Heat Stress Induced
Gain of Function in LHCII
and RubisCO Activase
By Juan Casas Doza
Background
● Global temperatures have increased and will continue to
increase if we continue to
○ Burn fossil fuels
○ Participate in deforestation
○ Farming
● Crops that can’t adapt to these climate changes will
eventually die of heat stress.
● Continuous population growth
○ massive agricultural loss could mean losing the lives of millions of
people due to famine.
● It is essential scientists and researchers continue finding
solutions and enhancements in agriculture to assure a
sustainable future.
Background
● Scientists have developed chemicals to combat heat stress
○ Calcium
○ Many more
● Plants do have numerous ways of resisting heat stress
related damage on their own
○ It is important to keep in mind that excessive heat stress can damage
plant cells eventually leading to its death.
● There are two important mechanisms that plants possess
that enables them to resist heat stress related damages and
it involves both:
○ RubisCO Activase
○ LHCII (PSII)
RubisCO Activase
Activates
How did they grow the plants?
● Spinach plants were grown in the same conditions
○ After 2 months of growing the plants were put
through various experiments.
●
●
How is CO2
assimilation
affected as
temperature
increases?
●
●
The figure describes the
level of CO2 assimilation
at a given temperature.
As temperature rises,
○ CO2 assimilation
decreases
significantly.
At around 25°C
○ CO2 assimilation is
high and slightly
decreases as the
temperature
increases to 35°C.
At 50°C
○ There is almost no
CO2 assimilation.
What does
RubisCO
Activase do?
● RubisCO Activase
regulates RubisCO
○ Releasing RuBP from
the active site of
RubisCO and allowing
it to fix CO2.
RubisCO
Activase is
related to
AAA family
● A class of chaperone - like
ATPases Associated with a
variety of cellular Activities
● Motifs conserved in AAA family
of proteins were also found in
rubisco activase.
RubisCO
Activase is
sensitive to
high
temperatures
● Structural changes
● Breakdown of association of subunits of RubisCO activase
●
○ Hydrophobic parts exposed
Binds to thylakoid membrane
○ Allows the enzyme to regain ATPase activity
○ Gains a new function as a chaperone.
RubisCO
Activase
How does RubisCO Activase Help during
Heat Stress?
● Protects ribosomes associated
with thylakoids from heat
stress.
○ Ribosomes can continue
translating in order for
essential thylakoid
proteins to be made during
heat stress.
● Apparently, RubisCO Activase
was proportional to the
amount of ribosome
polypeptide complexes
RubisCO
Activase
RubisCO Activase is a Hero.
● To conclude this section, RubisCO
Activase
○ Inactivates as a RubisCO activator
○ But, functions as a chaperone for thylakoidribosome associations
■ To enable the translation machinery to
function allowing it maintain active
protein synthesis to guarantee constant
repair of PSII
Review on photosynthesis
How can low CO2 fixation effect PSII During Heat Stress?
● Excess excitation energy in PSII
○ Could damage it
● CO2 fixation decreases during heat stress
● The maximal efficiency of PSII = Fv/Fm
○ Has only a slight decrease
○ Which means there is excess
excitation energy isn’t being used by
CO2 fixation
● Thus, heat-stressed plants are potentially
exposed to excess excitation energy
What happens to LHCII during heat
stress?
It aggregates.
Find LHCII using 77K
chlorophyll
fluorescence emission
spectra
● At 685 nm = PSII and 735 = PSI
● Normalized the wavelength at 680
nm
○ Effects of heat stress on PSII can
be viewed more clearly.
● 695 nm = peak for non heated.
● 698 nm = shoulder forming
○ This is LHCII aggregation
Finding LHCII
Figure shows
● LHCII
● CP 47
● and CP45
○ Increasing
wavelength
● LHCII
Aggregation
Finding LHCII
Figure shows
● ratio
increased
with
increasing
temperature
To Confirm Emission
bands were LHCII
Aggregates
● Native green gel was ran on
chlorophyll-protein complexes in
thylakoid membrane.
● There are proteins that passed
and others that didn’t
● Heat stress aggregated proteins
in stacking gel were analyzed
further using SDS gel and
immunoblotting
○ LHCIIb that was identified.
● Figure demonstrates aggregation
of PSII protein aggregation.
To Confirm Emission
bands were LHCII
Aggregates
● Figures demonstrate how PSII core
complex and LHCII were affected by heat
stress.
● These results are the same as the last
set of figures except instead they used
isolated PSII
● SDS electrophoresis and then
immunoblotting
○ LHCIIb was detected and increased
w/ increase in temperature.
● Heat stressed LHCII was separated
through Native green gel. The bands
contain: (1) trimer; (2) monomers.
The effects of heat stress on isolated
LHCII
What is Fluorescence quenching?
●
Fluorescence quenching
○ the process of lowering
intensity of emitted
light.
■ 2 different types of
Quenching
● Non Photochemical
Fluorescence
Quenching
● Photochemical
Fluorescence
Quenching
NPQ
qP
We know LHCII aggregates, but does it do
Fluorescence Quenching (lower the Intensity of
emitting light)?
● In short
○ Yes it does
● + Antimycin A inhibits LHCII
aggregation during heat stress.
○ resulting in decreased
fluorescence quenching
● - Antimycin A doesn’t inhibit LHCII
aggregation
○ Resulting in increased
fluorescence quenching.
● This means that aggregation of
LHCII does fluorescence
quenching.
V to Z increases NPQ (Heat dissipation)
Does aggregation of LHCII
increase NPQ?
○ They wanted to test this
but there is a problem
■ V to Z
● V to Z could increase NPQ
○ Zeaxanthin molecule
interactions dissipates
energy.
● So, they used DTT to blocks
conversion of V to Z.
●
High intensity light = VDE Activation
DTT
Does low levels of Z decrease NPQ during
heat stress?
● Yes
● When DTT was present:
○ Decreased NPQ
○ But NPQ is still
present with
temperatures over
35°C
■ Must be LHCII
aggregation
DTT decreases the levels of Z
● When there was DTT present it
lowered the levels of Z to almost
0.
● Now, they know they should
add DTT so it doesn't affect
NPQ
○ Now they can test LHCII
aggregation and if it does
participate in NPQ.
Higher ratio means
more zeaxanthin
If LHCII aggregation decreases, does NPQ
decrease as well?
● They wanted to see if there was
a correlation between LHCII
aggregation and NPQ.
○ There was
● Antimycin A decreased both
LHCII aggregation and NPQ
during heat stress.
● When LHCII Aggregation
decreased, NPQ decreased
DTT in Both
Does more LHCII Aggregation mean
Higher NPQ (Heat Dissipation)?
● Yes
● There was a clear
relationship found between
the ratio of F698/F680 and
NPQ.
● These results suggest that
LHCII aggregates was
associated with NPQ caused
by heat stress in the
presence of DTT
Conclusion
● In conclusion:
○ Heat stress induced an aggregation of LHCII (Not known how it forms)
■
LHCII aggregates induced by heat stress are reversible
○ There was a linear relationship between the formation of LHCII aggregates and
NPQ.
○ Heat stress decreased CO2 fixation rate but only slightly decreased Fv/Fm.
○ The results in this study says aggregation of LHCII may be a protective
Citations
1. Rokka, A., Zhang, L. and Aro, E.-M. (2001), Rubisco activase: an enzyme with a temperature-dependent dual function?. The
Plant Journal. 25, 463-471. https://doi.org/10.1046/j.1365-313x.2001.00981.x
2. Tang, Y., Wen, X., Lu, Q., Yang, Z., Cheng, Z., & Lu, C. (2007). Heat stress induces an aggregation of the light-harvesting
complex of photosystem II in spinach plants. Plant physiology. 143(2), 629–638. https://doi.org/10.1104/pp.106.090712
3. Bita, C. E., & Gerats, T. (2013). Plant tolerance to high temperature in a changing environment: scientific fundamentals and
production of heat stress-tolerant crops. Frontiers in plant science. 4, 273. https://doi.org/10.3389/fpls.2013.00273
Questions?