PLANT PHYSIOLOGY
Enquiry Class project:
Measurement of the Light Dependence of Photosynthesis:
Light response curves, photochemical efficiency and light
compensation points of C3 and C4 Plants
Photosynthesis is the most important energy-capturing process in both aquatic and terrestrial
ecosystems. As the name suggests, photosynthesis is a process by which light is converted to chemical
energy and is used to synthesize compounds within the organism. The green pigment chlorophyll is the
central light-absorbing pigment that makes this energy capture possible. Chlorophyll, along with
various other accessory pigments, is located in chloroplasts within the cells of the green portions of a
plant.
The leaves are the main organs of the plant housing chloroplasts and they are very well designed to
capture the maximum amount of light, as you will see when you examine their structure in detail. The
light energy captured by the chloroplast pigments is converted into chemical energy in the form of ATP
and NADPH2. These compounds can then be used to convert CO2 molecules into sugar molecules,
which are used to feed the plant and us. In the overall process of photosynthesis CO2 molecules are
absorbed by the leaf and O2 molecules are released as a waste product of the process, as seen in the
equation below:
6 CO2 + 12 H2O
C6H12O6 + 6 O2 + 6 H2O
Thus the rate of photosynthesis can be determined by measuring the rate of either O2 production or CO2
uptake.
The purpose of this experiment is to demonstrate that leaves produce O2 during photosynthesis
and to measure photosynthetic rate by measuring the rate at which O2 is evolved from a leaf at various
light intensities or wavelengths. In addition the results will show that the rate of photosynthesis
increases with light intensity until a light saturation point is reached. At that point, photosynthetic rate
is limited either by the ability of the leaf to convert the light energy it absorbs to chemical energy, or by
the supply of some other factor required for photosynthesis, such as CO2.
Materials
Leaves of a C3 and C 4 species of higher plant
Qubit Systems photosynthesis laboratory package with oxygen sensor
Plant Physiology Laboratory Exercise:
L a b 2 : Mineral Nutrition in Tomato Plants
Introduction:
At least 17 mineral elements are considered essential for plant growth. Plants acquire the
majority of these elements from the soil solution by absorption through the roots. Elements may also
enter plants via the stomata in gaseous states. Elements entering plants as gases may not only include
carbon as CO2, but also sulfur as gaseous SO2 and nitrogen as gaseous NO2 or NH3. Other elements
such as chlorine may also enter the plant via the atmospheric route carried on dust particles or water
droplets. In the case of essential micronutrients (those elements required in relatively small amounts; <
0.1 mg/g dry wt tissue), the atmospheric pathway could possibly supply most if not all of a plant's
requirements for that element. The required macronutrients (those elements required in relatively large
amounts; usually considerably more than 1 mg/g dry wt of tissue), except for carbon and oxygen,
typically cannot be supplied in sufficient quantity through the atmosphere to meet a plant's
requirements for those elements.
Low levels or the absence of a particular essential element will result in specific deficiency
symptoms in a given plant species. These deficiency symptoms can be useful for determining what
role(s) these elements play in the physiology of the plant. Colors, organ or tissue shapes, or excreted
products are examples of symptoms that may be clues about these essential roles. Whether the
deficiency symptoms first appear on the youngest or oldest portions of the plant also can provide clues
about the mobility and thus the identity of the element. Symptoms that first appear on older portions of
the plant such as fully expanded leaves are indicative of a mobile element. Plants can mobilize some
elements that are in short supply and transport them from older parts to actively growing (younger)
regions of the plant. Deficiency symptoms that first appear in younger regions of the plant are
indicative of non-mobile elements. A plant cannot remobilize these elements when they are in short
supply and therefore requires a constant supply of these elements to actively growing regions of the
plant.
Deficiency symptoms can best be studied using a water culture system (hydroponics), where
the presence or absence of essential mineral elements can be controlled precisely. In addition,
hydroponics allows the experimenter to observe deficiency symptoms that occur on the roots, which
cannot be observed easily in soil-grown plants. This experiment is designed to demonstrate the
deficiency symptoms caused by the deletion of specific macro- or micro-nutrients from the hydroponic
solution.
Students will also be given a chance to exercise their diagnostic skills by identifying nutrient
deficiencies in plants given to them as unknowns.
Materials
You will use 4-6 week old tomato plants (Lycopersicon esculentum) for all tests.
TOMATO (SOLANUM LYCOPERSICUM): A MODEL FRUIT-BEARING CROP
INTRODUCTION
Tomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It
originated in western South America, and domestication is thought to have occurred in Central
America. Because of its importance as food, tomato has been bred to improve productivity, fruit
quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as
food, but also as research material. The tomato plant has many interesting features such as fleshy
fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and
Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied
using other model plant systems. There are 13 recognized wild tomato species that display a
great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes
are important for breeding, as sources of desirable traits, and for evolutionary studies. Current
progress on the tomato genome sequencing project has generated useful information to help in
the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae
and is closely related to many commercially important plants such as potato, eggplant, peppers,
tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily
applied to these plants, which makes tomato important research material. Because of these facts,
tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited
plants.
PLANTING TOMATOES:
Tomato seed germinates fairly quickly, within 5-10 days. The plants also develop fast, so seeds
can be started 6-8 weeks before you intend to transplant outdoors.
Keep in mind that the warmer your seedlings are kept and the more light they are given, the
faster they will grow. You don't want them to get so large indoors that you have to keep moving
them to larger p
Preparing the Pots for Planting
Dampen the Potting Soil: It's easier to dampen the potting mix before you put it the containers.
Add some water and work it through. Keep adding water until the mix stays compressed in your
hand, but is not dripping wet. It should break apart when you poke it with your finger.
Fill the Pots: Fill your containers and gently firm the soil so that it is about an inch from the top.
Use your label to make a 1/4 inch furrow in the planting mix. Sprinkle 2–3 seeds into the furrow
and cover them with a sprinkling of potting mix. Gently firm the mix down, so the seeds make
good contact with the soil. You can spray the surface with water, if it doesn’t feel moist enough.
Be Patient: At this point, you should place your containers somewhere warm and check them
daily to make sure the soil is moist - not wet - and watch for germination. Putting containers
inside a plastic bag can create a mini greenhouse. Remove the bag when the seedlings emerge.
Caring for seedling
Once your tomato seedling has true leaves, it's time to start feeding it. Any good liquid fertilizer
can be used once a week. Dilute it to half the label recommended dose.
Light is critical now. Keep your tomato seedlings close to your grow lights and rotate the plants
if they seem to be growing or leaning I one direction.
Tomato stems grow sturdier if they are tossed about by the wind. You can simulate this indoors
by putting a fan on your plants for an hour a day or simply running your hand across them each
time you pass them.
When the tomato seedlings are 2--3 inches tall and have a couple of sets of true leaves, it's time
to pot them up or move them into larger pots of their own.
Potting Up Tomato Seedlings
You should transplant individual tomato seedlings into bigger pots, to continue growing stronger
indoors. Three to four inch containers are good for seedlings this size. You may need to move
them to larger pots later, if you can't move them outdoors.
Fill the new pots with moist potting mix, just as you did when you started the seeds. If more than
1 seed germinated in your containers, you will need to thin them. Either gently jiggle entangled
roots apart or simply snips off unwanted seedlings at soil level. This ensures that you won't
damage the seedling you want to keep.
Plant the tomato seedling in the new pot, a little deeper than it was in its original container. If it
is tall and leggy, you can plant it right up to its top most leaves. Firm the soil gently around the
seedling.
Materials:
- Walz Imaging-PAM M-Series MINI Chlorophyll Fluorescence System
Background:
Chlorophyll Fluorescence is a method used to determine photosynthetic activity. Chlorophyll
Fluorescence utilizes many parameters to obtain a conclusion regarding the rate of
photosynthetic activity in a plant. A few observable parameters that are useful for analysis
include, but are not limited to Electron Transport Rate, maximal Fluorescence yield, and
Effective Photosystem II yield. The Walz Imaging PAM MINI provides all this information in an
effective way.
Light Curve Analysis
Light Curve Analysis is done by exposing a plant leaf to increasing light intensities over a short
interval of time (20 seconds) and instant light-response curves are recorded. This method can be
criticized because photosynthesis will most likely not be in steady state, but instant light curves
can nevertheless provide reliable information about pivotal points of photosynthesis and can
provide essential details regarding the intrinsic properties of the plant leaves being measured.
The Light curve responses that we obtained were performed using the Walz Imaging-PAM MSeries MINI system. The leaf was exposed to increasing intensities of actinic light over a 2
minute interval in 6 steps, where each step followed each other after 20 seconds. The range of
actinic light was measured based on Photosynthetically Active Radiation (PAR) values that
ranged from 0µmol m⁻² s⁻¹ to 231µmol m⁻² s⁻¹. The Imaging system provides clear information
regarding the fluorescence yield parameters and Electron Transport Rate under the different
lighting conditions.
Electron Transport Rate
There is a positive linear relationship between the Electron Transport Rate (ETR) in Photosystem
II and the net photosynthetic rate of a plant leaf. The parameter ETR has been shown to correlate
well with linear electron flow calculated on the basis of O2 evolution rates (Flexas et al. 1999).
As we are well aware of the important role that the electron transport chain plays in the process
of photosynthesis in plants, the knowledge of the ETR under different lighting conditions can
help us understand the photosynthetic capacity resident in a plant leaf.
Maximal Fluorescence Yield
Chlorophyll fluorescence can be defined as the red to far-red light emitted by photosynthetic
tissues/organisms when illuminated by light of approximately 400–700 nm (photosynthetically
active radiation or PAR) (Kalaji.et al. 2016). Actinic Light at different intensities is flashed on a
leaf and the chlorophyll in the leaf supplies a light emission in response. Analysis of the
maximum amount emitted under the given conditions is useful in providing knowledge of the
growth rate capacity resident in the leaves at the time that the light curve analysis was
administered.
Effective Photosystem II Yield
Photosystem II (PSII) is a specialized protein complex that uses light energy to drive the transfer
of electrons from water to plastoquinone, resulting in the production of oxygen and the release of
reduced plastoquinone into the photosynthetic membrane. The key components of the PSII
complex include a peripheral antenna system that employs chlorophyll and other pigment
molecules to absorb light, a reaction centre at the core of the complex that is the site of the initial
electron transfer reactions, an Mn4OxCa cluster that catalyses water oxidation and a binding
pocket for the reduction of plastoquinone. PSII is the sole source of oxygen production in all
oxygenic photosynthetic organisms, which include plants, algae and cyanobacteria. In these
organisms, PSII operates in series with other protein complexes, including the PSI reaction
centre, to produce the reduced form of nicotenamide–adenine dinucleotide phosphate (NADPH)
and adenosine triphosphate (ATP), which is used in the Calvin–Benson cycle to produce
carbohydrates from carbon dioxide. The sensitivity of PSII activity to abiotic and biotic factors
has made this parameter an observable and essential one for understanding the photosynthetic
mechanisms in a plant. The yield recorded is the operating efficiency of PSII photochemistry,
Fq′/Fm′ (Genty et al., 1989), is calculated from (Fm′–F′)/Fm′ (and does not require a darkadapted measurement). This parameter is often termed ϕPSII or ΔF/Fm′ (Maxwell and Johnson,
2000; Baker, 2008). It gives the proportion of absorbed light that is actually used in PSII
photochemistry (Genty et al., 1992) and can therefore be used to estimate the rate of electron
transport through PSII with knowledge of light absorptance by the leaf and photosystems.
Procedure:
1.
2.
3.
4.
Select a leaf from around the middle of the stem
Attach to the Imaging-PAM system with the leaf holder.
Utilizing the computer software, run Light Curve Analysis.
Repeat for the remaining plant species/treatments.
Data:
PAR vs ETR
PAR:
MJ 0.1
MJ 0.25
0
2
32
80
137
231
MJ 0.5
0.0
0.4
2.0
0.0
0.0
0.0
0.5
0.5
5.2
12.5
5.6
0.0
MJ 0.75
Unknown
0.1
Unknown
0.25
Unknown
0.5
Unknown
0.75
SA 0.1
SA 0.25
SA 0.5
SA 0.75
References:
1. Flexas J, Escalona JM, Medrano H. Water stress induces different levels of
photosynthesis and electron transport rate regulation in grapevines. Plant Cell Environ.
1999;22:39–48. doi: 10.1046/j.1365-3040.1999.00371.x.
2. Kalaji HM, Schansker G, Brestic M, et al. Frequently asked questions about chlorophyll
fluorescence, the sequel [published correction appears in Photosynth Res. 2017
Apr;132(1):67-68]. Photosynth Res. 2016;132(1):13–66. doi:10.1007/s11120-016-0318-y
3. Genty B Goulas Y Dimon B Peltier G Briantais JM Moya I . 1992. Modulation of
efficiency of primary conversion in leaves, mechanisms involved at PS2. In: Murata N,
ed. Research in photosynthesis, Vol. IV: Proceedings of IXth International Congress on
Photosynthesis . Nagoya, JapanAugust 30–September 4, 603–610.
4. Maxwell K Johnson GN . 2000. Chlorophyll fluorescence—a practical guide. Journal of
Experimental Botany 51, 659–668.
5. Baker NR . 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual
Review of Plant Biology 59, 89–113.
6. Genty B Goulas Y Dimon B Peltier G Briantais JM Moya I . 1992. Modulation of
efficiency of primary conversion in leaves, mechanisms involved at PS2. In: Murata N,
ed. Research in photosynthesis, Vol. IV: Proceedings of IXth International Congress on
Photosynthesis . Nagoya, JapanAugust 30–September 4, 603–610.
Soaking
Before the tomato seeds were planted in soil they first soaked in their respective elicitor for 24
hours. A total of 192 tomato Meyer seeds were soaked in petri dishes, 16 seeds soaked in MJ 0.1,
16 seeds soaked in MJ 0.25, 16 seeds soaked in MJ 0.50 and 16 seeds soaked MJ 0.75. This same
process was done for Salicylic acid and the unknown elicitor with the same varying
concentration of 0.1, 0.25, 0.50 and 0.75.
Planting February 19, 2019
After the seeds were soaked for 24 hours, twelve 12inch planting pot were then labeled as MJ
0.1, MJ 0.25, MJ 0.50, MJ 0.75, SA 0.1, SA 0.25, SA 0.50, SA 0.75, Unk 0.1, Unk 0.25, Unk
0.50 and Ukn 0.75; the numbers correspond to the concentration. The 12in pots were then filled
with planting soil then thoroughly soaked with water for 10-15 seconds. Four holes were then
made in the soil of pot along the and with equal space; roughly 1/4-1/2 inch deep. The soaked
seeds were place onto a paper towel to soak the excess liquid, then planted into their respective
labeled pots and left in the Bowie State University greenhouse with controlled environment.
Maintenance 8 weeks
Two weeks after planting treatment began, 1000L of each elicitor was poured into their
respective pot and respective concentration. A 500ml graduated cylinder was used for treatment
and were thoroughly rinsed out after each treatment; this was done twice a week until April 30th,
2019.
Photosynthetic Activity Analysis
After the 10-week experiment of varying elicitor treatments the plant were then analyzed for
photosynthetic activity with the Walz Imaging-Pam M-Series Mini Chlorophyll Fluorescence
System. This system will be used to provide quantitative analysis for the Electron Transport
Rate, maximum Fluorescent yield and effective Photosystem II yield. A leaf was selected
(remained attached) from all twelve of the treated plants, then clamped to the Imaging-PAM
system, the leaf was then scanned with Light Curve Analysis for 2 minutes and varying
parameter. All data collect was recorded and is presented in the tables and figures below.
Result:
Table 1 Photosynthetically Active Radiation vs Electron Transport Rates
PAR:
0
2
32
80
MJ [0.10]
0.0
0.6
4.4
5.1
MJ [0.25]
0.0
0.6
1.0
0.0
MJ [0.50]
0.0
0.7
3.9
0.0
MJ [0.75]
0.0
0.6
2.2
0.0
Unknown
0.5
0.5
3.1
0.0
[0.10]
Unknown
0.0
0.6
3.6
0.0
[0.25]
Unknown
0.0
0.7
4.5
2.1
[0.50]
Unknown
0.3
0.4
1.4
0.0
[0.75]
SA [0.10]
0.4
0.4
1.2
0.0
SA [0.25]
0.5
0.5
3.4
0.0
137
0.0
0.0
0.0
0.0
0.0
231
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
SA [0.50]
SA [0.75]
0.5
0.3
0.5
0.4
2.9
1.4
0.0
0.0
Table 2 Photosynthetically Active Radiation vs Photosystem II
PAR:
0
2
32
80
MJ [0.10]
0.472
0.493
0.318
0.149
MJ [0.25]
0.537
0.483
0.076
0.0
MJ [0.50]
0.543
0.536
0.278
0.0
MJ [0.75]
0.486
0.470
0.162
0.0
Unknown
0.383
0.378
0.221
0.0
[0.10]
Unknown
0.494
0.485
0.257
0.0
[0.25]
Unknown
0.599
0.548
0.322
0.061
[0.50]
Unknown
0.450
0.475
0.096
0.0
[0.75]
SA [0.10]
0.305
0.305
0.087
0.0
SA [0.25]
0.426
0.428
0.244
0.0
SA [0.50]
0.392
0.379
0.211
0.0
SA [0.75]
0.258
0.278
0.103
0.0
Table 3 Photosynthetically Active Radiation vs Fluorescent Yield
PAR:
0
2
32
80
MJ [0.10]
0.1755
0.1711
0.1265
0.0824
MJ [0.25]
0.2828
0.2618
0.1554
0.1353
MJ [0.50]
0.4824
0.4750
0.3172
0.2446
MJ [0.75]
0.6142
0.6029
0.3510
0.2804
Unknown
0.6574
0.6525
0.4892
0.4343
[0.10]
Unknown
0.4544
0.4475
0.3765
0.2980
[0.25]
Unknown
0.4515
0.4426
0.2907
0.2255
[0.50]
Unknown
0.5618
0.5510
0.4456
0.3520
[0.75]
SA [0.10]
0.6574
0.6471
0.4490
0.4034
SA [0.25]
0.5426
0.5377
0.4475
0.3917
SA [0.50]
0.5176
0.5265
0.4407
0.3299
SA [0.75]
0.4912
0.4887
0.4377
0.3314
0.0
0.0
0.0
0.0
137
0.0
0.0
0.0
0.0
0.0
231
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
137
0.0721
0.1265
0.2206
0.2569
0.3858
231
0.0691
0.1191
0.1931
0.2431
0.3211
0.2680
0.2328
0.2064
0.1936
0.3069
0.2701
0.3539
0.3515
0.2843
0.3059
0.3142
0.3206
0.2613
0.3279
Table 4. Final Height of Tomato Plants after 8 weeks of elicitor treatment
Elicitor
Final Height
(cm)
MJ [0.10]
MJ [0.25]
MJ [0.50]
MJ [0.75]
Unknown
[0.10]
Unknown
[0.25]
Unknown
[0.50]
Unknown
[0.75]
SA [0.10]
SA [0.25]
SA [0.50]
SA [0.75]
55.88
66.04
94.06
91.77
91.44
101.6
81.28
76.2
83.82
93.98
92.71
88.9
Figure 1. Graph of Maximum ETR in varying concentration of MJ, SA and Unknown
Photosynthetically Active Radiation vs Maxima Electron
Transport Rates
6
5
4
[0.10]
3
[0.25]
[0.50]
2
[0.75]
1
0
MJ
SA
Unknown
Fig 1. In this graph the maximum ETR were plotted for each concentration for the three elicitors
during the imaging of PAM in the two-minute period.
Figure 2. Graph of Maximum Photosystem II in varying concentration of MJ, SA and Unknown
Photosynthetically Active Radiation vs Photosystem II
0.7
0.6
0.5
[0.10]
0.4
[0.25]
0.3
[0.50]
0.2
[0.75]
0.1
0
MJ
SA
Unknown
Fig 2. In this graph the maximum Photosystem II were plotted for each concentration for the
three elicitors during the imaging of PAM in the two-minute period.
Figure 3. Graph of Maximum Fluorescent Yields in varying concentration of MJ, SA and
Unknown
Photosynthetically Active Radiation vs Fluorescent Yield
0.7
0.6
0.5
0.1
0.4
0.25
0.3
0.5
0.2
0.75
0.1
0
MJ
SA
Unknown
Fig 3. In this graph the maximum Fluorescent Yields were plotted for each concentration for the
three elicitors during the imaging of PAM in the two-minute period.
Figure 4. Final Growth Measurement of Tomato Plants at 10 Weeks
Final Growth Measurements of Varying Ellicitor Treatments
120
Length in Centimeters
100
80
0.1
0.25
60
0.5
0.75
40
20
0
MJ
SA
Unknown
Fig 4. These are the measurement of the tallest plants in each of the experimental pots which
were taken April 30th, 2019
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