thus, animals have lungs or gills whereas plants have leaves with extensive spongy mesophyll (it may be time to review your leaf anatomy).
  2. A large surface area for exchanging gases offers a large surface area for desiccation. 
        Animals solve this problem by placing the absorptive surface inside a humid cavity (lung) opened with a small exit pore(s). Plants put the absorptive surface (spongy mesophyll) inside the leaf and cover it with a water impermeable layer (cuticle) peppered with a series of pores (stomata). The cuticle is comprised of waxes, which are an assortment of long chain hydrocarbons and, in particular, cutin (C16-C18 hydroxylated fatty acids). Note that even though these strategies minimize desiccation from the absorptive surfaces of plants and animals, they don’t completely eliminate it.
  3. Placing the absorptive surfaces inside the organism to reduce desiccation presents a problem - getting the gases to the absorptive surface.
        Animals use an active pumping mechanism (e.g., lungs/diaphragm) to move gases inside the organism by bulk flow. The gases are circulated by another pumping system (heart). The distances needed to move the gases are too great to be accounted for by simple diffusion. Plants do not have a pumping mechanism for moving gases; they rely on diffusion (and to a lesser degree, bulk flow). In either case, plants do not actively move gases. This is one reason why leaves are so thin (recall our previous discussion) - diffusion is not efficient over long distances (i.e., diffusion is inversely related to the square of distance).  For example, it would take about 2.5 seconds for glucose to diffuse the distance across a cell membrane (0.5 μm) but approximately 32 years to go one meter!
  4. Paradox and Compromise.
        In order to obtain carbon dioxide for photosynthesis plants needed to evolve a large, thin absorptive surface (leaves with spongy layer) and then protect it from desiccation. Thus we can consider this the photosynthesis/transpiration "paradox". Actually, it might better be considered a "compromise" since that is what a plant needs to do - strike a compromise or balance between the amount of carbon dioxide absorbed and the amount of water lost by transpiration.

IV. Further complications
    Not only is water loss a "necessary evil" of photosynthesis, but to make matters worse, the tendency to loose water is greater than the tendency of carbon dioxide to diffuse into the plant. As evidence, let's calculate the transpiration ratio, which is a measure of the the amount of water loss relative to the amount of carbon fixation.  

transpiration ratio =  mol water transpired / mol CO2 fixed

If carbon dioxide uptake (or fixation) and water loss are equal, this ratio should be one. In reality, experiments show that this ratio is closer to 200!  Thus, for every 200 kg of water transpired, 1 kg of dry matter is fixed by a plant. Let’s see why:

A. Diffusion and gradients
    Recall that during diffusion molecules move from an area of higher chemical potential (or concentration or chemical energy) to an area of lower chemical potential (or concentration or free energy) and that the driving force for diffusion is the gradient from one area to another. We can express this relationship mathematically using Fick’s Law:   Jv = (c1 - c2)/r    where   Jv = flux density, (mol m-2s-1); c1 - c2 = concentration gradient, and  r = resistance (a function of distance, medium viscosity, membrane permeability, etc.).  We can simplify this equation to:

diffusion = gradient/resistance

Now let’s compare the rates of diffusion for both water and carbon dioxide.  Since resistance, or the distance that either carbon dioxide or water must diffuse into/out of the leaf is the same, then diffusion is directly proportional to the gradient.

  1. Carbon dioxide - has a very shallow, or small, gradient from inside to outside of the leaf. Ambient carbon dioxide concentrations are approximately 0.03% (= 0.36 mmol mol-1).  The internal concentration cannot be less than zero.  Thus, the gradient is no larger than 0.36 mmol mol-1 (0.36 - 0).
  2. Water - has a very steep gradient from inside to outside. At a relative humidity of 50% and 25 C the water potential of water in air is ca. -100 MPa (= 32 mmol mol-1). The air in the substomatal cavity of a leaf is typically fully saturated, with a RH near 100%, and has a water potential near zero. Thus, the water potential gradient is 100 MPa (or 32 mmol mol-1).

Conclusion: based on gradient alone, the water has approximately 100x greater tendency to diffuse out of the leaf than carbon dioxide to diffuse into the leaf.

B. Diffusion and molecular weight. 
    Recall that diffusion is inversely related to molecular weight.  Simply put, the heavier the molecule the more slowly it will diffuse.  No big surprise.  Or, to express this mathematically: 
                        rate = 1 / sq rt MW

The relationship between the rate of diffusion of two molecules can be summarized by the following relationship:
                        Rate A / Rate B  =  sq rt B / sq rt A

Thus to calculate the ratio of water loss to carbon dioxide uptake:
                        H20 loss/CO2 uptake = sq rt 44 / sq rt 18 = 1.56

Conclusion:  Based on molecular weight alone, the tendency for water to diffuse out of the leaf is 1.56 times greater than the tendency for carbon dioxide to diffuse into the leaf.

V. The photosynthesis/transpiration compromise revisited
    Although it seems as though water loss is a serious, intractable problem for a plant, it is NOT. The reason - plants continually compromise between the amount of carbon dioxide absorbed and the amount of water loss. This compromise is mediated by the stomata, whose function is to regulate gas exchange.

VI. Stomatal Structure

  1. Anatomy of a stoma (stomata, plural) - guard cells, subsidiary cells, substomatal cavity, cuticle, ledge (or lip), stomatal apparatus. The subsidiary cells are epidermal cells that may be specialized and different from the other epidermal cells. The function of the ledge is to prevent liquid water from seeping into the pore. Interestingly, cutin covers most of the cells in the substomatal cavity; only regions near the actual opening are free of cuticle and most water is lost from this area.  For images of various guard cells/stoma click here.
  2. Types of guard cells: (1) elliptical or kidney-shaped. These are characteristic of eudicots and other non-grasses; and (2) dumb-bell or dog-bone shaped - characteristic of grasses (called graminaceous type)
  3. Common features - (1) thickened inner walls; and (2) radial micellation - the cellulose microfibrils radiate out around the circumference of the pore; (3) chloroplasts - these are the only epidermal cells with chloroplasts; and (4) connected end-to-end.

VII. The beauty of stomata
    The evolution of a water-impermeable covering of the absorptive surface that was peppered with oodles of pores was a great idea. The stomata are ideal structures for regulating gas exchange because:

  1. There are lots of them on any plant surface.   In fact, there can be as many as 1000 mm-2. Obviously, the larger the number of pores, the greater the amount of total diffusion that can occur (see accompanying data.  Plot: total diffusion (μg hr -1) vs. total pore number, and total diffusion (μg hr-1) vs. pores sq cm-1. A large number of pores is necessary so that plants are able to absorb enough carbon dioxide for photosynthesis.
  2. The pores comprise a large area of the surface of a plant. In fact, stomata occupy as much as 2-3% of the total leaf surface area. As expected, the greater the pore area the greater the rate of diffusion [plot total diffusion vs. total pore area (cm-2) see accompanying data] which is advantageous for maximizing carbon dioxide uptake.
  3. The pores are small.   On average, stomata are about 14 μm in diameter. Although more total diffusion occurs through large pores [total diffusion (μg hr-1) vs. pore dia. (μm)], small pores are more efficient than larger ones [diffusion rate (μg hr-1 cm-2) vs. pore dia (μm) see accompanying data]. This is due to the edge effect (related to surface-to-volume ratios).  Smaller pores have a greater proportion of edge.  As molecules reach the edge they "spill over" and in effect have a shorter diffusion distance to get away from the pore.
  4. The pores are optimally spaced.  A significant boundary layer of humid air forms around leaf surfaces. This humid area reduces the rate of further transpiration. It occurs because diffusion shells from adjacent pores fuse. If the pores were much farther separated, they wouldn't form a nice boundary layer. The thickness of the boundary layer is further affected by: (1) wind speed; (2) presence of hairs; and (3) sunken chambers.
  5. The pores are optimally located.   In grasses, the stomata are distributed approximately equally on both sides of the leave whereas in herbaceous eudicots there are generally more on the underside (abaxial) than the upper (adaxial) side. In woody eudicots there are usually few stomata in the upper surface, whereas aquatic plants with floating leaves have most stomata in the upper surface. These modifications are important to minimize/control water loss.  Conifers and xeric plants often put the stoma in sunken chambers.
  6. The degree of opening/closing of the pore is closely regulated by the plant in response to the environment. In effect, any factor that can impact the rate of photosynthesis or overall water status of the plant will influence the action of the guard cells (see below).  I like our textbook description that stomata are "multisensory hydraulic valves."
  7. Test these ideas by studying the data supplied (click here for Diffusion from a standard leaf)

VIII. Mechanics of Guard Cell Action
    Guard cells open because of the osmotic entry of water into the GC. In turn, this increases the turgidity (water pressure) in the GC and causes them to elongate. The radial orientation of cellulose microfibrils prevents increase in girth. Since GC are attached at the ends and because the inner wall is thicker, the guard cells belly out with the outer wall moving more pulling open the guard cell. Guard cell closure essential involves reversing this process.  We can summarize the mechanics of GC action as follows:

stoma closed (GC flaccid)   water uptake (osmosis)  increase pressure stoma open (GC turgid)

IX. Physiology of Guard Cell Action. Part I
    Since water is the driving force for GC action, this means that there must be a gradient in water potential between the GC and the surrounding cells (subsidiary cells). Thus, to open a stoma, there must be a mechanism to generate a water potential gradient.

A.  Hypotheses for how the water potential gradient is established include:

  1. There is a rapid decrease in pressure in surrounding cells (i.e., subsidiary cells shrink which would result in the GC expanding and taking up water).  Using a fine needle transducer, Mary Edwards and Hans Meidner showed that there is some decrease in surrounding cell P, but this is not a major factor.
  2. There is an increase in the stretchability of the GC cell wall. This would result in an expansion of the GC with a concomitant uptake of water. Little evidence exists for this idea.
  3. There is a decrease in the osmotic potential in the GCMuch evidence supports this hypothesis. For example, Humble and Raschke (1971) showed that the solute potential of turgid GC (open stoma) of broad bean is -3.5 MPA but when the guard cells are flaccid (stoma closed), the osmotic potential is -1.9 MPa. Thus, the solute potential of the GC decreases (becomes more negative) when open. Since the GC volume increases, this must mean that there is an accumulation or synthesis of solute. 

Thus, we can modify our schematic diagram:

stoma closed (GC flaccid)    add solute lower Ψs  decrease Ψw water uptake (osmosis) increase pressure stoma open (GC turgid)

B.  What is the solute and where does it come from?

  1. Carbohydrates, such as sucrose
        Since guard cells are the only epidermal cells with chloroplasts, plant physiologists have long hypothesized that sucrose and related carbohydrates are osmotic regulators of guard cells. For example, the starch content of guard cell chloroplasts decreases as the stomata open. This idea, the "starch-sugar hypothesis", was the first postulated mechanism for guard cell activity. It lost popularity after the role of potassium ions was discovered, but most now agree that both sugar and potassium ions play a role in guard cell regulation. Sucrose seems to be especially important in closing guard cells.    see graph in text/class
  2.     Where does the sucrose come from? (a) hydrolysis of starch in the GC chloroplasts. In other words, an indirect product of photosynthesis (evidence: starch grains disappear during opening); or (b) a direct product of carbon fixation (photosynthesis).

  3. Malate
        Malate is an organic acid (C4).  You may already be familiar with its role in the Kreb's cycle in the mitochondria.  In plants, malate is also derived from the hydrolysis of starches. The enzyme phosophoenolpyruvate carboxylase (PEPase) binds carbon dioxide (actually bicarbonate ions) to phosphoenolpyruvate (PEP; 3-carbon atoms; an intermediate in glycolysis) to produce oxaloacetate (C4) which is then reduced to malate and stored in the vacuole.
  4. Chloride ions
        Chloride ions are transported into the cell from the apoplast via a Cl-/H+ symport in which a proton gradient is used to "drag" the chloride into the cell.
  5. Potassium ions
        This appears to be the primary osmotic agent, especially for opening the GC in the morning. The potassium comes comes from surrounding cells. Evidence: (a) if you strip epidermis from a leaf, which breaks many epidermal cells but not the more resistant GC, the GC will only open if K+ is provided in the medium; (b) potassium concentrations increase in the guard cells upon opening (see Table 1)
Table 1: Potassium in the stomatal aperture of Commelina communis



K+ (mol) in GC



K+ (mol) in epidermal cells



Thus, we can modify our original scheme:

stoma closed (GC flaccid) → sucrose/potassium/malate/chloride ions → lower Ψs  → decrease Ψw  → water uptake (osmosis) → increase pressure → stoma open (GC turgid)

To close the stoma, the reverse process occurs. However, time course studies indicate that potassium uptake is associated with opening of the stomata in the morning, but sucrose loss is more closely associated with closure in the afternoon. Thus, the final modification to our scheme:

stoma closed (GC flaccid) → potassium and chloride ion uptake, malate synthesis → lower Ψs  → decrease Ψw  → water uptake (osmosis) from subsidiary cells → pressure increases → stoma open (GC turgid) → ||||| → sucrose (potassium, chloride, malate) decreases → Ψs increases → Ψw increases → water loss → pressure decreases → stoma closed (GC flaccid)

X. Environmental Control of GC Action
    Whatever physiological mechanism we finally postulate for the GC, it must also be compatible with the action of various environmental factors that are known to regulate stomatal activity.   Since guard cells respond to their environment, especially any factors that impact the photosynthesis/transpiration compromise. We expect any factor important in photosynthesis to exert regulatory control on GC. And, we expect water to have the "final word" on control since if a plant dries out too much it's as good as dead!

A. Light - exerts strong control. In general: light = open; dark = closed (except CAM plants).

What kind of light is important?

  1. Red & blue light
        The action spectrum for the process suggests that both red and blue light are important regulators of guard cell activity and that their action is, at least partially, mediated by photosynthesis (recall red and blue light are used in photosynthesis).  Further evidence that photosynthesis is important - DCMU (an inhibitor of PS) prevents stomatal action.
  2.     So, what is photosynthesis doing? (a) provides sugars (sucrose and glucose) for osmotic regulation; (b) provides ATP (via photophosphorylation) to power ion pumps (see below); (c) reduces internal CO2 levels which stimulates opening (see below); and (d) reduces the pH in the lumen of the thylakoid that stimulates the synthesis of the blue light receptor pigment (see below).

  3. Blue light
        There is an additional effect of blue light on stomatal activity that is irrespective of its role in photosynthesis. The evidence: (a) blue light is 10x more effective than red light; (b) in saturating levels of red light, treatment with blue light will cause additional GC opening; (c) the action spectrum for blue light is similar to other "blue light responses", a "three-finger pattern"; (d) photosynthesis inhibitors block the red light effect but not the blue light effect.

What is blue light doing?

  • Blue light activates a H+-ATPase in the membrane (recall the proton pump for cell elongation?). Evidence: (a) potassium accumulates in isolated GC protoplasts treated with blue light and causes them to swell; (b) blue light causes the acidification of the medium of GC protoplasts under saturating red light conditions; (c) fusicoccin, which stimulates proton pumping, also stimulates stomatal action; (d) vanadate (VO3-, blocks the proton pump) and CCCP (carbonyl cyanide m-chlorophenylhydrazone, an ionophore that makes the membrane leaky to protons) both inhibit stomatal opening.
  • Blue light stimulates starch breakdown and malate synthesis.
  • Blue light stimulates cellular respiration (which among other things may be required to produce ATP for the proton pump).

 What is the receptor for the blue light?       

    The action spectrum for the blue light response shows a "three finger pattern," which is characteristic of other blue light responses (i.e., phototropism). Absorption spectra of potential receptor pigments show a good match between zeaxanthin, a carotenoid pigment (C40) that occurs in the chloroplast thylakoid, and the action spectrum.  Further – zeaxanthin levels are directly correlated with stomatal aperture.

How does blue light cause stomatal closure?

    Photosynthetically active radiation (red and blue light) cause an acidification of the chloroplast lumen. This activates the synthesis of zeaxanthin, which in turn, zeaxanthin activates a calcium-ATP pump in the chloroplast membrane that decreases calcium concentrations in the cytosol. This, in turn activates the proton pump in the cell membrane.

B. Low oxygen levels → GC open

C. Carbon dioxide - intracellular level is an important regulatory control.

D. pH effect

This effect seems mediated by:

1. Carbon dioxide Concentration. 
    Recall the interaction of carbon dioxide and water:

CO2 + H2O  →  CO2 (aq)  → H2CO3 (aq)  → H+ + HCO3-  →H+ + CO3-


low CO2 = hi pH = open
hi CO2 = lo pH = closed

2. H+/ATPase proton pump.
    The pump is required for stomatal opening (see above). Protons are transferred from the cytosol into the apoplast. As protons are removed from the cytosol, the pH increases. 

E. Water - protects against excessive water loss. 
    This is the prevailing and overriding control mechanism. There are two mechanisms by which water loss regulates stomatal closure, one of which is active and the other passive.

  1. Hydropassive Control - simply put, as the plant looses water, the turgidity of the leaf cells, including guard cells, decreases and this results in stomatal closure. The plant is not "intentionally" closing the stoma, it is simply a consequence of drying out.
  2. Hydroactive Control - this mechanism is one in which the plant actually seems to monitor its water status. When the water potential drops below some critical level, it engages a cascade of events that close the stomata. Presumably the plant is measuring pressure (turgor) and then synthesizes or releases an anti-transpirant that is translocated (moved) to the GC to cause closure.

    The anti-transpirant is abscisic acid (ABA), one of the major plant growth regulators. It is active in very low concentration (10-6 M) and appears very rapidly after water stress (within 7 minutes). After 4-8 hours, the [ABA] increases nearly 50x. ABA comes from two sources: (a) root – in response to water stress, the xylem sap pH increases which in turn stimulates the release of ABA into the xylem sap for transport to the leaves. This seems to be a root signal to the leaves that "water stress is coming"; and (b) leaves – water stress stimulates a synthesis of new ABA and redistribution of existing ABA.

Mechanism of Action: 
    Treatment with ABA results in decrease of potassium, chloride and malate levels in the guard cells which in turn increase the water potential resulting in water efflux.  Evidence suggests that there is an ABA receptor in the cell membrane. The receptor: (a) activates calcium channels in the membrane causing calcium uptake from the apoplast; (b) activates calcium channels in the tonoplast causing calcium release from the vacuole into the cytosol; (c) activates chloride (and malate) efflux channels; (d) inactivates potassium ion "in" channels; (e) inactivates the cell membrane proton pump; and (f) causes an increase in pH that activates potassium efflux channels. Thus, in short, ABA treatment causes an increase in cellular calcium levels which in turn results in decreases potassium and chloride levels and turns off the proton pump.

F. Temperature
    Increased temperatures usually increase stomatal action, presumably to open them for evaporative cooling. If the temperature becomes too high the stomata close due to water stress and increased CO2 that results from respiration.

G. Wind
    Often causes closure because it:  (a) brings CO2 enriched air; and  (b) increases the rate of transpiration that causes water stress which causes the stomata to close. In some cases, wind causing stomatal opening to increase transpiration for cooling.

XI. Physiology of Guard Cell Action - Part II
    Now, let's pull everything we've learned together to hypothesize a mechanism of action. First, there are a couple of other observations that we also need to reconcile with our mechanism:

  1. Starch disappears in open stomata. 
        Alkaline conditions favors the starch hydrolysis and acidic conditions favors starch synthesis. Starch hydrolysis is activated by blue light; and
  2. PEPcase activity is high in GC.
        Phosphoenolpyruvate carboxylase catalyzes the reaction: phosphoenolpyruvate (PEP) + HCO 3- → oxaloacetate (OAA).

XII. Grand Model    (see diagram;  we will discuss in class)

XIII. Why does transpiration occur?

  1. Transport in plants. This is important to a small degree. Transpiration is certainly not a necessity.
  2. Heat loss (latent heat of vaporization)
  3. Carry nutrients in the soil to the plant
  4. Perhaps plant cells need to maintain some optimal level of turgidity and this helps them do so.
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Last updated:  02/24/2009     Copyright  by SG Saupe