https://doi.org/10.

1038/s41477-021-00947-5

Supplementary information

Impact of ion fluxes across thylakoid

membranes on photosynthetic electron
transport and photoprotection
In the format provided by the
authors and unedited
Supplementary Information for

Mechanistic insights into ion fluxes across thylakoid membranes

during photosynthesis

Meng Li a,b, Vaclav Svoboda a, Geoffry Davis c, David Kramer c,d, Hans-Henning Kunz e,
Helmut Kirchhoffa,1

a
, Institute of Biological Chemistry, Washington State University, PO Box 646340,
Pullman, WA, 99164-6340, USA.
b
, Current Address: School of Oceanography, University of Washington, Box 357940,
Seattle, WA 98195-7940, USA.
c
, Department of Energy Plant Research Laboratory, Michigan State University, 612
Wilson Road, East Lansing, MI 48824-6406.
d
, Department of Biochemistry, Michigan State University.
e
, School of Biological Sciences, Washington State University, PO Box 644236 Pullman,
WA 99164-4236, USA.

1
, Corresponding author: Helmut Kirchhoff, Institute of Biological Chemistry, Washington
State University, PO Box 646340, Pullman, WA, 99164-6340, USA. T: +1 509 335 3304,
F: +1 509 335 7643
Email: kirchhh@wsu.edu

1
SI Methods
Documentation about the deltapsi_leaf.py model describing photosynthetic light
harvesting and Clavin Benson cycle.
deltapsi_leaf.py is a program for simulating effects of ion transporters on photosynthetic
parameters, modified from DeltaPsi.py (1). The code is deposited in Github:
https://github.com/limengwsu/Ion_NPQ/blob/master/delta_psi_py/deltapsi_leaf.py. The
following describes the key updates and changes made for our purpose.
1) The model was adjusted to simulate parameters such as NPQ, ΦPSII, and qL that
can be measured for leaf samples. This was realized by several changes including
calculation of how much light, or how many photons/s, is available per simulated
area (normalized to one PSII).
2) Some kinetic constants are updated, such as PC to PSI rate, lumen-buffering
capacity, relative quantity of ATP synthase, Cytb6f, etc.
3) An approximation of CBC, with activation kinetics overtime, and different
maximum CBC rate under different light intensities. The NADPH consumption
rate is used as the proxy for simulated CBC.
4) ATP synthase rate is simulated using a sigmoidal model/data, dependent on PMF
and reduction level (2). The kinetics of ATP synthase activation is also presumed
light intensity dependent.
5) Ion transporters, VCCN1, CLCe, KEA3, are added to the model to simulate and
explain the observed differences between WT and mutants.
6) NPQ calculation is updated.
PSII reactions
𝑃𝑆𝐼𝐼%&'()*_,*-'('./01, = PSII6789776_:;<9 × photonC9DEFGG × ΦPSII
Difference from DeltaPsi.py (Davis et al. 2017):
PSII per leaf area is calculated based on measured Chl content per leaf area, 210 µmol
Chl/m2, and PSII content in relation to Chl, ~300 Chl/PSII, which gives a PSII content
per leaf area of 0.7 µmol PSII/m2. With given light intensity of x µmol photon/m2/s,
photonC9DEFGG is 0.84*x/0.7, where 0.84 is the fraction of light a typical leaf absorbs.
This translates into Phython code as:
light_per_L=0.84 * PAR/0.7
It is assumed that half of the photons go to PSII and the other half go to PSI, or
PSII6789776_:;<9 = 0.5. Note that photonC9DEFGG is the total photons received for the
simulated system, normalized to one PSII. PSII6789776_:;<9 determines how much fraction
of it goes to PSII for PSII charge separation, NPQ, fluorescence, etc.

Cyt b6f Content

0.433/PSII (3)

PSI reactions
PC to P700 kinetic constant: 𝑘IJ_.0_IKLL = 5000/s
PSI to PSII ratio = 0.7 (3)

Mehler reaction

2
As described in Lyu and Lazár 2017 (4):
[UVWXY ]
𝑉P* = 𝑘P* × [𝑂S ] × [UV ]Z[UV
WXY [\ ]
In Python code: V_me = 4*0.000265*Fd_red/(Fd_red+Fd_ox)

NDH reactions
The NDH reaction takes the work done by Strand et al. 2017 (5) into account.
Forward reaction
Z Z
𝐹𝑑(*V + 0.5 𝑃𝑄 + 3𝐻,.(0P' − −𝑘efg −→ 𝐹𝑑0i + 0.5 𝑃𝑄𝐻S + 2 𝐻klP*1
Reverse reaction
Z Z
𝐹𝑑(*V + 0.5 𝑃𝑄 + 3𝐻,.(0P' < − − 𝑘efgWXnXWoX − 𝐹𝑑0i + 0.5 𝑃𝑄𝐻S + 2 𝐻klP*1
The electrical potential of PQH2 is adjusted based on stromal pH:
𝐸𝑚IrgS = 𝐸𝑚IrgS_-gK − 0.06 × (𝑝𝐻,.(0P' − 7.0), 𝑢𝑛𝑖𝑡 𝑖𝑛 𝑣𝑜𝑙𝑡𝑠
∆𝐸𝑚 = 𝐸𝑚IrgS − 𝐸𝑚UV
So,
∆𝐺efg = 𝑧 × 𝐹 × ∆𝐸𝑚 + 2 × 𝐹 × 𝑝𝑚𝑓
where z = -1 as one electron transferred per reaction, and 2 is he number of protons
pumped by NDH.
∆‰PŠS×-P‹
𝐾𝑒𝑞efg = 10 L.LŒ
𝑘efg
𝑘efg (*•*(,* =
𝐾𝑒𝑞efg

𝑣efg = 𝑘efg × [𝐹𝑑(*V ] ∗ [𝑃𝑄] − 𝑘efg (*•*(,* × [𝐹𝑑0i ] ∗ [𝑃𝑄𝐻S ]

𝑘efg 𝑖𝑠 𝑓𝑖𝑡𝑡𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑣𝑎𝑙𝑢𝑒: 1000 𝑠 Š’

NADPH oxidation/ CBC cycle

The kinetics of NADPH oxidation can involve other reactions besides CBC cycle. For
simplification, the oxidation rate of NADPH is used as a proxy for CBC cycle.
For different light intensities, kCBC_max_light can be different (6, 7):
𝑃𝐴𝑅
𝑘J“J_k/)&._P'i = 𝑘J“J_P'i ×
𝑃𝐴𝑅 + 250
𝑘J“J_P'i (photosynthetic NADPH oxidation rate) is estimated at 60/PSII/s, equivalent to
total leaf CO2 assimilation rate at 21 µmol/m2/s;
The units for PAR and 250 in the equation are in µmol photons/m2/s.
From dark to light, kCBC is time dependent (8):
Š.
𝑘J“J– = 𝑘J“J_k/)&._P'i × —1 − 𝑒 ˜ ™
T is the lifetime of CBC activation, T500uE = 900 s and T100uE = 600 s; t is the duration of
illumination of a constant light.
NADPH/NADP ratio regulating NADPH oxidation rate:
• In the dark adapted state: NADPH/NADP ~ 1.25 (9),
𝑘J“J
= 0
𝑘J“J ,.*'Vš
• At steady state: NADPH/NADP ~3.5 (9),

3
 𝑘J“J
= 1
𝑘J“J ,.*'Vš
• Since CBC is also a reaction of electron flow, the rate can be normalized as:
[𝑁𝐴𝐷𝑃𝐻]
ln — [𝑁𝐴𝐷𝑃] ™ − 𝑙𝑛1.25
𝑘J“J›œ•žŸ/›œ•ž = 𝑘J“J_. ×
𝑙𝑛3.5 − 𝑙𝑛1.25
Altogether,
[𝑁𝐴𝐷𝑃𝐻]
𝑘J“J = 𝑘J“J_P'i × 𝑓(𝑃𝐴𝑅, 𝑡, )
[𝑁𝐴𝐷𝑃]

ATP synthase activity

The ratio of ATP synthase per PSII is 0.367~0.5 (9, 10). ATP synthase maximum rate is
400 ATP/s per ATP synthase (2). The normalized maximum ATP synthase rate per PSII
is 200/s. The rate of ATP synthesis is PMF dependent (2).
1
𝑉¤˜I = 𝑉¤˜I_P'i × (1 − ’.¨ )
10(I¥UŠ-P‹¦.§ )×L.LŒ + 1
For oxidized and reduced ATP synthase, the pmf0.5 are different (2):
𝑝𝑚𝑓L.¨ = 0.132 𝑉 𝑓𝑜𝑟 𝐴𝑇𝑃𝑎𝑠𝑒(*V
𝑝𝑚𝑓L.¨ = 0.204 𝑉 𝑓𝑜𝑟 𝐴𝑇𝑃𝑎𝑠𝑒0i
From dark to light, ATP synthase is activated/reduced overtime, and the kinetics of this
activation is not clear. Using data provided by Dr. Davis and Dr. Kramer, we use gH+ as
a proxy for the activation of ATP synthase from oxidized form to its reduced form.
𝑡 ¯
𝐴𝑇𝑃𝑎𝑠𝑒(*V -𝑇 ®
= 0.2 + 0.8 ∗
𝐴𝑇𝑃𝑎𝑠𝑒.0.'k 𝑡 ¯
-𝑇 ® + 1
For different light intensity:
When PAR = 100 µmol/m2/s, calculated T = 165 s;
When PAR = 500 µmol/m2/s, assumed T = 60 s.
So,
𝑉¤˜I = 𝑉¤˜I',*_(*V + 𝑉¤˜I',*_0i

Proton leakage
Z
𝑉g ° k*'± = 𝑘k*'± × 𝑝𝑚𝑓 × [𝐻klP*1 ]
K Š’ Š’ Š’
𝑘k*'± = 3 × 10 𝑠 𝑀 𝑉

NPQ calculation
𝑁𝑃𝑄 = 0.4 × 𝑁𝑃𝑄P'i × 𝑃𝑠𝑏𝑆𝐻 × 𝑓´i + 0.1 × 𝑁𝑃𝑄P'i × 𝑓´i
+ 0.5 × 𝑁𝑃𝑄P'i × 𝑃𝑠𝑏𝑆𝐻
Maximum NPQ 𝑁𝑃𝑄P'i = 3
𝑃𝑠𝑏𝑆𝐻 is the protonated PsbS fraction.
𝑓´i is the degree of de-epoxidation, (Zx+0.5*Ax)/(Zx+Ax+Vx), value 0~1.
The NPQ (mostly qE) is simulated based on the observation that NPQ (qE) is PsbSH
dependent. When fzx is 0, as in npq1 mutant, the NPQ is about half of its maximum (11,

4
12). When PsbS is absent, the NPQ is close to zero (11), and qZ is mostly PsbS
dependent.

Ion Transporters
K+ movement across the membrane:
We kept the voltage-gated K+ channel in our model with updated kinetic constant
“Perm_K”. For KEA3, the K+/H+ antiporter, we changed the kinetic constant k_KEA
from zero so that the model can simulate WT and other mutants which have intact KEA
function. Meanwhile, we implemented some regulation factors for KEA activity. It has
been speculated that KEA3 has an NADP/NADPH binding domain(13, 14) and the
sequence analysis, such as BLAST, of this domain does point to its homologs binding to
NADP/NADPH. Our model does not describe the accurate level of NADPH/NADP due
to the lack of information about NADPH oxidation through other pathways. Using an
alternative proxy, 𝑄¤Š ,we were able to simulate the delayed KEA3 activity under the
higher light intensity, 500 µmol/m2/s. The result suggests a negative correlation between
𝑄¤Š (possibly through regulation by NADPH) and KEA3 activity. Besides, pH regulation
of KEA3 activity was implemented as well.
𝑞𝐿 = 1 − 𝑓rœ¶ , 𝑓rœ¶ is the fraction of 𝑄¤Š
KEA3 regulation by NADPH, calculated from qL:
·¸¹
𝑞𝐿'%. = ·¸¹ ZL.’¨¹
KEA3 regulation by lumen pH:
1
𝑝𝐻'%. =
10-gº»¼X½ ŠŒ.L +1
KEA3 activity fraction:
𝑓¾‰¤_'%. = 𝑞𝐿'%. × 𝑝𝐻'%.
Š
𝐶𝑙 movement across thylakoid membrane:
VCCN1 has been reported as the major voltage-gated chloride channel (15), the
Š
𝐶𝑙 flux rate in relation to voltage was described in earlier study. By fitting the
experimental data (Supplementary figure 7 in ref(15)) to an empirical equation, the
activity was normalized to the conductance at 0.1 V.
Relative 𝐶𝑙 Š flux through VCCN1 in relation to driving force:
𝐶𝑙‹kliWXºÀ–ÁnX = 332𝑣  + 30.8𝑣 S + 3.6𝑣
𝑤ℎ𝑒𝑟𝑒 𝑣 𝑖𝑠 𝑡ℎ𝑒 𝑑𝑟𝑖𝑣𝑖𝑛𝑔 𝑓𝑜𝑟𝑐𝑒 𝑓𝑜𝑟 𝐶𝑙 Š 𝑓𝑙𝑢𝑥 𝑎𝑐𝑟𝑜𝑠𝑠 𝑡ℎ𝑒 𝑡ℎ𝑦𝑙𝑎𝑘𝑜𝑖𝑑 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒, 𝑣
Š
[𝐶𝑙,.(0P' ]
= ∆𝜓 + 0.06 × log’L ( Š ), 𝑢𝑛𝑖𝑡 𝑖𝑛 𝑣𝑜𝑙𝑡𝑠.
[𝐶𝑙klP*1 ]
𝐶𝑙 Š flux through VCCN1:
Š
[𝐶𝑙,.(0P' Š
] + [𝐶𝑙klP*1 ]
𝑉ÊJJe’ = 𝑘ÊJJe’_L.’Ê × 𝐶𝑙‹kliWXºÀ–ÁnX ×
2
where 𝑘ÊJJe’_L.’Ê 𝑖𝑠 𝑡ℎ𝑒 𝐶𝑙 Š 𝑓𝑙𝑢𝑥 𝑟𝑎𝑡𝑒 (𝑝𝑒𝑟 𝑃𝑆𝐼𝐼) 𝑤ℎ𝑒𝑛 𝑣 = 0.1 𝑉, 𝑢𝑛𝑖𝑡: 𝑠 Š’ 𝑀Š’ .
CLCe is also postulated as one of the 𝐶𝑙 Š channels/transporters (16), the
differences observed between clce-2 and WT were either small or value based on our
experimental results and others(16, 17). Here, we treat CLCE as a 𝐶𝑙 Š /𝐻 Z antiporter,
with 2 𝐶𝑙 Š moving into the lumen and one H+ out.
Š
[𝐶𝑙,.(0P' Š
] + [𝐶𝑙klP*1 Z
] [𝐻,.(0P' Z
] + [𝐻klP*1 ]
𝑉J¸J‰ = 𝑘J¸J‰ × (𝑣 × 2 + 𝑝𝑚𝑓) × ×
2 2
5
 where 𝑘J¸J‰ 𝑖𝑠 𝑡ℎ𝑒 𝑘𝑖𝑛𝑒𝑡𝑖𝑐 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝑜𝑓 𝐶𝐿𝐶𝑒 𝑤𝑖𝑡ℎ 𝑢𝑛𝑖𝑡 𝑖𝑛 𝑠 Š’ 𝑀ŠS 𝑉 Š’
To simulate a mutant, the kinetic constant(s) of the corresponding ion
channel/transporter(s) is set to zero. Now the script also exports the simulated results to
csv files for comparison.

Other constants
Changed constants or initial values
Constansts in Python code Value in Updated value Unit Notes and
deltapsi.py in references
deltapsi_leaf.py

VDE_max_turnover_number 1 0.08 𝑠 Š’ A slower de-

epoxidation rate is
estimated.
pKvde 5.8 5.65
kZE 0.03 0.004 𝑠 Š’
pKPsbS 6.4 6.2 Hill coefficient is
changed to 3 in the
updated model
ATP_synthase_max_turnover 1000 200 𝑠 Š’ ATP synthase: PSII
= 0.5, and maximum
turnover rate for
ATP synthase is
400/s (2), so per
PSII, 200/s.
Volts_per_charge 0.033 0.047 𝑉𝑜𝑙𝑡 See calculations
below
perm_K 6000 150 𝑠 Š’ 𝑉𝑜𝑙𝑡 Š’
b6f_content 1 0.433 𝑃𝑆𝐼𝐼Š’ Normalized to PSII
max_b6f 500 300 𝑠 Š’ (18)
pKreg 6.5 6.2
lumen_protons_per_turnover 1.4 × 10Š¨ 0.000587 M (or mol/L) See calculation
below
buffering_capacity 0.03 0.014 𝑀 (10, 19, 20) and see
∙ (𝑝𝐻 𝑢𝑛𝑖𝑡)Š’ calculation below
PSI_antenna_size 1 0.5
k_PC_to_P700 500 5000 𝑠 Š’
k_KEA 0 2500000 𝑠 𝑀ŠS
Š’

PSII_antenna_size 1 0.5
k_CBC 3000 60 𝑠 Š’ Normalized and
estimated maximum
NADPH
photosynthetic
oxidation rate (per
PSII).
Klumen_initial 0.04 0.1 𝑀
pHlumen_initial 7.01 7.8 Assuming PMF = 0
in dark adapted state
Dy_initial 0.474 0 𝑉𝑜𝑙𝑡 Assuming PMF = 0
in dark adapted state
Phi2_initial 0.8 0.83
PQ_content_initial 6 7 𝑃𝑆𝐼𝐼Š’
P700_red_initial 1 0.7 𝑃𝑆𝐼𝐼Š’ (3)
ATP_pool_initial 30 4.15 𝑃𝑆𝐼𝐼Š’

6
 NADPH_pool_initial 0 1.5 𝑃𝑆𝐼𝐼Š’ (9)
NADP_pool_initial 1 3.5 𝑃𝑆𝐼𝐼Š’ (9)

Constant calculations:
Our modified model is based on the observations that thylakoid PSII/Chl ratio 3
mmol/mol (3, 10), and thylakoid area per Chl is 1.7 nm2 (10). Thus, when normalized to
per PSII basis, the thylakoid area simulated is
1.7 𝑛𝑚S 3 𝑚𝑚𝑜𝑙𝑃𝑆𝐼𝐼
÷— ™ = 567 𝑛𝑚S /𝑃𝑆𝐼𝐼
𝐶ℎ𝑙 𝑚𝑜𝑙 𝐶ℎ𝑙

Capacitance and “Volts_per_charge”

So, with 0.6 µF/cm2 capacitance for thylakoid membrane, the total capacitance (per PSII)
is
0.6 µF 567𝑛𝑚S
× = 3.4 × 10Š’Ð 𝐹
cmS 𝑃𝑆𝐼𝐼
As a result, for each charge (H+, e-, Cl-, K+) moving across the normalized thylakoid
membrane area (per PSII), the voltage change induced is
3.4 × 10Š’Ð 𝐹
1.6 × 10Š’Ñ 𝐶 ÷ Ò Ó = 0.047𝑣𝑜𝑙𝑡𝑠
𝑃𝑆𝐼𝐼

Lumen volume and “lumen_protons_per_turnover”

Assuming lumen space width between two layer of thylakoid membrane 10 nm, the
volume of the lumen/PSII will be
567 𝑛𝑚S
× 10𝑛𝑚 ÷ 2 = 283 𝑛𝑚Â /𝑃𝑆𝐼𝐼 = 2.83 × 10ŠS’ 𝐿/𝑃𝑆𝐼𝐼
𝑃𝑆𝐼𝐼
The factor 2 is due to the fact that two layers of thylakoid membrane enclose one layer of
lumen.
So, the “lumen_protons_per_turnover” term, or concentration change per one
molecule/ion moving into lumen (per PSII) is
1
— ™ ÷ (2.83 × 10ŠS’ 𝐿) = 0.000587 𝑚𝑜𝑙/𝐿
6.02 × 10SÂ /𝑚𝑜𝑙

Buffering capacity
Per Chl the lumen volume is
1.7 𝑛𝑚S 8.5 𝑛𝑚Â 8.5 × 10ŠS¯ 𝐿
× 10𝑛𝑚 ÷ 2 = = = 5.1 𝐿/(𝑚𝑜𝑙 𝐶ℎ𝑙)
𝐶ℎ𝑙 𝐶ℎ𝑙 1
- 𝑚𝑜𝑙 𝐶ℎ𝑙®
6.02 × 10ŠSÂ
According to references (19, 20), buffer capacity of lumen is 70 mmol / (mol Chl • pH),
so the “buffering_capacity” when expressed in relation to proton concentration change:
70mmol 5.1𝐿 𝑚𝑚𝑜𝑙
÷ = 14 /𝑝𝐻 = 0.014𝑀/𝑝𝐻
mol Chl • pH 𝑚𝑜𝑙 𝐶ℎ𝑙 𝐿

7
Supplementary Figures
SI Fig. 1
a
Genotype Testing insertion Testing WT
kea3_as5: caggccttgaatgaaagaggaattc kea3_as5: caggccttgaatgaaagaggaattc
kea3-1
Sail_LB3: tagcatctgaatttcataaccaatctcgatacac kea3_s1: caccatggcaattagtactatgttagg
vccn1_rvs: catgtcatgtgaagtgaagtgaag vccn1_rvs: catgtcatgtgaagtgaagtgaag
vccn1-1
LBb1.3: attttgccgatttcggaac vccn1_fwd: gctgcaatgtaacgaagaagc
clce_rvs: cccgggcaaagcaagaccatttacagatac clce_rvs: cccgggcaaagcaagaccatttacagatac
clce-2
LBb1.3: attttgccgatttcggaac clce_fwd: ggatccaaaatggcagccacgcttccactc

SI Fig.1. Genotyping of ion transporter mutants. (a) the primers used for identifying the
corresponding ion transporter mutants. For kea3-1 and vccn1, all three primers were
mixed in one PCR reaction since the product of WT and insertion DNA can be resolved
on a single gel. (b) Examples of agarose gel electrophoresis of genotyping PCR.
Genotypes are labeled on top of each lane. Gel images are inverted for clarity. The
genotypes of Arabidopsis plants that were used for propagation and phenotypical
analyses were checked twice from genomic DNA extraction to PCR test. For the PCR
more than 100 runs were done for each genotype test. (c) Uncropped gels used for (b).
(A) PCR products of vccn1 and WT. (B) PCR products of checking WT background
(left) and kea3-1 T-DNA insertion (right). (C) PCR products of checking WT background
(left) and clce-2 T-DNA insertion (right). The color of the images is inverted for clarity.

8
 SI Fig. 2
a b c
PSII cyt b6f complex SDS PAGE, thylakoids
kDa
76
52
38
31
24

17
cv
ck

cv
ck
k

k
t

vk

vk
c

c
v
k

v
k
w

cv
ck

k
vk
c
vc

vc

v
k

t
w
vc
Neo b-Car
d PsbS
e

total Xan Lut

ck
cv

k
vk
c
t
v
k
w

vc

cv
ck
vc k
t
v
k

k
w

SI Fig. 2. Compositional analysis of thylakoid membranes. (a, b) PSII and cyt b6f
complexes were quantified (relative to Chl) by difference absorption spectroscop of cyt
b559 (marker for PSII) and cyt f and b6 (markers for cyt b6f complex). Data show the mean
with standard deviation of n=3 biological replicates. (c) Coomassie stained SDS PAGE
gel showing similar protein content in thylakoid membranes of all genotypes. The gel
was repeated for three biological independent samples given very similar results. (d)
Semi-quantitative analysis of Western blots against PsbS. Data were normalized to the
WT signal that run on the same gel. Data show the mean with standard deviation of n=3
biological replicates. (e) Pigment analysis by rp-HPLC. Data show the mean with
standard deviation of n=9 biological replicates. Two-sided t-test analysis of data in (a, b,
d, e) gives p-values >0.05 (no statistical differences).

9
 SI Fig. 3

c v k cv
total leaf area / cm2

ck vk cvk * * * fluc.
total leaf area / cm2

con.

ck
cv

vk
v
k
t

k
w

vc
SI Fig. 3. Growth rates of ion transporter/channel mutants and WT plants. Plant growth
was measured as change in total leaf area determined by image analysis of Chl
fluorescence pictures recorded in Phenomics chambers (see Methods for details). Data
show the mean with standard deviation of n=5 biological independent samples. Open
symbols indicate constant light, closed symbols fluctuating light conditions, respectively.
Time axes give days after sowing. From linear regressions (solid and dashed lines)
growth rates were calculated summarized in the histogram to the lower right. Horizontal
lines show the growth rates for WT under constant (black) or fluctuating (grey) light. *,
p-value of two-sided t-test: for k, p=0.0500; for ck, p=0.0015, for vck, p=0.0127.

10
 SI Fig. 4
DpH change at ~7.5 min
illumination relative to wt

DDpHECS, a.u.

DDpH
SI Fig. 4. To further validate the DDpH changes in thylakoid ion transporter/channel
mutants we used the fluorescent DpH indicator 9 aminoacridine (9-AA, 22). In contrast to
the previous measurements performed on intact leaves, the 9-AA fluorescence must be
measured with isolated thylakoid membranes. The DDpH derived by 9-AA (grey squares)
is plotted together with the DDpHECS on leaves (black circles) for comparison. DpH
measured by 9-AA in thylakoid in the presence of the PSI acceptor methylviologen was
measured at 3 min illumination that gives a stable signal. ECS data represent the mean of
seven plants with standard deviation, the 9-AA of 11 to 15 independent thylakoid
preparations. The 9-AA data are in good agreement with the DDpHECS measurements with
the exception of the vccn1 mutant. A possible explanation for this discrepancy is that the
Cl- concentration used in the thylakoid membrane experiment does not match the
concentration in intact leaves.

11
 SI Fig. 5
Single mutants
a c v k
WT, 100 µmol quanta
2 2 2
m-2 s-1

NPQ
NPQ

NPQ
WT, 500 µmol quanta
1 1 1
m-2 s-1
mutant, 100 µmol quanta
0 0 0
m-2 s-1
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
mutant, 500 µmol quanta
time / min time / min time / min
m-2 s-1

b Double/triple mutants
cv ck vk vck
2 2 2 2
NPQ

NPQ
NPQ

NPQ
1 1 1 1

0 0 0 0
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
time / min time / min time / min time / min

SI Fig. 5. NPQ data measured on intact WT and mutant plants. Light intensities are
indicated to the right. Light period was from 0 to 20 min. The data were used to calculate
the DNPQ given in Fig. 1. Data show the mean with standard deviation of n=13
biological independent samples.

12
 SI Fig. 6 !O#P'Y 1

]
= 4 O#P' F 4 N! 4 5QR→S2TB − 5J\J_1,A 4

4 [Y,?/
F
!% 2

]−
O#P'Y

] 4 [YXì1/°
'#^ ln − ln(1.25)

= @•JJS_M.u• 4 EíoXìArsîïñóòs 4 [Eí,?/

3=c O#P'
4 (1 − b defg ) 4

= 5JXJ/ 4 (2 4 8õ: + úõ:) 4 Eí,?/

F
'#^ + 250`a ln 3.5 − ln(1.25)

3
CO2
!N!

]
= 5çÄ2ë 4 :çÄ2ïüñ 4 ([†m=-01,

= 5n/-1_ç 4 5õ: 4 [†,?/

= '()*+,-./ − O#P' F 4 N! 4 5QR→S2TB −

F
!%
Calvin Benson

F
@STC − @BUV − @W/+X/-

] 4 [Ym=-01, ])
!'700 +
cycle

F
= '()*+,-./ − 'E 4 '700F 4 5BJ→BLMM
!%

[†Xì1/°
!(89% :)
= <:,*=>?/ 4 @1,A 4 :B6CD 4 'E F − @-/?

F
!%

û
!Eí•JJS

!†•3ç
!Eí JXJ/

!%
!"# −

!%

F
3

Rçàãfù
3

!%
= '())*+,-./ − '())-/*01 − "23 4 '" 4 562

R=
û
!%
R NADP+ NADPH ADP ATP
PG Cl- Cl- K+ K+
H

Fd
ND

VCCN1
NPQ

FNR

KEA3
PsbS

V-K+
ClCe
VDE PSII PQH2
b6f PSI ATP
ase
PC
H+
H2O H+ H+ H+ H+
!#l'1,R/ 1
1 = @2dB_1,A 4 :2dB,m/_-/R 4 1 − +
'|<(Y = !% BWQ3n1op.qrst 4u.vcM.Mw
10 nCÉÑÖsÜ 3nçeéèê 4ë +1 10 +1
1
@2dB_1,A 4 :2dB,m/_0A 4 (1 − )
BWQ3n1op.q_xy 4u.vcM.Mw
Ro~y u 10 +1
= :•A 4 5•TÄ 4 - :}A 4 5}Ä
R= uM ÅÇÉÑÖsÜáÅàâäã 4å Fu
F F F F F F F F
!úYXì1/° !YB¢££ + !Y§wo + !YSTC − !Y2dB,m/ − !YX/,• − !YçÄ2ë − !YJXJ/ Y=ì-°0?/-
O'" = 0.4×O'"1,A ×'|<(Y×:}A + = 4
!% !% <¶::bß 8®ú®8©%9
0.1×O'"1,A ×:}A + 0.5×O'"1,A ×'|<(Y

SI Fig. 6. Detailed mathematical model including full differential equations. For further
details see supplemental methods section. A reverse reaction from P700 to PC+ was not
included because of the high equilibrium constant for this reaction (K ~ 80), i.e. the back
reaction is 80-times slower than the forward reaction (PC to P700+). The apparent constant
can be significantly lower for dark- or low-light-adapted plants owing to kinetic effects
that prevent full local equilibrium. However, for the light intensities studied in our
manuscript we expect the system to approach local equilibrium, and thus the reduction of
P700 should be favored, and we can reasonably ignore the reverse reaction from P700 to
PC+.

13
 SI Fig. 7

SI Fig. 7. Comparison on simulated (lines) and measured (symbols) photosynthetic

parameters without implementation of cyclic electron transport around PSI in our model.
Data show the mean with standard deviation of n=16 biological replicates. The numbers
in the qL plot represent light intensities in µmol quanta m-2 s-1. Note the deviation of
simulated and measured data and the lack of this deviation in Fig. 3b (with cyclic electron
transport) indicating the need to include cyclic electron transport processes for a realistic
description of photosynthetic electron transport and light harvesting.

14
 SI Fig. 8
SI Fig. 5
100 µmol quanta m-2 s-1 500 µmol quanta m-2 s-1

a wt minus vck wt minus vck

0.004 0.004
0.008 0.008 0.008
1e-7 0.002 1e-7 0.002
0 0.006 0 0.006 0.006
0 0
-0.002 0.004 -0.002 0.004 0.004
-1e-7 -0.004 -1e-7
-0.004
0.002 0.002 0.002
-2e-7 -0.006 -2e-7 -0.006
-0.008 0 -0.008 0 0
-3e-7 -3e-7

0 1 2 3 4 5 min 0 1 2 3 4 5 min

b wt minus clce wt minus clce

3e-7 0.008 3e-7 0.008

0.008 0.008 0.008
0.006 0.006
2e-7 2e-7
0.004 0.006 0.004 0.006 0.006
1e-7 1e-7 0.002
0.002 0.004 0.004
0.004
0 0 0 0
0.002 0.002 0.002
-0.002 -0.002
-1e-7 -1e-7
0 -0.004 0 0
-0.004

SI Fig. 8. Simulated behaviors of lumen acidification, electrical gradient (Dy), as well as

potassium and chloride fluxes into the thylakoid lumen over 5 min of illumination with
100 (left) or 500 (right) µmol quanta m-2 s-1 for (a) the triple mutant (vck) and (b) the
Clce mutant. The data are expressed as WT minus mutant.

15
 SI Fig. 9 With KEA3 regulation Without KEA3 regulation
measured, 100 µmol quanta m-2 s-1 simulated, 100 µmol quanta m-2 s-1
measured, 500 µmol quanta m-2 s-1 simulated, 500 µmol quanta m-2 s-1

SI Fig. 9. Comparison measured DNPQ with simulated data for a scenario with KEA3
regulation (left) and without such regulation (right) for all mutants with KEA3 loss-of-
function. Note that measured data can be well described by simulation at 100 µmol
quanta m-2 s-1 irrespectively of KEA3 regulation (black points and lines). In contrast, the
simulation describes the experimental data much better with KEA3 regulation at 500
µmol quanta m-2 s-1 (red points and lines). This indicates that KEA3 activity is
downregulated at higher light intensities.

16
SI Fig. 10

100 10 1 0.1 0.01

SI Fig. 10. Dependencies of DNPQ on the abundance of KEA3 antiporter and VCCN1
chloride channel in thylakoid membranes for tow different light intensities.

17
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