Hacking photosynthesis

Hacking Photosynthesis
Sacrificing the photo-respiratory process to realize global food security
DOCTORAL SEMINAR II SUDERSHAN MISHRA ID - 51063

What are we going to talk about?
• The most important aspect of our survival; which is why it
happens to be a global concern- “FOOD”
• The process that is at the heart of careers and lives of all of us
sitting in the room in particular and the world in general-
“PHOTOSYNTHESIS”
• The protein which happens to be the most abundant protein
on earth, singularly responsible for entry of more than 99% of
inorganic carbon in the living system – “RUBISCO”
2

Overview
• Scale of yield enhancement required
• Theoretical framework for analyzing yield
• Is there scope for yield enhancement?
• Can improvement in photosynthesis help?
• Potential RUE of crops
• Opportunities for Hacking Photosynthesis
• Enabling technologies
• Brief discussion about each of the opportunities including the
latest research idea
• In-depth discussion about the photorespiratory bypass
• Conclusion
• Future perspectives
3

Scale of yield enhancement required to
realize global food security
Current Levels Future (2050) Levels Options at hand
Population stands at 7.7
Billion
Population will be 9.4
Billion
• Increase the land area
under agriculture
• Find alternate sources
of food
• Increase the
proportion of light
energy harvested by
crops
• Increase the harvest
index
• Increase the
photosynthetic
efficiency
Current production level
has stagnated after rapid
increase in 1960s and is
increasing annually at a
rate of 1.2 percent; this
rate is decreasing every
year
2.2 percent increase in
yield is required annually
i.e. 83 to 91% yield
enhancement is required
Overall agricultural
resources are shrinking
by 0.95% annually
22-28% decrease in
agricultural resources
compared to current
levels would have
occurred by 2050
Walker et.al., 2016
4

Theoretical framework for analyzing yield
increase
• Yp = η·Pn; Pn = St·εi·εc/k
• Yp = Yield potential
• η = harvest index/ efficiency with which biomass is
partitioned into harvestable product
• Pn = primary productivity
• St = Annual integral of incident solar radiation (MJ m-2)
• εi = efficiency with which radiation is intercepted by the crop
• εc = efficiency with which intercepted radiation is converted
into biomass
• k = energy content of plant mass 5
Long et.al., 2018

Theoretical framework for analyzing yield
increase Contd..
• Yp is therefore determined by the combined product of
three efficiencies, each describing broad physiological
and architectural properties of the crop
• εi
• εc
• η
• εi is determined by the speed of canopy development
and closure, canopy longevity, size and architecture.
• εc is determined by the combined photosynthetic rate of
all leaves within the canopy, less crop respiratory losses.
6
Long et.al., 2018

Is there any scope for yield enhancement?
• Over the past 50 years, increase in Yp has been successfully achieved
largely through increase in η. Grain in the modern cultivars of
cereals can represent 60% of the total above-ground biomass at
harvest. While some opportunities for further increase in η remain,
it seems unlikely that a η much greater than 0.6 may be realized
• Increased Yp also results from increased εi through earlier canopy
development and ground cover, and selection of cultivars able to
respond to additional nitrogen fertilization without lodging. With
these cultivars achieving an εi of 0.9 over the growing season,
again, scope for further improvement is very limited
• Further increase in Yp can only be achieved by an increase in εc
which is determined by the efficiency of photosynthesis corrected
for respiratory losses, summarily called as Radiation Use Efficiency
(RUE) 7
Long et.al., 2018

Will improvement in photosynthesis help?
The arguments against
• There is lack of correlation
between crop yield and
photosynthetic rates (Evans &
Dunstone, 1970)
• Photosynthesis is limited by
sink capacity (Borras et al. ,
2004)
Supporting Facts
• European Stress Physiology and
Climate Experiment (ESPACE)
project grew a single genotype of
spring wheat under FACE 650
µmol mol−1) at 7 sites in Germany,
Ireland, the UK, Belgium and the
Netherlands, over 3 consecutive
growing seasons
• Across these sites, photosynthesis
of the flag leaf the major source of
assimilate for the grain – was on
average increased by 50%, and
grain yield was increased by 35%
8
Long et.al., 2018

What is the potential RUE (εc ) of crops
Table 1 - Efficiency of the transduction of intercepted solar radiation into plant carbohydrate
through photosynthesis of crop leaf canopies
9
Long et.al., 2018
Observed maxima- 0.034 (C3) and 0.042 (C4)
i.e. only 70% of the theoretical

Opportunities for hacking photosynthesis
Major Issue Strategy Approach
Theoretical
Increase
Light
Saturation
of leaves
Can be improved by a
canopy architecture that
provides better distribution
of light by maintaining the
maximum efficiency of
photosynthesis under light-
limiting conditions and by
increasing photosynthetic
rate at light saturation
Modifying canopy
architecture
0.051
Improved regeneration of
acceptor molecule
Improving RUBISCOs for
higher rates of catalysis of
carboxylation
Relaxing Photoprotection
Photo-
respiration
Bypassing Photorespiration
Improving RUBISCO to be
specific for carboxylation
0.060
Converting C3 plants to C4
Algal Mechanisms
0.073
Photorespiratory Bypass
Table 2 – Specific opportunities for increasing photosynthesis
10
Long et.al., 2018

Enabling Technologies
Technology/Tools Applications
Bacterial transformation Engineering photosynthesis in cyanobacteria
Nuclear transformation
Engineering of nucleus-encoded components of the
photosynthetic apparatus; expression of novel genes and
pathways. Development of synthetic chromosomes.
Plastid transformation
Engineering of plastid-encoded components of the
photosynthetic apparatus; expression of novel genes and
pathways of carbon metabolism.
Mitochondrial
transformation
Engineering of mitochondrially encoded components of
the respiratory chain to minimize respiratory losses;
expression of novel pathways of carbon metabolism.
Multigene engineering
Engineering of protein complexes in the electron
transfer chain; engineering of carbon fixation pathways.
11
ort et.al., 2018
Table 3 – Specific molecular biology and systemic techniques for hacking photosynthesis

Enabling Technologies
Technology/Tools Applications
Protein design
Redesign of the electron transfer chain; Rubisco
engineering; redesign of carbon-fixing enzymes.
Synthetic genomics
Radical redesign of the photosynthetic apparatus via
synthetic plastid genomes and/or artificial
(mini)chromosomes in the nucleus.
Design of logic circuits;
development of sensors for light
intensity, light quality,
temperature, and CO2
concentration
Smart canopy concept.
Phenotyping in the field
Evaluation of design concepts under field conditions
and further optimization through mutagenesis.
12
ort et.al., 2018

Modifying crop canopies to increase εc
• Leaf photosynthesis responds non-linearly to increases in solar
energy
• A mature crop may have 3 or more layers of leaves (i.e. a leaf
area index of ≥ 3). If the leaves are roughly horizontal, the
uppermost layer would intercept most of the light at midday,
while about 10% may penetrate to the next layer and 1% to
the layer below that
• PPFD intercepted per unit leaf area by an almost horizontal
leaf at the top of a plant canopy would be 1400 µmol m−2 s−1
i.e. about 3 times the amount required to saturate
photosynthesis .
• 2/3rd of the energy intercepted by the upper leaves is wasted
• Upper layers must intercept less light making way for the
lower layers
13
Andrew et.al., 2018

• Upper leaves should be vertical while lower leaves should be
horizontal . A leaf with a 75° angle with the horizontal
intercepts 700 µmol m−2 s−1, just sufficient to saturate
photosynthesis
• Remaining direct light (1300 µmol m−2 s−1) penetrates to the
lower layers of the canopy.
• This distribution almost doubles the efficiency of such as
canopy as compare to a canopy with horizontal leaves
• Although this is only about half the increase that would occur
if the sun remained directly over-head , it nevertheless
suggests considerable improvement may still be achieved by
manipulation of canopy architecture
14
Andrew et.al., 2018

Research Insight/Case Study
Chlorophyll Can Be Reduced in Crop
Canopies with Little Penalty to
Photosynthesis
Berkley J. Walker, Darren T. Drewry, Rebecca A. Slattery, Andy VanLoocke, Young
B. Cho, Donald R. Ort
Journal- Plant Physiology
Published February 2018.
DOI: https://doi.org/10.1104/pp.17.01401
15

Concise review-
• Hypothesis- Reducing Chl content in upper leaves and
promoting it in lower leaves could increase canopy
photosynthesis
• Methodology- Relationship among leaf Chl, optical properties
and photosynthetic capacity was measured in 67 different
soybean varieties and integrated into a biophysical model
(WIMOVAC) of canopy-scale photosynthesis to simulate the
intercanopy light environment and carbon assimilation
capacity of canopies with varying chl content, as well as
among wild types and chl deficient mutants
• One line Summary- An empirically parameterized model of
canopy photosynthesis in soybeans reveals that leaf
chlorophyll can be reduced in upper leaves with significant
nitrogen savings and only minor reductions in daily carbon
gain.
16
Walker et.al., 2018

Figure 1 – Variation in canopy photosynthesis with canopy architecture, A- Light interception as
affected by two type of architecture X & Y, B- Accumulative LAI vs PPFD for X and Y, C- PPFD vs CO2
assimilation rate for X & Y, D- comparison of Diurnal PPFD and Photosynthetic rate for X & Y.
17
Walker et.al., 2018

Smart Canopy Concept-
1. Transitioning from vertical leaves in high light in the upper
canopy to horizontal leaves in low light deeper in the canopy
2. Deploying a Rubisco with a high catalytic rate in the upper
leaves, (even at the expense of specificity for CO2 over O2), &
replacing Rubisco with a high specificity form in the lower
canopy where light is limiting to minimize photorespiration
3.In upper leaves small antennas in large numbers, in lower
leaves larger antenna systems maybe fewer in number
4. Repositioning floral organs and panicles inside the canopy
5. Decreasing leaf chlorophyll content in sun-exposed leaves
6. Engineering of a switchable system where a leaf in a nascent
canopy initially operates a light-driven CO2-concentrating
mechanism and later conducts C3 photosynthesis after it is
shaded during canopy development 18
Walker et.al., 2018

Triple penalty of RUBISCO-
• Penalty 1- Catalyses oxygenation of RuBP leading to
photorespiration which translated into loss of fixed carbon by
about 25 to 40 %
• Penalty 2- maximum catalytic rate of Rubisco (kc
cat) is
remarkably slow compared with most plant enzymes, such
that large amounts of the protein are required to achieve the
photosynthetic rates necessary to support high productivities
in C3 crops. It is already 50% of total leaf protein hence
increasing it is not an option on v/v basis
• Penalty 3- The forms with higher CO2 specificity (τ) over O2
have very poor catalytic rates (kc
cat).
19
Andrew et.al., 2018

Improving catalysis and/or specificity of
RUBISCO
• Increased τ will result in increased leaf and canopy
photosynthesis If
1. A fixed inverse relationship between kc
cat and τ implied
from measurements is assumed,
2. Increasing concentration of Rubisco per unit leaf area
is not an option
20
Andrew et.al., 2018

Engineering chloroplasts to improve Rubisco
catalysis: prospects for translating
improvements into food and fiber crops
Robert E. Sharwood
Journal- New Phytologist
New Phytologist (2018) 213: 494–510
doi: 10.1111/nph.14351
21

Concise review-
• Hypothesis- Using plastid transformation for bioengineering of
improved forms of RUBISCO in terms of specificity and/or
catalysis can enhance photosynthetic efficiency
• Methodology- Saturation state kinetics of RUBISCO from
varied sources like Bacteria, Cyanobacteria, Red algae, Diatom
C3 and C4 plants was studied to delineate the specificity and
carboxylation properties and candidate RUBISCO was
modelled using biophysical simulative transformation models
• One line Summary- The model generates much evidence in
favor of a strong negative correlation between specificity and
carboxylation in case of RUBISCO. The study suggested that
ideally, a crop should express a high kc
cat RUBISCO in the upper
canopy leaves exposed to full sunlight and a high τ RUBISCO
in the shaded lower canopy leaves 22
Sharwood, 2018

Exploring natural diversity of RUBISCO
23
Sharwood, 2018
Fig 2a - Natural diversity of Rubisco catalysis exists among photosynthetic organisms as shown in the
relationships between (a) Kc (Michaelis constant for CO2), (b) specificity for CO2 opposed to O2 (Sc/o) and
the carboxylation speed (kcat
c). Solid lines represent the exponential relationship y = 4.9e0.377x r2 = 0.5 and
y = 134.5e 0.137x r2 = 0.4

Exploring natural diversity of RUBISCO
24
Sharwood, 2018
Fig 2b – (C)Modelling at 25°C of the influence of rice (C3 monocot), tobacco (C3), Griffithsia monilis (red
alga) and maize (C4 monocot) Rubisco catalytic parameters on CO2 assimilation for C3 photosynthesis in
response to chloroplastic CO2 partial pressures (CC) under Rubisco activity limiting and under electron
transport limiting conditions. (d) Comparing the influence of rice, tobacco, G. monilis, Synechococcus
PCC6301, Rhodospirillum rubrum, Cyanobium spp. PCC7001 and maize Rubisco catalysis in CO2 conditions
experienced by plants operating a Kranz-type C4 photosynthetic pathway under Rubisco activity limiting
conditions

Significant Findings
• Following the steady-state biochemical model of leaf
photosynthesis, the C3 photosynthetic CO2 uptake rate (A) is
either limited by the maximum Rubisco activity (Vc,max) or by
the rate of regeneration of RuBP which is determined by the
rate of whole chain electron transport (J).
• If J is limiting, increase in τ would increase net CO2 uptake
because products of the electron transport chain would be
partitioned away from photorespiration into photosynthesis
• If Rubisco from the non-green algae Griffithsia monilis can be
expressed in place of the present C3 crop RUBISCO, then
canopy carbon gain can be increased by 27%.
25
Sharwood, 2018

Significant Findings
Note-
The final row represents simulation of the gain that can be achieved if a form of Rubisco with a high kc
cat (A.
edulis) can be expressed in the sunlit leaves and if a form with high τ (current C3 average) can be expressed in the
shade leaves.
kc
c, maximum catalytic rate of Rubisco; τ , specificity of Rubisco for CO2 relative to O2; Asat, maximum rate of
photosynthesis; Rubisco, ribulose 15-biphosphate carboxylase/oxygenase.
26
Sharwood, 2018
Table 4 - Reported values for kc
cat and AC’ of species under study

Improved regeneration of Acceptor Molecule
• The progenitors of modern crop plants evolved in, and are thus
adapted to, an atmospheric [CO2] of about 240 ppm.
• The accelerated rate of Rubisco-catalyzed carboxylation at today’s
[CO2] of >400 ppm has led to a kinetic limitation in the
regeneration of the CO2 acceptor molecule ribulose-1,5-
bisphosphate (RuBP), which will become increasingly limiting as
[CO2] increases further
• If the rate of carboxylation at Rubisco is increased, then Jmax
should also be increased to gain maximum benefit From kinetic
data, it may be calculated that as a result, Jmax/Vc,max would need
to increase by 30% to maintain an optimal distribution of
resources 27
Andrew et.al., 2018

Identifying the key limiting points
• Unlike Vc,max, regeneration of RuBP does not depend on the
amount or the properties of any single protein, but on the
complete photosynthetic electron transport chain and on all the
enzymes of the Calvin cycle except Rubisco
• Transgenic plants with small decreases in the quantities of specific
proteins produced by antisense technology in tobacco suggest
that two points in this chain limit Jmax and strongly control the rate
of RuBP synthesis
• 1. The cytochrome b6/f complex in the electron transport chain
• 2. Sedoheptulose-1:7-bisphosphatase (SbPase) in the Calvin cycle
28
Andrew et.al., 2018

Simultaneous stimulation of sedoheptulose 1,7-
bisphosphatase, fructose 1,6 bisphophate aldolase
and the photorespiratory glycine decarboxylase-H
protein increases CO2 assimilation, vegetative
biomass and seed yield in Arabidopsis
Andrew J. Simkin1, Patricia E. Lopez-Calcagno1, Philip A. Davey1, Lauren
R. Headland1, Tracy Lawson1, Stefan Timm2, Hermann Bauwe2 and
Christine A. Raines1*
Journal- Plant Biotechnology Journal
Plant Biotechnology Journal (2017) 15, pp. 805–816
doi: 10.1111/pbi.12676
29
Simkin et.al., 2017

Concise review-
• Hypothesis- To explore the possibility that the simultaneous
increase in the activity of enzymes of both the CB cycle and the
photo respiratory pathway could lead to a cumulative positive
impact on photosynthetic carbon assimilation and yield
• Methodology- SBPase, FBPA and GDC-H either alone or in
combination were over expressed in Arabidopsis plants using
tissue specific promoters and T3 plants were analyzed for
chlorophyll fluorescence, photosynthetic efficiency, carbon
assimilation and overall growth and yield
• One line Summary- Simultaneous over expression of CB cycle
enzymes along with PR pathway enzymes lead to an enhancement
in dry weight by 39 to 45%
30
Simkin et.al., 2017

Increased photosynthetic efficiency in
young over expressing seedlings
Fig 3 -Photosynthetic capacity and
leaf area in transgenic seedlings
determined using chlorophyll
fluorescence imaging. (a,b)- Fq’/Fm’
(maximum PSII operating efficiency)
values of the whole plant at 200
lmol/m2/s and (c)- leaf area at time
of analysis. Azygous controls (A)
recovered from a segregating
population. Lines over-expressing
SBPase (S), FBPA (F), GDC-H protein
(H), SBPase and FBPA (SF) and
SBPase, FBPA and GDC-H (SFH) are
represented. Significant differences
between lines(P < 0.05) are
represented as capital letters
indicating whether each specific line
is significantly different from another
(i.e. SBPase lines (S) are significantly
bigger than wild type (WT) and
azygous lines (A)). Numbers indicate
% increases over WT.
31
Simkin et.al., 2017

Enhanced Photosynthetic CO2 fixation rates
Fig- 4 Photosynthesis
carbon fixation rates
determined as a
function of light
intensity in developing
leaves. Red arrow
indicates optimum
growth light intensity.
Lines over-expressing
SBPase (S), FBPA (F),
GDC-H protein (H),
SBPase and FBPA (SF),
and SBPase, FBPA and
GDC-H (SFH) are
represented. Results are
based on 4 to 7 plants
per line
32
Simkin et.al., 2017

Growth analysis
Fig 5- Growth analysis of the transgenic and control plants after 38 days. Lines over-expressing
a single transgene, SBPase (S), FBPA (F), GDC-H protein(H), two transgenes, SBPase and FBPA
(SF), or three transgenes, SBPase, FBPA and GDC-H (SFH) are shown.
33
Simkin et.al., 2017

Growth analysis Contd..
Fig 6a Growth analysis of C and transgenic lines grown in low light (a) Leaf area per plant evaluated over the
first 38 days. (b) Final dry weight (g) after 38 days of development and statistical differences between lines.
% increases over C are indicated within the columns. Lines over-expressing SBPase (S), FBPA (F), GDC-H
protein (H), SBPase and FBPA (SF) and SBPase, FBPA and GDC-H (SFH) are represented. Significant
differences between lines (P < 0.03) are represented as capital letters indicating whether each specific line is
significantly different from another.
34
Simkin et.al., 2017
a b

35Fig 6b- GDC-H and GDC-H with SBPase and FBPA overexpression in Arabidopsis
differentially impact biomass and seed yield. (a, c) Dry weight and (b, d) seed weight
were determined at seed harvest.

Relaxing Photoprotection
• When light intensity is too high or increases too fast for photo
chemistry to use the absorbed energy, many photoprotective
mechanisms are induced to protect the photosynthetic
antenna complexes from photo-oxidation
• Excess excitation energy in the photosystem II (PSII) antenna
complex can be harmlessly dissipated as heat, which is
observable as a process named non-photochemical quenching
of chlorophyll fluorescence (NPQ). The rate of NPQ relaxation
is slower than the rate of induction
• This slow rate of recovery of PSII antennae from the quenched
to the unquenched state implies that the photosynthetic
quantum yield of CO2 fixation is transiently depressed by NPQ
upon a transition from high to low light intensity 36
Andrew et.al., 2018

Basics of NPQ
Fig 8- The highly quenched state of photosystem II is associated with zeaxanthin, the
unquenched state with violaxanthin. Enzymes interconvert these two carotenoids, with
antheraxanthin as the intermediate, in response to changing conditions, especially changes in
light intensity. Zeaxanthin formation uses ascorbate as a cofactor, and violaxanthin formation
requires NADPH
37
Plant Physiology, Taiz and Zeiger, 3rd 3d.

Basics of NPQ
Fig- 7- Nonphotochemical quenching regulates light harvesting by PSII. In limiting light, LHC proteins
efficiently transfer excitation energy to the reaction center of PSII. In excess light, when the rate of
photosynthesis is saturated and protons accumulate to a high concentration in the thylakoid lumen, a
flexible type of nonradiative dissipation is induced in the PSII antenna on a timescale of seconds to
minutes. Proton binding to the PSBS protein and accumulation of zeaxanthin (not shown) causes a
conformational change or reorganization of PSII that switches the antenna into a dissipative state that
prevents overexcitation of chlorophyll and overreduction of the electron transport chain.
38
Biochemistry and Molecular Biology of Plants, Buchannan et al. 2nd ed., 2015

The Problem
Fig 8- Photoprotection and CO2 fixation during sun-shade transitions. When leaves are exposed to high light, the
rate of CO2 fixation is high, and excessive excitation energy is harmlessly dissipated through NPQ. NPQ is
correlated with the abundance of PsbS and further stimulated by the de-epoxidation of violaxanthin to
zeaxanthin, catalyzed by VDE. Upon transition to low light, CO2 fixation becomes limited by the reduced form of
NADP and ATP derived from photosynthetic electron transport, which in turn is limited by high levels of NPQ. The
rate of CO2 fixation therefore remains depressed until relaxation of NPQ is complete. This can take minutes to
hours and is correlated with the rate of zeaxanthin epoxidation, catalyzed by ZEP.
39
Andrew et.al., 2018

Improving photosynthesis and crop productivity by
accelerating recovery from photoprotection
Johannes Kromdijk,1* Katarzyna Głowacka,1,2* Lauriebeth Leonelli,3
Stéphane T. Gabilly,3 Masakazu Iwai,3,4 Krishna K. Niyogi,3,4† Stephen
P. Long1,5†
Journal- Science
18 Nov 2016:
Vol. 354, Issue 6314, pp. 857-861
DOI: 10.1126/science.aai8878
40

Concise review-
• Hypothesis- By accelerating the xanthophyll cycle
intermediates and increasing PsbS, NPQ would decline more
rapidly on transfer of leaves to shade
• Methodology- Nicotiana tabacum was transformed with the
coding sequences of Arabidopsis VDE, ZEP, and PsbS under the
control of different promoters for expression in leaves.
Suitable traansformants were testes for faster relaxation of
NPQ and recovery of CO2 fixation rate
• One line Summary- Transgenic expression of Arabidopsis VDE,
PsbS, and ZEP (VPZ) in combination in tobacco led to a marked
and statistically significant acceleration of NPQ relaxation on
transfer of leaves from high light to shade. This led to a more
rapid recovery of the efficiency of photosynthetic CO2
assimilation in the shade.
41
Kromdijk et.al., 2016

Faster relaxation of NPQ and recovery of
CO2 fixation rate
Fig 9- Transient adjustment of NPQ and net CO2 assimilation. (A) Dark relaxation of NPQ after exposure to
alternating high and low light in young seedlings of wild-type N. tabacum (WT) and three lines expressing AtVDE,
AtPsbS, and AtZEP (VPZ). (B) Time course of net CO2 fixation rate in fully expanded leaves in response to a
decrease in light intensity of 2000 to 200 mmol photons m−2 s−1 at time zero, indicated by the black arrow.
42

Effects of fluctuating light on the efficiency of
photosynthetic CO2 assimilation
Fig 10- Photosynthetic efficiency and
NPQ under steady-state and
fluctuating light. (A) Quantum
efficiency of leaf net CO2 assimilation
(FCO2max) under steady-state light. (B)
FCO2max under fluc-tuating light. (C)
Quantum efficiency of linear elec-
tron transport (FPSIImax) under
steady-state light. (D) Quantum
efficiency of linear electron transport
(FPSIImax) under fluctuating light. (E)
Average NPQ corresponding to (A)
and (C). (F) Average NPQ
corresponding to (B) and (D
43

Productivity under field conditions
Fig 11- Productivity of field-grown N. tabacum plants. Lines expressing AtVDE, AtPsbS, and AtZEP
(VPZ) produced 15% larger plants than did the WT. (A) Total dry weight. (B) Leaf area. (C) Plant
height. Data were normalized to WT. Error bars indicate SEM (n = 12 blocks), and asterisks indicate
significant differences between VPZ lines and WT (a = 0.05).
44

Abundance of Xanthophyll Cycle
Intermediates
45
Table 5- Xanthophyll cycle pigment concentrations and De-epoxidation state

Incorporating Algal CCM Mechanisms
Fig 12- Cyanobacterial CCM components for improved photosynthesis Carboxysomes of Cyanobium PCC7001,
used in this study, consist of many thousands of polypeptides, arranged in an icosahedral structure. In this model,
a single layer of shell-bound Rubisco (CbbLS, green) is shown, with carboxysomal CA (orange). CsoS2
(yellow/brown) interlinks Rubisco and the shell made predominantly of CsoS1A hexamers (light blue). These and
ancillary shell proteins (CsoS1D and CsoS1E, dark blue) enable substrate transport via central pores. Pentameric
vertex proteins (CsoS4AB, purple) complete the structure
46
Long et.al., 2018

Carboxysome encapsulation of the CO2-fixing
enzyme Rubisco in tobacco chloroplasts
Benedict M. Long 1, Wei Yih Hee 1, Robert E. Sharwood2, Benjamin
D. Rae 2, Sarah Kaines 1, Yi-Leen Lim 1, Nghiem D. Nguyen 2, Baxter
Massey 1, Soumi Bala2, Susanne von Caemmerer
Journal- Nature Communications
September 2018
Nature Communications volume 9,
Article number: 3570 (2018)
47

Concise review-
• Hypothesis- Targeted chloroplastic expression of Cyanobium
carbozysomes assembly proteins may lead to carboxysome
assembly in tobacco chloroplasts. The latter shall result in
enhanced photosynthetic efficiency and growth
• Methodology- Nicotiana tabacum was transformed with genes for
2 carboxysome assembly proteins CsoS1A and CsoS2
singularly(CyLS) and in combination (CyLS-s1s2). The assembled
structure was isolated and parametrically compared with
cyanobium carboxysomes using TEM and immunogold
localization. The transformants were screened for growth
parameters
• One line Summary- A structure empirically similar to
carboxysomes was assembled and it could be clearly visualized
within the chloroplast. However the native functionality was not
observed.
48
Long et.al., 2018

Carboxysome assembly and visualization
a
Fig 13a- Carboxysomes are synthesized in tobacco chloroplasts from four proteins. (a) a model (b)TEM of
chloroplasts from tobacco expressing Cyanobium Rubisco (CyLS plants) and (c) tobacco expressing Cyanobium
Rubisco along with the shell proteins CsoS1A and CsoS2 (CyLS-S1S2 plants), The inset in c at higher magnification
(d). Negatively stained carboxysomes purified from CyLS-S1S2 plants (e) and carboxysomes purified from
Cyanobium cyanobacterial cells (f).
49
Long et.al., 2018

Carboxysome assembly and visualization
50
Long et.al., 2018
Fig 13b (g) Diameters of carboxysomes from wild-type Cyanobium cells (cyan line) and carboxysomes purified
from CyLS-S1S2 plants (magenta line) determined using a Nanosight particle analyser. (h) TEM of ultrathin sections
through CyLS-S1S2 plant chloroplasts reveal the presence of elongated structures (arrowheads) associated with
the more regular carboxysome structures. These elongated structures co-purify with the plant-expressed
carboxysomes.
h

Plant Growth Analysis
Fig. 14 Form-1A Rubisco-dependent
plant growth. CO2 assimilation rates
of wild-type and transgenic tobacco
expressing Cyanobium PCC7001
Form-1A Rubisco (CyLS) and
expressing Cyanobium Rubisco
together with the carboxysome
genes csoS1A and csoS2 (CyLS-S1S2),
determined by gas exchange of
attached leaves. Rates are expressed
on a leaf area basis (a) with an
expanded scale for the same data
presented in b to show assimilation
rates in transformed plants. Fitted
lines (WT, black; CyLS, cyan; CyLS-
S1S2, yellow)
(C) CO2 assimilation rates of leaf discs
from each plant line from plants
grown at 2% (v/v) CO2 in membrane
inlet mass spectrometer (MIMS)
assays. Solid lines (WT, magenta;
CyLS, cyan; CyLS-S1S2, yellow) The
dashed lines for CyLS and CyLS-S1S2
are modelled assimilation rates using
the same parameters as a, b. (d)
Growth measured as plant height
post
51

Plant Growth Analysis
Fig 15- Growth phenotypes at days after germination (e–g) and at maturity (h–j) of wild-type (e, h), CyLS
(f, i) and CyLS-S1S2 (g, j) plants grown in soil at 2% (v/v) CO2 in 20 cm pots. Note the delayed germination
and time to reach maturity in both transformant lines
52
Long et.al., 2018

Loss due to photorespiration
Fig 16- Calculated actual and potential rates of crop canopy photosynthesis versus temperature, where potential
is defined as the rate in the absence of photorespiration. The difference represents the loss caused by
photorespiration. Calculation assumes a crop with a leaf area index of 3 and a photon flux above the canopy of
1800 µmol m−2 s−1 (i.e. full sunlight)
53
Walker et.al., 2016

Is photorespiration actually required?
• Photorespiration can dissipate excess excitation energy at high PPFD, involves the
synthesis of serine and glutamate, and transfers reductive power from the
chloroplast to the mitochondrion. This has led some to suggest that
photorespiration is essential for normal plant function
• However, xanthophylls provide a far more effective means of dissipating excess
energy. Unlike photorespiration, this dissipation mechanism is not a significant drain
on the ATP and NADPH produced by the light reactions. Further, dissipation of
energy as heat through xanthophylls is reversible.
• In addition, the photosynthetic cell has pathways besides photorespiration for
amino acid synthesis and transfer of reductive energy to the cytosol Hence the
supposed ‘beneficial’ functions of photorespiration are redundant within the cell.
• Further, photorespiration can be eliminated without detriment to the plant by
growing plants in a very high concentration of CO2, a competitive inhibitor of the
oxygenase activity of Rubisco. For example, wheat can grow normally and can
complete its life cycle under these unusual conditions. Commercial growers of some
greenhouse crops increase [CO2] to three or four times the normal atmospheric
concentration
54
Walker et.al., 2016

Synthetic glycolate metabolism pathways stimulate
crop growth and productivity in the field
Paul F. South, Amanda P. Cavanagh, Helen W. Liu, Donald R. Ort*
Journal- Science
Feb 2019
Vol. 363, Issue 6422, eaat9077
DOI: 10.1126/science.aat9077
55

Concise review-
• Hypothesis- Installing a non native, synthetic glycolate metabolism
pathway in tobacco stimulate crop growth and productivity in the field
• Methodology- Nicotiana tabacum cv. Petite Havana was transformed
with three different photorespiratory alternative pathway (AP) designs
using multigene constructs assembled by Golden Gate cloning . Native
photorespiratory pathway was suppressed using a long hairpin RNA
interference (RNAi) construct. The transformants were analyzed for
resistance to photorespiratory stress and greater biomass accumulation
as a result of Photorespiratory bypass.
• One line Summary- AP3 plants show increased photosynthetic rates,
quantum efficiency, and biomass accumulation in replicated field trials
particularly due to have an altered photorespiratory metabolite profile
that forced a greater flux of glycolate through the synthetic pathway 56
South et.al., 2019

The Three SyntheticAlternate Pathways
Fig 17- Model of three alternative photorespiration pathway designs. AP1 (red) converts glycolate to glycerate
using five genes from the E. coli glycolate pathway encoding the enzymes glycolate dehydrogenase, glyoxylate
decarboxylase, and tartronic semi- aldehyde reductase. AP2 (dark blue) requires three introduced genes
encoding glycolate oxidase, malate synthase, and catalase (to remove hydrogen peroxide generated by glycolate
oxidase). AP3 (blue) relies on two introduced genes: Chlamydomonas reinhardtii glycolate dehydrogenase and
Cucurbita maxima malate synthase.
57
South et.al., 2019

Gene and protein analysis confirm
chloroplast-localized transgene expression
Fig 18- A. qRT-PCR analysis of the two transgenes in AP3 and the target gene PLGG1 of the RNAi construct.
Results for three independent transformation events are shown with (1, 5, and 8) and without (8, 9, and 10)
PLGG1 RNAi. (B) Immunoblot analysis from whole leaves and isolated chloroplasts, including the insoluble
membrane fraction, using custom antibodies raised against the indicated target genes, cytosolic marker actin,
and chloroplast-specific marker platoglobulin 35 (PGL35). Five micrograms of protein was loaded per lane. Arrows
indicate detected protein based on molecular weight.
58
A B
South et.al., 2019

AP plants are resistant to photorespiration
stress
Fig. 19- AP plant lines are more photoprotective under photo-respiration stress. (A) Representative photos of 9-
day-old T2 transgenic tobacco lines during the chlorophyll fluorescence photoprotection screen for AP pathway
function showing AP3 protecting photosystem II from photodamage under severe photorespiratory conditions.
(B) Combined values of the three AP construct designs with and without RNAi targeting the glycolate-glycerate
transporter PLGG1.
59
South et.al., 2019

AP plants show enhanced biomass
accumulation in greenhouse growth studies
Fig 20- Photorespiration
AP lines increase biomass
under greenhouse
conditions. (A) Photos of
6-week-old AP3 and WT
plants grown in the
greenhouse. Individual
plant lines are indicated
in the labels below the
plant. (B) Percent
difference in total dry
weight biomass of the
indicated combined plant
lines.
60
South et.al., 2019

AP3 plants have an altered
photorespiratory metabolite profile
Fig 21-
Photorespiratory and
AP3 metabolic
intermediates. (A to
F) Relative amount of
the indicated
metabolite detected
from ~40 mg of leaf
tissue (fresh weight;
FW) sampled in the
late morning.
Metabolite
concentrations were
reported as
concentrations
relative to the
internal standard
61
South et.al., 2019

AP3 plants exhibit increased
photosynthetic rate and chloroplast [CO2]
Fig 22- Photosynthetic efficiency of field grown
plants. Data are the combined result of three
independent trans-formants (hereafter referred
to as combined) with and without PLGG1 RNAi.
(A) CO2 assim-ilation based on intercellular
[CO2] (Ci).
(B) Combined apparent CO2 compensation
point: Ci* (C) Combined maximum rate of
RuBisCO carboxylation (Vcmax).
62
South et.al., 2019

Summary
• The yields need to be improved while the plants are woking at
only 65 to 70 % of their photosynthetic capacity
• Can Hacking Plants Feed the World? Well at least the
Research Looks Good
• Different pathways have been tried, but with limited success
• Bypassing of Photorespiratory pathway has been by far the
most satisfactory hack in terms of field based performance
64

Future Perspectives
• The performance of the synthetic glycolate pathway in staple
food crops needs to be evaluated (where seed yield is
economically more important)
• The attempts at combining more than one hack need to be
undertaken for realizing better increment in photosynthetic
efficiency
65

Hacking photosynthesis

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