LSM2231 – General Physiology

Lecture 1

Physiology: science of the function of living systems which includes how organisms, organ systems, organs, cells and biomolecules carry out the chemical or physical functions that exist in a living system.

Life began on earth ~3.5 to 4 billion years in a unicellular, anaerobic form where archean microbes oxidize dissolved iron in ocean water to generate energy

Evidence as hematite in rock. Initial form of photosynthesis was anaerobic as there was no free oxygen in the

atmosphere. Almost 99.99% of the oxygen was locked up in rocks and minerals. Earliest photosynthesizing microbes did not release oxygen and use hopane as

their metabolite. When photosynthesis evolved and oxygen was released (cyanobacteria), the oxidizing

atmosphere caused a massive extinction of species. Archaea moved to the depths of the oceans to avoid oxygen.

When oxygen concentration increased slowly, aerobic life evolved further and flourished while many anaerobic organisms went extinct.

Oxygen allowed for more efficient harnessing of energy through respiration which allowed for multicellular animals to evolve.

UV radiation from the sun reacted with oxygen in the stratosphere and formed ozone which filters out harmful UV light – this helped to spread life out from the deep oceans.

Bioenergetics: energy flow through living systems through different cellular processes (respiration, photosynthesis etc) which result in the production and utilization of energy in forms such as ATP.

Basic properties of energy (3 laws of thermodynamics): Energy can neither be created nor destroyed, but it can be converted from one

form to another. In any transfer of energy, there is always a loss of useful energy to the system,

usually as heat. Processes that proceed spontaneously result in increased randomness and

disorder unless energy is added into the system. Living things are divided into two groups:

Photosynthetic organisms (plants, algae, bacteria) – Autotrophs, take up CO2 and release O2

Non-photosynthetic organisms – Heterotrophs (with the exception of chemoautotrophs), take up O2 and release CO2

Light has both a wave and particle character and each photon contains an amount of light energy that is called a quantum. Energy is delivered in discrete packets called quanta.

Energy of a particle of light is inversely related to the wavelength of light. Short wavelength = high energy, long wavelength = low energy.

Energy flow: sun is the ultimate source of energy for life on earth. Solar energy reaching Earth’s surface 4.3 1020 J/hr Solar energy consumed on the planet 4.1 1020 J/yr

LSM2231 – General Physiology

Why did plants evolved to use visible light but not other parts of the electromagnetic spectrum?

Earth’s atmosphere is relatively opaque at most wavelengths, only ‘visual window’ and ‘radio window’ allows light and radio waves to reach the surface.

Cells of leaves have chloroplasts (1 to >200 per cell) which are membrane-bound vesicles containing chlorophylls.

Chlorophylls are located on thylakoid membranes in the chloroplasts which produce ATP and NADPH.

ATP and NADPH are cofactors which are necessary for the Calvin Cycle which occurs in the stroma (which contains all the enzymes necessary for the cycle).

LSM2231 – General Physiology

Plants are primary producers, using elements to synthesize thousands of compounds through photosynthesis.

Natural compounds include: carbohydrates, amino acids, lipids, nucleotides Energy is stored as chemical bonds (ATP provides energy when it transfers

phosphate moieties to more strongly bonded glucose or fructose phosphates). Plant structure: above-ground (stem, leaves, flowers and fruits) and under-ground

(roots) system. Node: point of leaf attachment on a stem Internode: portion of stem between two adjacent nodes

Photosynthesis is aided by enzyme RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) which uses the redox reaction to reduce carbon atoms of CO2

and synthesize sugars. There were at least 5 different pathways of photosynthesis, but the plants today appears

to be the combination of two ancient pathways – Photosystem I and Photosystem II. Without photosynthetic oxygen evolution, atmosphere would have taken a far longer

time to accumulate oxygen to appreciable levels due to splitting of H2O via UV radiation. First land plants (475mya) were lacking root systems and had no leaves, by 400mya

vascular plants evolved and had roots. Evolution of first tiny leaves occurred by 360mya in ferns/horsetails.

Leaves vary in shape and size but most are green. It is the major site of photosynthesis and transpiration.

Simple Leaf Compound Leaf with leafletsCross section of leaf:

LSM2231 – General Physiology

Chloroplast has 3 membrane system:

Envelope: Outer membrane Inner membrane

Thylakoid membrane, thylakoid has stacked membrane regions called the grana (granum plural).

The matrix of the chloroplast is called the stroma which contains the enzymes necessary for the Calvin cycle.

Photosynthesis has two major phases:

Phase 1: light reactions Phase 2: Calvin cycle

LSM2231 – General Physiology

Lecture 2 Photosynthesis phase 1: light reactions of photosynthesis where light energy is converted to

chemical energy. Each photosystem is composed of several hundred chlorophyll and carotenoid

molecules that make up a light harvesting ‘antenna’ along with a number of associated proteins.

When light strikes a pigment molecule, the resonance energy is transferred and funnelled into a reaction centre.

Only at the reaction centre (photosystem II) can the energy be used to split water.

Radiative transitions of energy: Excitation Transfer (Resonance Energy Transfer): Transfer of energy to a nearby

unexcited molecule with similar electronic properties due to overlapping of molecular orbital energy levels (energy transfer to neighbouring chlorophyll molecules)

Fluorescence: Photons emitted when an electronically excited molecule decays to a lower state of the same multiplicity.

Phosphorescence: same as fluorescence but transition is between states of different multiplicity, lifetime is longer.

All chlorophylls have: Lipid-soluble hydrocarbon tail, flat hydrophilic head with Mg ion at its centre and

the tail and head is linked by an ester bond. Chlorophyll a and Chlorophyll b differs in the CH3 and CHO group which changes the ability

of light absorption. Having both chlorophyll a and b and carotenoids can allow for multiple absorption of light,

maximizing the light intake of plants. Chlorophylls a and b absorb blue and red light while carotenoids absorb blue and some green – 400 to 700nm of visible light.

Note that carotenoids are not as efficiently mobilized to capture light as chlorophyll.

LSM2231 – General Physiology

When plants get too much sunlight, the chlorophyll molecules gets oxidized and start to die off, resulting in the leaves turning orange/brown which are the colours of the pigments (carotenoids).

Non-cyclic photosynthetic electron transport chain: Two cooperating photosystems (PS) work together Absorbed light energy is transferred to electrons and are captures by electron

carriers PS II and PS I act in conjunction and results in the ‘Z’-scheme of electron flow. PS II facilitates generation of ATP via splitting of water while PS I facilitates

generation of NADPH. The splitting of water at PS II (photolysis) maintains the flow of electrons

through both photosystems and O2 is released.

Pq: Plastoquinone, Pc: Plastocyanin, Fd: Ferredoxin Plastoquinone is a mobile organic molecule while ferredoxin (Fe-S) and plastocyanin (Cu2+)

are two mobile proteins on the membrane of the chloroplast stroma. Plastoquinone functions to transfer the protons across the membrane.

LSM2231 – General Physiology

PS I can also function in a cyclic fashion where instead of reducing NADP, the electrons can be passed back to P700 to generate ATP via cyclic photophosphorylation

The electron transport complex are not uniformly distributed where PS II are in the grana stacks while PS I & ATPase complex are along the edges of grana and stromal thylakoids.

Despite segregation, the complexes are linked with each other through mobile carriers.

The energy released when electrons move from one carrier to another is used to transport H+ from stroma into thylakoid interior. H+ is unable to leak through the thylakoid membranes and can only be transported out via ATP synthase in ATP production.

This process is known as chemiosmosis (ATP synthesis in chloroplast) Phase 2 of photosynthesis is the Calvin cycle where CO2 is fixed and reduced to form CH2O

(sugars) For every 6 moles of CO2 that enters the cycle, 1 six-carbon sugar is produced. ATP and NADPH used up during the cycle comes from the light reaction Glucose formed can be converted into other sugars and eventually stored as starch. RUBISCO (Ribulose Bisphosphate Carboxylase/Oxygenase) is the most abundant

protein on earth which contributes to 50% of the soluble protein in leaves. 8 active sites where CO2 can be fixed, the active site are arranged around an Mg ion. RUBISCO is a slow and steady enzyme, plants compensate by making lots of enzyme.

LSM2231 – General Physiology

RUBP is regenerated and used in the next cycle.

Lecture 3 Auto-fluorescence of chlorophyll can be monitored via carbon-mapping satellite and this

fluorescence is the energy that cannot be absorbed by the plants. Photosynthesis levels change over the months where it is the lowest in winter and highest in

summer. This can be correlated with the actual CO2 level in the atmosphere where it peaks in winter and drops during summer.

Variations of photosynthesis: C3 pathway (Rubisco enzyme), C4 pathway (PEP Carboxylase) and CAM pathway (PEP Carboxylase).

C3 plants: Rice, Rose, Apple, Tomato (~85% of all plant species) C4 plants: Corn, Sugarcane, Sorghum CAM plants: Pineapple, Cactus, Orchid

C4 plants have a special “Kranz anatomy” which is a layer of mesophyll cells surrounding the bundle sheath cells around the vascular bundle which prevents O2 from diffusing in.

CO2 is fixed to PEP to form oxaloacetic acid (4 carbons) and then to malic acid in the mesophyll cell. The malic acid (with fixed carbon) is then transported into the bundle sheath cells which is then decarboxylated and releases CO2 for Calvin cycle to take place.

Note that PEP fixation can also happen in the cytoplasm other than the chloroplast.

LSM2231 – General Physiology

The malic acid is then converted into pyruvic acid and moves back into the mesophyll cell.

Lack of oxygen in the bundle sheath cells removes the oxygenase function (photorespiration) of the Rubisco and increases the production of sugar.

The chloroplasts in the mesophyll cells can take up PGA transported from the bundle sheath cells & can synthesis triose phosphate. Note that CO2 cannot be fixed in the mesophyll cell chloroplasts.

The chloroplasts in the bundle sheath cells lack granal stacks of the thylakoid membrane and thus are deficient in PSII – results in low O2 evolution rate. However they have high amounts of PSI which can support a high rate of cyclic electron flow.

Overall rate of photosynthesis in C4 plants are higher than that in C3 plants as photorespiration is negligible in C4 plants. This results in higher average crop yield.

Note that the change in CO2 concentrations would significantly affect the yield of C3 plants but not for C4 plants.

CAM plants grow in deserts and climates with poor water availability conditions – they experience periods of water stress and need to keep their stomata closed in the day to conserve water. Since CO2 cannot be taken in during the day, the carbon fixation can only take place at night.

LSM2231 – General Physiology

At night, the CO2 is fixed by PEP carboxylase and converted into oxaloacetic acid and then to malic acid and is stored in the vacuole – this is to prevent the pH in the cytoplasm from dropping.

During the day, the malic acid is decarboxylated into CO2 and the Calvin cycle can take place, however the extent is limited by the amount of malic acid stored in the vacuole.

Up to 90% of the cell volume in CAM plant cells are the vacuole – succulent cells Note that CO2 can be taken in by the plant and be fixed by C3/C4/CAM modes, but sugar

production only takes place when CO2 is assimilated by the Calvin cycle (C3) CO2 fixation efficiency: CAM<C3<C4

Plants use the energy from photosynthesis for cellular respiration via Glycolysis and Krebs cycle: C6H12O6+6O2→6C O2+6H2O+36 A TP

Glycolysis occurs in the cytoplasm and forms a net 2ATP + 2NADH. PEP generated in Glycolysis is used for the fixation of CO2 in C4/CAM plants.

Krebs cycle takes place in the mitochondria where pyruvate (3 carbons) from glycolysis is transported into the mitochondria and converted into Acetyl-CoA (2 carbons) which gets fixed to oxaloacetate (4 carbons) to form Citrate (6 carbons).

All the CO2 released in respiration comes from the Krebs cycle: pyruvate (3C) acetyl-CoA (2C), citrate (6C) -KG (5C), -KG (5C) Succinate (4C).

Overall Reaction: 8NADH + 2 FADH + 2 ATP + 6CO2

NADH and FADH2 are deprotonated releasing H+ into the intermembrane space and electrons are passed along carriers in the ETC. The final electrons are accepted by O2 to form water and the energy released drives the formation of ATP.

In mitochondria, H+ accumulates in the intermembrane space during electron transport and H+ is translocated back to the matrix via ATP synthase. In the chloroplast, H+ translocated into the thylakoid compartment (lumen) during the light reaction, and also back to the stroma via ATP synthase during respiration.

Lecture 4 Photorespiration in C3 plants is also called the photosynthetic carbon oxidation (PCO) cycle.

It is a competing reaction for Rubisco where the oxygenase part of the enzyme is used. When Rubisco reacts with O2 and RuBP, one 3-PGA molecule and one phosphoglycolate (2C)

molecules is formed.

LSM2231 – General Physiology

Phosphoglycolate is metabolised by the C2 glycolate pathway which releases CO 2 and forms 3-PGA. Production of CO2 is due to the oxygenase activity of Rubisco in the light and results in the formation of phosphoglycolate.

Photorespiration is negligible in C4 plants as there is limited oxygenase activity of Rubisco in C4 plants.

The C2 glycolate cycle (photorespiration) involves three organelles – chloroplast, peroxisome and mitochondria.

Photorespiration is a competing reaction with the first step of photosynthetic carbon reduction with a ratio of carboxylation to oxygenation = 3:1.

Factors such as increased temperature and decreased CO2 concentrations favour oxygenation.

Physiological benefit to this pathway: PCO scavenges the glycolate, returning it to the carbon pool Pathway may contribute the amino acids glycine and serine to metabolism Pathway may help to minimize photo-oxidative damage

Result of photorespiration is the loss of CO2 from the plant and thus is a “wasteful” process as it leads to reduced crop yield.

Limits on photosynthesis: will the rate of photosynthesis continue to increase if we keep shining brighter light on the plants – No.

Enzymes in respiration has a slightly higher temperature tolerance and thus the optimum rate is at a higher temperature – due to mitochondrial exothermic reactions the enzymes has evolved to operate at a slightly higher temperature.

LSM2231 – General Physiology

With global warming, photosynthetic rate would decrease. Annual O2 output by photosynthesis is about 300 billion tons, annually about 20% of photosynthetic biomass is consumed by animals.

There are some creatures including jellyfish, flatworms, bivalve molluscs and salamanders also make use of photosynthesis due to a symbiotic relationship with photosynthetic algae.

Plants are indispensable to human society and they have many direct and indirect effects on individuals, society, ecosystem, health and the economy (trillion of dollars per year)

Plants provide most of the calories and protein for the human diet as they tend to store excess nutrients in the following form:

Corms are thickened and compressed stems that grow underground (e.g. Water chestnut)

Bulbs are highly compressed underground stems to which numerous storage leaves are attached to form a compact structure (e.g. onions)

Stem tuber: enlarged fleshy tips of underground stems. (e.g. potato, tapioca) Taproots (e.g. carrots)

A quarter of all prescription drugs in Western society contain plant-derived ingredients while 80% of the world’s population still relies mainly on herbal medicine.

Timber/Wood is another important plant product.

LSM2231 – General Physiology

Due to the cell being very crowded, there is high resistance for movement of contents and thus plants have xylem and phloem to transport water/minerals from the soil to the leaves and sugars from the leaves to the rest of the plant.

Water is “v-shaped” and polar and thus is a super solvent and can pull apart many molecules.

Water is the most common constituent in cells and takes up 80-95% weight in most plant cells and even up to 5% in dry evens.

Water is necessary for transpiration (dissipation of heat), maintaining the structure of proteins and nucleic acids, and is necessary for uptake, distribution and transport of nutrients.

Xylem is a complex tissue made up of tracheids, vessel elements and parenchyma cells. Tracheids and vessel elements are the actual conducting tubes which are dead at

maturity but have highly thickened cell walls with holes called pits which allow for lateral transfer of water.

Phloem is also a complex tissue made up of sieve tube, companion, fibres and parenchyma cells.

Sieve tube cell has no nucleus and gets the nucleus function from the companion cell.

Plasmodesmata establish connections from cell to cell and permit the passage of materials. Plasmodesmata goes through the cell wall and thus able to connect the two cells

together. Transport can occur via bulk flow, diffusion and selective uptake.

LSM2231 – General Physiology

The transport of water occurs by the energetically passive processes of diffusion and bulk flow.

Bulk flow is driven by pressure difference while diffusion is driven by the concentration difference of water.

Diffusion is the random movement of individual molecules along a concentration gradient (high to low) and results in an equal distribution of molecules at the end.

Osmosis is a special kind of diffusion which involves the movement of water across a selectively permeable membrane (restrict the movement of dissolved solutes)

Passive movement of water or other substances across a membrane is dictated by chemical potential () of that substance on each side of the membrane.

Chemical potential is the free energy per mole of substance and is a measure of the capacity of a substance to move

Pure water has a chemical potential of zero and the addition of solutes decreases the mole fraction of water and thus the chemical potential decreases.

For two sides of a membrane with different chemical potential, water will move passively from the higher chemical potential to the lower chemical potential.

Since water only moves passively, plants control the movement of water indirectly by transporting ions to later the w.

Ion transport to regulate water movement may require ions to be moved against their gradient (active transport) – osmotic adjustment.

Ions can be actively transported into the cells and this establishes a gradient of lower chemical potential of water inside than that of outside and this allows water to diffuse passively into the cell.

Water potential: Ψ=ΨP+Ψ S where Ψ P is the pressure potential and Ψ S is the

solute/osmotic potential. Pressure potential is the pressure exerted on the cell by the cell wall. Water flows from more positive value to more negative values. Units are in Pascals and are relevant to the soil-plant-atmosphere system.

When plant cells gain water, they exert an outward pressure called turgor pressure (>0) The amount of water vacuole results in the cell being turgid or flaccid. With turgor pressure against the call, there is an equal and opposite wall pressure. Plasmolysis is the condition where the protoplast has lost water and the plasma

membrane has retracted from the cell wall.

Lecture 5

LSM2231 – General Physiology

In a hypotonic solution, the cell would gain water as Ψcell is more negative than the solution while in a hypertonic solution, the cell would lose water and undergo plasmolysis because Ψcell is more positive than the solution.

Note that a cell can only come into equilibrium with its surrounding environment. The flow of water from one cell to another depends on the water potential where the water

potential decreases as the cell gets further and further away from the xylem.

Water can enter cells either directly through the lipid bilayer, or through special membrane channel proteins called porins.

Porins are non-selective cation channels found in the plasma membrane and tonoplast (membrane bounding the vacuole) and mediate the transport of water and neutral solutes like glycerol.

Plant aquaporins are intrinsic proteins found in the plasma membrane (PIP) and the tonoplast membrane (TIP)

Each aquaporin channel is formed from four protein subunits, and each subunit has 6 membrane spanning regions. The four subunits together form membrane spanning gated channels that allow water flow at higher rate than across the lipid bilayer.

Although water can diffuse directly through the cell membrane between the phospholipid molecules, aquaporins provide a more rapid rate of water movement and thus make a greater contribution to osmoregulation.

Aquaporins are gated channels and can be selectively opened and closed, gating is controlled by dephosphorylation (close during drought - S115 & S274) or protonation (close during flooding – H193).

LSM2231 – General Physiology

Water movement through plants relies on an integrated connection of water from the soil through the roots, stem and leaves and then into the atmosphere.

In the soil, the size of the soil particles determines the characteristics of the soil such as the retention of water

40-60% of soil volume is composed o large pores and capillary pores. When the pore space is filled with water, the soil is at field capacity.

The water potential of the soil and the roots determines water movement where root uptake of water occurs only if the water potential in the root cells is more negative than the water potential in the soil.

The soil water percentage at which the root is unable to obtain water corresponds to the permanent wilting percentage. The water content between field capacity and permanent wilting percentage is available water.

The roots need to be extensive in length and surface area in order to provide for the following functions:

Anchoring the plant in the soil Storing carbohydrates and other molecules Synthesis of some hormones and secondary metabolites Uptake and transport of water and minerals

Root hairs are present to increase surface area for absorption of water and nutrients and water is taken up primarily near the tips.

Uptake of water occurs along other root sections as a function of age, physiological condition and water status.

The bulk of water is taken up along the root hair zone. Root hairs are outgrowths of individual epidermal cells and have small diameters

(200m) to allow for root hairs to explore smaller pore spaces in the soil. The cell wall of root cells has a waxy thickening called the ‘casparian strip’ on the radian

surface which prevents water movement. Thus water and minerals must pass through the endodermal cells (symplastic) and

not between them (apoplastic) before entering the stele (xylem+phloem). Cell surfaces in contact with the cortex and stele does not have the casparian strip.

Building the casparian strips requires the construction of extracellular lignin structures that encircle cells within the cell wall and that are anchored to the plasma membranes of adjacent cells to form tight seals between them.

Bulk of the water moves between the cells in the roots initially along

the porous cell walls until it reaches the endodermis surrounding the central vascular tissue of the roots.

Water moves radially across the diameter of the root to the stele by two pathways: The apoplast represents the continuity of the cell wall space outside of the cells The symplast represents the connection of cellular cytoplasm between cells

provided by the plasmodesmata. Water will move along the path of least resistance – the cell wall space outside of

the cells (apoplast)

LSM2231 – General Physiology

The radial movement of water in roots creates a lag between uptake and transpiration If transpiration exceeds root uptake of water, there may be a midday closure of the

stomata while the balance is restored.

As atmospheric pressure can only raise a column of water only about 10m, the xylem would require other mechanisms to transport water and they include:

Root pressure: is a positive pressure at the root level that pushes water up the xylem.

Root pressure is generated when mineral ions are taken up, thus decreasing the water potential and inducing water uptake.

The water then moves radially to the endodermis which has the casparian band preventing the backflow of water to the cortex.

The continual influx of water causes a positive pressure to develop and forces water up the step in the xylem.

Guttation is when water oozes out at the tips of intact leaves of herbaceous plants when the soil and atmosphere are saturated with water.

Water secretion occurs through modified stomata called hydathodes (tips of veins) during guttation and root pressure provides the motive force for this process.

LSM2231 – General Physiology

Colocasia plants can lose from 10-100ml of water per day. Capillarity occurs because water shows adhesion to solid surfaces. And this

contributes to the rise of sap in the xylem Surface tension of water also contributes to capillary rise. For the radius of a typical xylem element, capillarity would raise the water

column < 1m. Cohesion/transpiration pull – currently accepted hypothesis describing sap ascent.

Water evaporation (transpiration) from leaves is the driving force which creates a tension in the water column.

Since water is cohesive, the tensile strength that develops pulls the continuous network of water up from the soil to leaves.

Plants lose about 95% of water absorbed through transpiration. When stomata are open, both transpiration and photosynthesis occur as H2O

diffuse out and CO2 diffuse in. When guard cells are turgid (take in water via osmosis), stomata are open.

When guard cells are flaccid (lose water), stomata are closed. This cohesive pull will be exerted on the whole water column in the plant

down to the roots. Translocation of sugar via pressure flow hypothesis:

Products of photosynthesis move from source to sink via phloem. At the source (leaves), sugar molecules are loaded into phloem with the help of

companion cells. As sugar concentration increases, water moves in from adjacent xylem and pressure builds up, forcing sugar solution through the plant to the sink (fruit/roots).