Week 07 Lab Prep - Photosynthesis and Respiration PDF

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Lab 6: Photosynthesis and Cell Respiration LAB PREPARATION EXERCISES As part of your lab preparation, work through this document and answer the bolded questions in the spaces provided. You’ll submit this assignment as a pdf, with a deadline the night before your lab. It will be uploaded into a Canvas quiz.

Useful Tips Need more information? Try checking out textbook Chapters 9 (Cellular Respiration) and 10 (Photosynthesis). Reading these sections may help give some context and details if you need them.

Overview Photosynthetic organisms capture the energy of the sun into chemical energy of sugar molecules, by the process of photosynthesis. When they require energy, they can tap the stored energy in sugar by a process called cellular respiration. The process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. This process is often summarized by the following reaction: 6 H2O + 6 CO2 + light energy → C6H12O6 + 6 O2 Cellular respiration refers to the process of converting the chemical energy of organic molecules into a form immediately usable by organisms. In this lab, Cell Respiration (abbreviated as CR) here refers to complete aerobic cell respiration of glucose. Glucose may be oxidized completely if sufficient oxygen is available by the following equation: C6H12O6 + 6 O2 → 6 H2O + 6 CO2 + energy All organisms, including plants and animals, oxidize glucose for energy. Often, this energy is used to convert ADP and phosphate into ATP. Your pre-lab will focus on the major components, inputs and outputs of both photosynthesis and cellular respiration, and the similarities between the two processes. You will then play with online simulations of both processes, to get more familiar with how manipulating certain factors affects reaction rate. All SFU BISC101 lab materials © SFU course instructors, 2020. Week 7 Lab Prep

A note on being efficient for time: Parts A and B have lots of overlap with some lecture topics. I’d suggest first starting to answer the questions based on what you know already, then when stuck, reach out to other resources (course notes, textbook or video). This will save you spending time on re-reading material you already are familiar with. Ideally, you want to spend no more than 25 minutes on parts A + B total.

Part A: Exploring the process of Photosynthesis Use your own background knowledge, as well as your textbook and/or the video, to answer the questions below in your pre-lab. Video (~10 minutes) on photosynthesis: https://media.hhmi.org/biointeractive/click/photosynthesis/ Question A1: Draw a rough sketch of a chloroplast and identify the following parts: inner & outer membranes, intermembrane space, thylakoid, stroma, granum

Inputs of the Light-Dependent Reactions (LDR) Question A2: What are the major inputs required for the LDR to take place? Light Energy (Sun), H2O, ADP and NADP+ molecules Question A3: Where do the LDR take place, specifically? Thylakoids All SFU BISC101 lab materials © SFU course instructors, 2020. Week 7 Lab Prep

Electron flow through Photosystems II & I Question A4: Photosystems II & I are large proteins complexes that are key players in the electron transport chain of chloroplasts. Describe what they do using the following terms: electron, photon, excite, chlorophyll, pigment molecules Light Energy which travels in small packets of energy called Photons is absorbed by Photosystems II which excites the electrons in the chlorophyll. The excited electrons move through Cytochrome complex and then is passed to Photosystem I to release H+ ions in the complex. Question A5: Photosystem II has the special task of supplying the electrons that will flow through the ETC. Describe the process of how it obtains electrons using the following terms: water, oxygen, hydrogen ions. The Photosystem II obtains electrons when water splits into oxygen and hydrogen ions. Question A6: Electrons become excited in PSI, what molecule do they get passed on to? They get passed onto an electron carrier which carries it a molecule called NADP+ to form NADPH. Question A7: As electrons move from PSII to PSI, H+ ions (a.k.a. protons) accumulate in the lumen. Describe how these ions are used to generate ATP, using the following terms: ATP synthase, ADP, ATP, electrochemical gradient, chemiosmosis, lumen, stroma. H+ ions that are accumulated in the lumen diffuse into an enzyme called ATP synthase. The H+ ions diffuse into the stroma by ATP synthase by a process called chemiosmosis. This ATP synthase uses the potential energy of the electrochemical gradient to combine ADP with inorganic phosphate to form ATP which is released into the stroma.

Outputs of the Light-Dependent Reactions (LDR) Question A8: What are the major outputs of the LDR? ATP, NADPH and O2

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Question A9: Identify the two electron carriers used in photosynthesis, and what happens to them as electrons flow through the enzyme complexes associated with the LDR. The first electron carrier is between the PSII and Cytochrome Complex. The main use here for the electrons is to be used as an energy source to transport H+ ions into the lumen. Later, electrons are passed onto PSII to either get recycled or for join NADP+ to form NADPH.

The Calvin Cycle (a.k.a. Light-Independent Reactions, or LIR) Question A10: Where do the reactions of the LIR take place? Stroma – the watery clear fluid surrounding the thylakoids Question A11: What happens to the electron carriers as they enter the LIR? They get recycled – sent back to Light Reaction Question A12: What are the major outputs of the LIR, and what is their fate? G3P is the sugar molecule that is produced as an output from the LIR which is used as a raw material to make glucose and other organic molecules. Question A13: Is photosynthesis an anabolic or catabolic process? Anabolic processes

Part B: Exploring the process of Cellular Respiration Use your own background knowledge, as well as your textbook and/or the video, to answer the questions below in your pre-lab. Video (~6 minutes) on Cellular Respiration: https://www.youtube.com/watch?v=YHKIlTiSodk&ab_channel=McGrawHillAnimations Question B1: Draw a rough sketch of a mitochondrion and label the matrix, inner membrane, outer membrane, and the inter-membrane space -This space is intentionally left blank-

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Question B2: Identify the four main processes of cellular respiration, and identify the location (cellular compartment) where they take place Process

Major Input(s)

Major Output(s)

Location

Glycolysis

Glucose

Pyruvate, ATP and Cytoplasm NADH

Pyruvate Oxidation Pyruvate

Acetyl CoA, CO2 and NADH

Mitochondrial matrix

Kreb’s Cycle

Acetyl CoA

ATP, CO2, NADH and FADH2

Mitochondrial matrix

Oxidative Phosphorylation

NADH, FADH2

ATP

Inner membrane of mitochondria

Question B3: Identify the electron carriers involved in cellular respiration. NAD+ - Nicotinamide adenine dinucleotide and FAD – Flavin adenine dinucleotide

All SFU BISC101 lab materials © SFU course instructors, 2020. Week 7 Lab Prep

The Electron Transport Chain in Cellular Respiration Question B4: Complexes I & II have the special task of supplying the electrons that will flow through the ETC. Describe how each complex obtains electrons. NADH obtained from Glycolysis and Pyruvate Oxidation FADH2 from the Kreb’s Cycle pumps electrons to Complex II. Question B5: As electrons move from Complex I & II to Complex IV, H+ ions (a.k.a. protons) accumulate in the intermembrane space. Describe how these ions are then used to generate ATP, using the following terms: ATP synthase, ADP, ATP, electrochemical gradient, chemiosmosis, intermembrane space, matrix. During Chemiosmosis, the free energy from the series of reactions that make up ETC is used to pump H+ ions into the membrane – making up the electrochemical gradient. Hydrogen ions in the matrix space can only pass through the inner mitochondrial membrane through a membrane protein called ATP synthase. As protons move through ATP synthase, ADP is turned into ATP. The production of ATP using the process of chemiosmosis in mitochondria is called oxidative phosphorylation.

Comparison! Question B6: Identify three similarities between the light dependent reactions of photosynthesis and the ETC of cellular respiration. - In photosynthesis, ATP is produced via light energy (photophosphorylation) and used to make organic molecules. In cell respiration, ATP is produced by breaking down organic molecules (oxidative phosphorylation) - In photosynthesis, electrons are donated by chlorophyll and protons accumulate within the lumen of the thylakoid. In cell respiration, electrons are donated by hydrogen carriers and protons accumulate in the intermembrane space

Part C: Photosynthesis Simulation Let’s explore the inputs and outputs of photosynthesis in a simulation! Using online simulations can help us understand how all these components work together, and how manipulating certain variables can affect the outcomes or process in general, in a few minutes or less! All SFU BISC101 lab materials © SFU course instructors, 2020. Week 7 Lab Prep

Click the link below, which will take you to a photosynthesis simulation. The page below explains the simulation variables/options. https://sites.google.com/site/biologydarkow/photosynthesis-simulation

How to use the simulation: variables and controls explained If "In the Dark" is ON (green), then the entire experiment is without light. If "In the Dark" is OFF (grey), then the experiment will vary by the type and amount of light. If "full light" is ON (green), then it DOES NOT MATTER what the "Light Wavelength" is set to because "Full Light" = All Wavelengths. If "full light" is OFF (grey), then the experiment will receive the "Light Wavelength" only. Therefore, if you want to test a particular wavelength, turn "Light Wavelength" OFF. The “Distant from Light” (the distance the plant experimental group is from the light source) is in centimeters. The “Initial pH of Stroma” is the starting pH of the stroma in the plant experimental group. The stroma is the fluid space between the chloroplast membrane and the thylakoids. Atrazine is one of the most widely used herbicides used to control weeds in agriculture. The “Atrazine” slider controls the relative amount atrazine added to the plant experimental group. The “Light Wavelength” is the wavelength of light from the light source in nanometers. To manipulate the “Light Wavelength” the “full light” must be OFF (grey). NADPH can be synthesized during photosynthesis. “added NADPH” includes an addition of NADPH concentration (㎛/gram) to the plant experimental group at the start of the experiment. Temperature (degrees Celsius) is the environmental conditions of the experiment.

Run allows you to run the simulation once with the paramet ers set in place. If you make changes, you can hit run again to see the outcome Live shows what is happening in real time as you make changes to the settings Reset will take you back to the initial settings chosen

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The buttons on the right manipulate the reaction rates, viewed in the two boxes on the left side of your screen. The top output box presents 4 variables, graphed together: ATP, NADPH, Glucose and Oxygen concentrations. The bottom box allows you to select one variable for a clearer observation of the data. NOTE: The scales on the Y-axis change as the variables are manipulated they do not stay the same! If the graph changes shape, make sure you understand what, if anything, changed. First, play around with the settings and do a few runs to get a feel for the simulation. Then, hit “reset” to return to initial conditions before you begin answering the questions below. Question C1: There is a lot going on in this data! Take a moment to look at the graph axes and the colors, to help you interpret what the graph is showing. In the table below, Identify the cellular processes X and Y in the graph below, and give your reasoning why you made this identification.

Process within Photosynthesis

What, in the graph, helped you figure out what cellular process this is?

X

Light Dependent Reactions

Since this reaction produces ATP and NADPH – it is evident that the concentration graph is going to show a linear increase

Y

Calvin Cycle

This reaction uses ATP and NADPH which means that a decrease is to be depicted. Oxygen is a common by product hence a linear increase is always to be expected.

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Question C2: Why do NADPH and ATP levels spike up & down as time goes on? The reason why NADPH and ATP levels spike up and down is because the Calvin Cycle and Light Dependent reactions are mutually dependent which means that when ATP and NADPH is produced in the Light reactions, the Calvin Cycle is going to use the ATP and NADPH. ADP + Inorganic Phosphate and NADP+ are some of products from the Calvin cycle which are going to be used by the light reactions to produce ATP and NADPH. Run some simulations, and use these to answer the questions below: In the simulation, perform three runs of photosynthesis: with the CO2 concentration set at 0 umol, at 5 umol, and at 25 umol. Look and see what happens to the response variables’ concentrations in these runs. (CO2 concentration here is given as μmol, which is here a short-hand for “micromoles of CO2 per mole of air. Note that “umol” should technically be written as “μmol” – the Greek letter μ representing micro or 10-6 – but is often written with a regular “u” to avoid mistakes from font-conversion issues.)

Question C3: Why does a big increase or decrease of CO2 levels not change the amount of oxygen available? Because of NADPH molecules. Question C4: Why does ATP decrease, overall, as CO2 concentration rises? As CO2 concentration increases, more ATP would be required in the reduction process of Calvin Cycle to produce G3P molecules. Question C5: As CO2 concentrations increase from 0, ATP levels appear as a straight line, then they become spiked. Explain why this trend occurs. Not too sure about this one. Question C6: Let’s find out how relevant this simulation is to reality. Do a little light google/Wikipedia research (no sources/references necessary) – what is the average concentration of CO2 in earth’s atmosphere? 409.8 umol Given this, which simulation run is most relevant for a photosynthetic organism in an average environment? All SFU BISC101 lab materials © SFU course instructors, 2020. Week 7 Lab Prep

(You may find that CO2 concentration is often reported parts per million (ppm), which is numerically equivalent to umol CO2 per mole of air.)

Challenge Question (not optional, but won’t be graded for correctness): In the simulation, can you make glucose in the dark? Turn off the light source, modify the initial variables, and try to make glucose happen. Explain what settings are necessary, and why this can occur in the absence of light energy. I believe that light is a crucial for Light dependent reactions to produce ATP. That said, the Calvin Cycle can produce ADP+, Inorganic Phosphate and NADP+ for some time after the plant is taken from light to dark. That production would also eventually stop once the light reaction would stop to provide NADPH and ATP due to absence of light.

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Part D: Cellular Respiration Simulation The cellular respiration simulation uses a similar setup as the photosynthesis simulation, with some slight changes. Here we are observing the effects of 1 mole of glucose being metabolized. Follow the link below to open up the simulation. https://sites.google.com/site/biologydarkow/cellular-respiration-accounting-model Tabs along the top of the graph allow you to focus on specific variables, making changes easier to observe. Question D1: There is a lot going on in this data! Take a moment to look at the graph axes and the colours, to help you interpret what the graph is showing. In the table below, Identify the cellular processes A-D in the graph below, and give your reasoning why you made this identification.

Process A:

Process B:

Process C:

Process D:

What is the cellular process What, in the graph, helped you figure taking place here? out what cellular process this is? Initial production of NADH and ATP Glycolysis along with no increase in CO2 Pyruvate Oxidation Citric Acid Cycle

Oxidative Phosphorylation

Increase in CO2 and 2nd time increase in NADH Almost exponential increase in NADH and CO2 Decrease in NADH, Oxygen and the fluctuation in membrane potential

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Run some simulations: In the next few questions, you’ll be running a few simulations, and comparing them to understand the relationships between the inputs and outputs of cellular respiration. Question D2: Using the simulation to see how NADH affects glucose metabolism. First run the model at initial (“reset”) conditions. Notice that the amount of oxygen starts decreasing after 9 milliseconds. In the table below, first fill out the first two columns to describe what happens to the concentration of the other measured variables. Then complete “Run 2” as described in the table below, and fill in the right-hand column. Run 1: Default initial settings Run 2: Reset to initial settings, then set NADH to 40, & run. At 9 milliseconds, Looking just at Run 1, At 9 milliseconds, this concentration… explain why each this this concentration… Response (increases, doesn’t variable responds the (increases, decreases, variables: change, or decreases) way it does. or doesn’t change) ATP Constant supply from No change

Glycolysis and Kreb’s

Increases

Cycle – prior to 9 ms NADH

Consumption by Decreases

Oxidative

Decreases

Phosphorylation O2

Combines with H+ to Decreases

form H2O in Oxidative

Decreases

Phosphorylation CO2

Constant supply from No change

Pyruvate oxidation and Kreb’s Cycle – prior to

No change

9 ms Comparing Run 1 and Run 2: What changes do you see? Why does the initial presence of NADH cause these changes? NADH is required by Oxidative Phosphorylation process to produce ATP – ATP levels increase as we increase the in flow of NADH to 40 mol.

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Question D3: Using the simulation to see how intermembrane pH affects glucose metabolism. Complete runs 2 and 3, filling out the table below. Run 3: Reset, then set Run 4: Reset, then set intermembrane pH to 5, & run. intermembrane pH to 7, & run. This concentration… (increases, This concentration… (increases, Response doesn’t change, or decreases) variables: doesn’t change, or decreases) ATP Increase Decreases NADH No change No change O2 No change No change CO2 No change No change Based on your Run 3&4 simulation results, explain how the different pH values in the intermembrane space affect chemiosmosis/oxidative phosphorylation. Low pH means higher H+ ions pumping. The high external acid concentration causes an increase in H+ in the inter membrane space leading to increased ATP production by ATP synthetase. Question D4: Set the model at initial condition...