Photoprotection and photoinhibition

Question 1

Photoprotection (qE and qT) & Photoinhibition (ql)

Answer 1

photoprotection qE:
Energy-dependent quenching: The xanthophyll cycle

Photoprotective qT:
State transition: Conformational changes in LHCII

Photoinhibition qI:
Light-induced reduction in quantum yield as a consequence of damage

^all are non-photochemical quenching qE and qT manage electron energy qI results in damage

Question 2

Photoprotection (qE):
Damage avoidance and repair: Acclimations to light stress

Answer 2

At low light intensities, light intensity is the dominant factor limiting photosynthesis

At high light intensities, all photosystems are maxed out and excess light can damage photosynthetic machinery

The metabolic and physiological state of the plant or cell determines how much light is “too much”

Question 3

Photoprotection (qE):
Excess excitation energy can lead to photo-oxidative damage

Answer 3

Photon-excited chlorophyll forms excited singlet chlorophyll 1Chl*

1Chl* can return to its ground state through:
- Photochemistry
- Fluorescence
- Dissipation (e.g., heat, NPQ)

Alternatively, it can convert to the excited triplet state 3Chl* which can transfer energy to oxygen to produce singlet oxygen with subsequent damage due to reactive oxygen species

Question 4

Photoprotection (qE):
There are protective strategies to avoid high-light induced damage

Answer 4

Some pigments absorb light for photosynthesis and some reflect it – lutein and zeaxanthin are two reflective pigments

Antenna complexes:
Decrease light incidence through dynamic changes to antenna complex
Release excess energy as heat or fluorescence

lutein and zeaxanthin:
Detoxify reactive oxygen side-products of excess excitation energy (e.g., antioxidant production)

A plant defective in energy dissipation is susceptible to high-light induced bleaching and death

Question 5

Photoprotection (qE):
Excess light energy is dissipated via non-photochemical quenching

Answer 5

“Non-photochemical quenching”
encompasses several components:

qE = Energy-dependent quenching: The xanthophyll cycle
qT = State transition: Conformational changes in LHCII
qI = Photoinhhibition: Light-induced reduction in quantum yield as a consequence of damage

Question 6

Photoprotection (qE):
Energy-dependent quenching (qE) usually is dominant form of NPQ

Answer 6

(see diagram: note: Lumen acidification in this diagram refers to lumen over-acidification)

Unquenched:
High efficiency transfer of light energy to PSII reaction center

Energy-dependent quenching:
1. Lumen acidification activates Violaxanthin De-epoxidase
2. VDE converts violaxanthin to zeaxanthin, leading to light energy dissipation in LHCII

Question 7

Photoprotection (qE):
Xanthophyll cycle: reversible interconversion of carotenoids

Answer 7

High light / low luminal pH induces VDE, which catalyzes the conversion of violaxanthin to zeaxanthin
In low light / high luminal pH, the reaction is reversed by ZE

Xanthophyll = generic name for these three pigments
( viola/anthera/zeax-anthin)

These pigments span the thylakoid membrane

Violoxanthin epoxidation reduces delocalised electron density in the molecule preventing it from receiving electrons

But when the lumen is over acidified de-epoxidase activates violaxanthin converting it to antheraxanthin able to accept electrons

We don’t fully understand what zeaxanthin does

Question 8

Photoprotection (qE):
Zeaxanthin promotes structural changes & heat dissipation

Answer 8

Zeaxanthin accumulation (due to VDE activation) causes a rearrangement of LHCII and RCII, which decreases light transfer to RCII

The structural changes cause more light energy to be dissipated as heat

Chl = chlorophyll; Car S1 – carotenoid singlet excited state
We lack full understanding of zeaxanthin function
Excitonic = excited photon

Question 9

Photoprotection (qE):
Zeaxanthin and lutein also have roles as antioxidants and in photoprotection

Answer 9

Chlamydomonas mutants deficient in zeaxanthin and lutein production are more susceptible to photo-oxidation

Interestingly, these two xanthophyll pigments (obtained from dietary sources) also protect human eyes from phototoxic damage by accumulating in the macula (orange color)

see diagram based on green algae in notes

comparatively:
- Brown seaweeds occur in the intertidal zone able to dessicate and rehydrate twice a day following the tides, they have additional pigments that allow this
- Red algae also possess some of these pigments

Question 10

Photoprotection (qT):
The redox state of PQ pool contributes to state transitions

Answer 10

• When PSII, PSI and downstream metabolism are balanced, the PQ pool is distributed between PQ (oxidized) and PQH2 (reduced
• High light (or light that favors PSII) or conditions that decrease downstream metabolism lead to an over-reduction of the PQ pool

State transitions are triggered by the second change overreduction of the plastoquinone pool
If light levels are ok but nutrients are limiting then to slow down metabolism you need to reduce reduction in the plastoquinone pool

Question 11

Photoprotection (qT):
Reduced PQH2 activates LHCII kinase and promotes state transition

Answer 11

• Accumulation of PQH2 activates LHCII kinase
• LHCII kinase phosphorylates LHCII. Some LHCII relocates to PSI

Uncoupling the light harvesting mechanism from the
CEF =cyclic electron flux supercomplex

LHCII phosphorylation also prevents light energy from being passed to PSII

Does state transfer involve a transfer between photosystems or just an uncoupling?
Do some of the light harvesting aspects move from PS2 to PS1?

A current model indicates that state transitions balance PSII and PSI mainly by quenching LHCII energy transfer to PSII

• Argument for uncoupling thus slowing down photosystem 2 function
• Probably both happen: activating and moving/ uncoupling – this is ongoing work

Question 12

Photoinhibition (qI):
is caused by light damage to PSII

Answer 12

(see diagram)
The D1 protein of PSII is susceptible to photodamage, and when its rate of damage exceeds the rate of repair, photosynthesis is inhibited.

D1 is the part that holds the reactive chlorophyll centres which is the reason why protein D1 gets oxidative damage
If too much proteolytic damage occurs the plant detects this and breaks it down, then rebuild it
Hence an increase in photosynthetic components can be observed under stress

Slide diagram shows removing damaged D1 breaking it down and resynthesising it, however when coupled with nutrient stress (aka metabolic stress) regeneration may not be necessary

As usually plants are under polystress e.g. drought and high light levels are often in combination, ROS therefore inhibit the resynthesis signals for D1
Demonstration of evolution of use of ROS for signalling triggering cellular responses for protection

Tips of roots have higher ROS used as an endogenous signal using ROS as a developmental signal BECAUSE plants have had to evolve stress response to these chemicals they have adapted to utilise this for signalling

Question 13

Photoinhibition (qI):
Damage and repair of PSII are stress and environmentally sensitive

Answer 13

An area currently under much discussion/ research FTsH proteose particularly
Current work involves creating mutants

ROS inhibits translation of D1

Protein homeostasis in the thylakoid membranes is dependent on protein quality control mechanisms, which are necessary to remove photodamaged and misfolded proteins.

An ATP-dependent zinc metalloprotease, FtsH, is the major thylakoid membrane protease. FtsH proteases in the thylakoid membranes ofArabidopsis thalianaform a hetero-hexameric complex consisting of four FtsH subunits, which are divided into two types: type A (FtsH1 and FtsH5) and type B (FtsH2 and FtsH8). An increasing number of studies have identified the critical roles of FtsH in the biogenesis of thylakoid membranes and quality control in the photosystem II repair cycle.

Furthermore, the involvement of FtsH proteolysis in a singlet oxygen- and EXECUTER1-dependent retrograde signaling mechanism has been suggested recently.

FtsH is also involved in the degradation and assembly of several protein complexes in the photosynthetic electron-transport pathways.

Question 14

Photoinhibition (qI):
Metabolic demand for NADPH and ATP feed back into light harvesting

Answer 14

see diagram:
When supply > demand, elevated NADPH & ATP levels feed back and induce photoprotection (red arrows)

Metabolic imbalances, drought, cold, pathogen infection and other factors can decrease flux through the Calvin-Benson cycle

Question 15

Photoinhibition (qI):
Timescales of high light responses

Answer 15

see slide diagram + consider circadian rhythm

Question 16

Photoinhibition (qI): Arabidopsis NPQ and role of PsbS

Answer 16

See mutant screening studies e.g.
Li et al (2000). A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403:391-395.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that

exceeds a plant’s capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of

photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the

balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the

plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have

isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding

PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient

light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding

that has implications for the functional evolution of pigment-binding proteins.

See: Correa-Galvis et al. (2016). PsbS interactions involved in the activation of energy dissipation in Arabidopsis. Nature Plants 4:15225.

PsbS removal does not affect Photosynth under normal conditions but does affect stress response

Has a glutamate molecule making its pH ~4.1 similar to that of the lumen – so can act as a pH sensitive switch – so when overacidification occurs the glutamate becomes protonated resulting in a confirmational change uncoupling the photosynthetic systems

Question 17

Photoinhibition (qI): Algal photoprotection

Answer 17

Linear electron transport (LEF) starts with the light-induced oxidation of water catalysed by the oxygen-evolving complex (OEC) of PSII followed by electron transfer from PSII through the plastoquinone (PQ) pool to cytb6f, which oxidizes plastoquinol (PQH2) thereby reducing the soluble electron carrier plastocyanin (PC).

This so-called Q cycle pumps protons from the stromal to the luminal side of the thylakoid membrane. PSI acts as light-driven plastocyanin ferredoxin (Fd) oxidoreductase and reduced ferredoxin is re-oxidized by another oxidoreductase (FNR) which converts NADP+ to NADPH.

The energy needed for charge separation in PSII and water splitting is provided via light absorption in the peripheral antenna of PSII (LHCBM) and transfer of excitation energy (EET) to the special pair chlorophyll in the PSII reaction centre (P680).

In the cyclic electron flow (CEF) mode, electrons are recycled from either reduced Fd or NADPH to plastoquinone, and subsequently to the cytochrome b6f complex.

The Fd-dependent pathway requires the proteins PGR5 and PGRL1 while the NADPH-dependent pathway uses the enzyme NDA2 to re-inject electrons from NADPH into the PQ pool.

Like linear electron flow, the cyclic mode is coupled to proton translocation and formation of a pH gradient that drives ATP production via ATP synthase, but in contrast to LEF, the operation of CEF does not result in NADPH accumulation.

The ATP and NADPH generated by the photosynthetic light reactions is consumed by the Calvin–Benson cycle to produce carbohydrate from fixed CO2.

Oversaturation of photosynthetic electron transport at saturating light intensities when the rates of ATP/NADPH supply exceed their consumption by the Calvin–Benson cycle. Together with the accumulation of NADPH, kinetic bottlenecks such as slow PQH2 oxidation by the cytb6f complex cause an over reduction of the electron transport chain.

In this overexcited state of PSII a large fraction of absorbed light is re-emitted as fluorescence and singlet oxygen production (not shown) is increased. The reduced backflow of protons into the stroma caused by ATP accumulation leads to an acidification of the thylakoid lumen.

This activates energy-dependent quenching (qE) as one of the major photoprotective mechanisms by protonation of LHCSR3 and subsequent conformational changes in the antenna. In this quenched state, the yield of chlorophyll-excited states and energy transfer to the reaction centre are reduced, because light energy is mainly dissipated as heat in the antenna and not used for photochemistry. This prevents singlet oxygen formation but lowers photosynthetic efficiency.

Question 18

Photoinhibition (qI):
Heat, drought, & other stresses affect photosynthetic efficiency

Answer 18

• High temperatures increase membrane permeability and decrease proton-motive force
• Drought-induced stomatal closure prevents CO2 uptake
• Nutrient deficiency or toxicity affects electron transport
• Cold temperature slows enzyme-catalyzed reactions

Under polystress there is a lot of flexibility that we are still exploring

What stress may crops be exposed to?

Which parts of these super-systems should be focussed on, edited etc.

Question 19

Summary of photosynthetic acclimation mechanisms

Answer 19

(see diagram)

Although the photosynthetic machinery is susceptible to photo-inhibitory damage, it also has several strategies for photoprotection