PBIO 3110 - CROP PHYSIOLOGY
Photosynthesis Laboratory - Instrumentation
Introduction
Crop physiologists rely on a wide array of highly specialized instruments for making
measurements in the field and laboratory. The number of available tools has grown
steadily with advances in the technology (e.g., low-noise infra-red gas analyzer which
allow measurement of CO2 uptake by leaves) and understanding of basic physiological
process of plants (e.g., the relationship between chlorophyll fluorescence and the light
reactions of photosynthesis). In this lab we will consider the uses and basic principles of
operation of several instruments used by crop physiologist in the field and laboratory.
Many of these instruments are complex and difficult to handle. In order to ensure more
individual attention and to allow each student to handle the instruments, the class will be
divided into 6 groups.
The following instruments will be discussed and displayed:
1) LI-185B Quantum Radiometer/Photometer
2) Walz MiniPam Chlorophyll Fluorometer
3) LI-6400 Portable Photosynthesis System
a) net photosynthesis or leaf CO2 uptake measurement
b) stomatal conductance measurement
4) LI-3100 Leaf Area Meter
5) Minolta SPAD 502 Chlorophyll Meter
6) Whole Plant Respirometer
1) LI-185B Quantum Radiometer/Photometer
What does it measure?
Photosynthetic photon flux density (PPFD). This is a "count" of the total number of
photons in the photosynthetically-active waveband (400-700 nm) incident per unit area
per unit time (µmol photons m-2 s-1). Because plants utilize all absorbed photons from
400-700 nm with equal efficiency (i.e., one photon produce one photochemical event,
regardless of the photon's wavelength), a count of the total number of incident photons
(rather than the total energy of incident photons) is the most appropriate measure for
photosynthesis studies.
How does it work?
The detector (or quantum sensor) does not actually detect individual photons. It is a
silicon photodiode which measures total incident energy in the PAR. However, the
photodiode is covered by mosaic of colored glass filter that reduce the likelihood of some
photons being absorbed by the detector, relative to the likelihood of other photons being
absorbed. Remember that photon energy is inversely proportional to photon wavelength.
Thus, a photon of red light (680 nm) has only 0.617 the energy of a photon of blue light
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(420 nm). In an ideal quantum sensor, then, the likelihood of a photon of red light
reaching the photodiode will be 1/0.617 (i.e. 1.62) times the likelihood of a blue photon
reaching the photodiode. In other words, the design of the quantum sensor uses a
difference in absorptance to compensate for the difference in photon energy, thus creating
the illusion of counting individual photons (Fig.1).
How to take measurements?
Place the detector head at the top of the plant canopy and hold in a horizontal position.
Maintain the detector head as level as possible as the angle of the detector affect the
proportion of direct and diffuse radiation being received by the photodiode. When taking
measurement of the growth cabinet make sure that the reading are taken at the comers as
well as the centre of the cabinet in order to determine whether radiation levels are
constant throughout the cabinet.
A point quantum sensor (left) and a line quantum sensor (right)
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2) Walz MiniPAM Chlorophyll Fluorometer
What does it measure?
This instrument measures a number of parameters related to the light reactions of
photosynthesis. We will concentrate on a single parameter, the chlorophyll fluorescence
"efficiency ratio", Fv/Fm. This ratio is essentially equivalent to the fraction of absorbed
photons that are used to drive the light reactions of photosynthesis, as opposed to being
wasted. In "dark-adapted" measurements the sample of leaf tissue is left in the dark for
10-15 minutes in order to "open" all the reaction centers. Therefore, dark-adapted
measurements represent the maximum possible efficiency of the light reactions of
photosynthesis (i.e., the efficiency is lower for an illuminated sample). In an illuminated
reading it is possible to determine the actual efficiency of the light reactions under natural
conditions, as opposed to the maximum possible efficiency in a dark-adapted sample.
How does it work?
A pulse of light is delivered to the sample via a fiber optic probe. This pulse of light
initially induces a very low level of chlorophyll fluorescence, Of, since most of the
photons are being used to drive photochemistry, and therefore are not re-released as
fluorescence. As the pulse of light continues, the leaf became light saturated, and none of
the additional photons applied can be used photochemically. Instead, many photons are
re-released from the leaf as chlorophyll fluorescence, causing the fluorescence signal to
rise to its maximum, Fm. Variable fluorescence, Fv, is equal to Fm-Fo, and the
chlorophyll fluorescence efficiency ratio is calculated as:
Fv/Fm = (Fm-Fo) / Fm
In a healthy, dark-adapted sample, this ratio will be equal to about 0.84. If chloroplast
components that carry out the light reactions of photosynthesis have been affected by
environmental stress, the ratio will fall due to an increase in Fo, a decrease in Fm, or
both. The MiniPAM employs a Pulse Amplitude Modulation (PAM) system that allows
the detector to distinguish the small amount of red light, originating from chlorophyll
fluorescence, from the much larger amount of "background" red light, which is reflected
from the leaf surface when the leaf is illuminated.
Knowing the efficiency with which absorbed photons are being used to drive the light
reactions of photosynthesis (or, in this case, the reaction of Photosystem II), it is possible
to calculate the rate of electron transport (ETR) in the thylakoid membrane as:
ETR (electrons m-2 s-1) = Fv/Fm x PPFD x a x 0.5;
where a is the absorptance of incident PPFD by the leaf, and 0.5 is a factor which
assumes equal distribution of absorbed photons between Photosystem II and Photosystem
How to take measurements?
Attach the clamp to the second youngest fully expanded leaf (use the centre trifoliate in
soybean). After 10 minutes, take the photosynthetic efficiency measurement (Fv/Fm). By
using the Leaf clip-Holder on an illuminated sample, determine the actual efficiency of
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the light reactions at different values of PPFD. Take several measurements on each of the
two plants provided and report mean values for each of plants assigned to your group.
Walz MiniPAM Chlorophyll Fluorometer
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3a) LI-6400 Portable Photosynthesis System – Net photosynthesis measurement
What does it measure?
The LI-6400 will provide a measure of leaf net CO2 uptake (i.e., net photosynthesis) as
well as a measurement of stomatal conductance. In this section we will discuss the
measurement of net photosynthesis.
How does it work?
To make a measurement, a known area of the sample leaf is enclosed in a chamber where
air is pumped in a continuous, closed circuit through an infrared gas analyzer (IRGA),
where the current CO2 concentration of the air is measured. As the photosynthesizing leaf
draws CO2 Out of the circulating air, the drop in CO2 concentration is detected by the
IRGA. Since the total volume of the circulating air is known, the drop in concentration
can be used to calculate the total molar amount Of CO2 taken up by the leaf sample. This
can then be expressed as net photosynthetic rate (i.e., µmol CO2 m-2 s-1). Typically, the
drop in CO2 is monitored for about 30 s per measurement, and several measurements are
taken in succession per sample. During the measurement, a portion of the circulating air
can be diverted through a desiccant, so the leaf transpiration does not lead to a large
increase in RH within the chamber.
How to take measurements?
Place the leaf chamber over the second youngest fully expanded leaf. In soybeans use the
middle trifoliate leaf of the second youngest fully expanded leaf. Take several
measurements per plant for each of the plants provided and report mean values for each
of the two plants assigned to your group.
LI-6400 Portable Photosynthesis System
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3b) LI-6400 Portable Photosynthesis System – Stomatal conductance measurement
What does it measure?
Stomatal conductance. Transpiration from leaves is driven by the difference in vapour
pressure inside and outside the leaf. However, plants can regulate transpiration rates by
altering the stomatal aperture, which changes the resistance to diffusion of water vapour
from the substomatal cavity to the air surrounding the leaf. This resistance (and its
inverse, the stomatal conductance) can be quantified according to diffusion theory. In the
simplest possible treatment:
E = (VPleaf - VPair) / (P(rstom + rbl))
where:
E is the transpiration rate (mmol H2O m-2 s-1)
VPleaf is the vapour pressure inside the leaf (kPa)
VPair is the vapour pressure of the ambient air (kPa)
P is the barometric pressure (kPa)
rstom is the stomatal resistance (mmol-1 m-2 s-1)
rbl is the boundary layer resistance (mmol-1 m-2 s-1)
How does it work?
The equation is solved for rstom:
rstom = (VPleaf - VPair) /(E x P) - rbl
To make a measurement, a known area of the sample leaf is allowed to equilibrate with
the air in a small chamber. As the leaf transpires, the instrument automatically replaces
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some of the air in the chamber with dry air in order to keep the chamber relative humidity
(RH) constant. E can then be calculated from the flow rate of dry air required to keep the
RH stable. For the remaining variables in the above equation, vpleaf is a function of leaf
temperature and barometric pressure, VPair is a function of chamber temperature, chamber
RH and barometric pressure P entered by the user, and rbl is a constant dependent of the
chamber design
How to take measurements?
This measurement is made automatically as the photosynthetic measurement is made.
Take several measurements (simultaneously with the photosynthetic measurements) on
each of the two plants provided and report mean values for each of the two plants
assigned to your group.
4) LI-3100 Leaf Area Meter
What does it measure?
Detached leaves can be passed through the meter in order to rapidly and accurately
determine their area.
How does it work?
Leaves fed into the meter are held flat between two moving, transparent belts. As they are
carried along, they pass under a fluorescent lamp, causing some of the lamp's light to be
intercepted. A scanning camera determines the fraction of light intercepted, which is
proportional to the sample width. The speed at which the samples are moving under the
lamp (the conveyor speed) is known, and can be combined with the information from the
scanning camera to calculate the area of the leaf sample.
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How to take the measurements?
Remove the leaf blades from all the plants provide to your group. In soybeans all three
trifoliolates are used but the petioles are left on the stem. Pass all the leaf blades through
the transparent belts. The number on the display should be divided by the number of
plants that have been used for the measurement to get the average leaf area per plant.
LI-3100 Leaf Area Meter
5) Minolta SPAD 502 Chlorophyll Meter
What does it measure?
This meter measures leaf chlorophyll content. Leaf chlorophyll levels can be affected by
a variety of environmental and developmental factors. Crop physiologists might wish to
determine leaf chlorophyll contents when studying, for instance, the effects of soil
nitrogen of leaf greening, or the effects of temperature on leaf senescence. Unfortunately,
traditional methods of measuring leaf chlorophyll are destructive (require leaf sampling)
and time-consuming (grinding on samples in liquid nitrogen, solvent extraction of
pigments, spectrophotometric determination of pigments). The SPAD 502 chlorophyll
meter provides a very rapid, non-destructive measure of leaf chlorophyll content.
How does it work?
The SPAD 502 determines the transmittance of light through the sample leaf at two
different wavelengths: 650 nm, which is strongly absorbed by chlorophyll, and 920 nm,
which is not. By then comparing the transmittance at these two wavelengths, the
instrument calculates a value, X, which is linearly related to the leaf chlorophyll content:
Chl=a + m X
Unfortunately, the values of a and m vary with each individual SPAD 502 unit, and also
with each different plant species. Therefore, determining actual chlorophyll content (i.e.,
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µmol chlorophyll per m2 of leaf) with the SPAD 502 requires an initial calibration of the
machine against chlorophyll concentrations determined by solvent extraction of leaf
pigments. Nonetheless, the SPAD reading alone provide a reliable relative measure of
leaf chlorophyll. Also, it has recently been shown that SPAD reading can be used to
accurately estimate the absorptance of PAR by leaves (Fig. 3).
How to take measurements?
Ensure that the "eye" of the instrument is placed over the leaf blade and does not include
any large veins including the midrib. Take four measurements on the second youngest
fully expanded leaf of each plant and obtain an average value for all plants tested by your
group. Record the average value on the data sheet provided.
Minolta SPAD 502 Chlorophyll Meter
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6) Whole Plant Respirometer
What does it measure?
Respiration (CO2 release) of plant shoot, root, or whole plant.
How does it work?
In this system, you need to know the concept of molar volume. This is determined
according to the universal gas law:
PV = nRT
(1)
Where: P = pressure (kPa), V = volume (L), n = number of mols of gas (mol), R = the
universal gas constant (8.314 kPa L mol-1 K-1), and T is the absolute temperature (K). P
and T are measured by a pressure sensor and thermocouple, respectively.
Rearranging the formula (1),
n = PV / (RT)
(2)
So for example, for the large 117-L chamber, if T is 293 K and P is 100 kPa,
n = (100 kPa x 117 L) / (8.314 kPa L mol-1 K-1 x 293 K) = 4.80 mol
When a sample is in the chamber, the radial fans at either end stir the air so that it is well
mixed. As the sample respires, the CO2 concentration in the chamber increases. A pump
circulates a small fraction of the chamber air through an IRGA so that the [CO2] increase
can be measured. And then the slope of the [CO2] increase is determined by regression.
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Since ppm = µmol mol , units for the slope are µmol CO2 mol air s . The respiration
rate of the sample is then determined by multiplying the molar volume of the system by
the slope of the CO2 increase. In our example:
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Respiration rate = 4.80 mol air x 0.1549 µmol CO2 mol air
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= 0.744 µmol CO2 s .
How to take measurements?
Cut above-ground part of each plant and put it into a chamber and close it. Then you can
start to record data through a computer program. Data are usually collected for about 3
minutes, and then using collected data to determine the slope of the [CO2] increase
through regression. The last step is to calculate respiration rate by multiplying the molar
volume of the system and the slope of the CO2 increase. Report mean values for each of
the two plants measured in your group.
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Whole Plant Respirometer
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