Deletion of Proton Gradient Regulation 5 (PGR5) and PGR5-Like 1 (PGRL1) proteins promote sustainable light-driven hydrogen production in Chlamydomonas reinhardtii due to increased PSII activity under sulfur deprivation

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Continuous hydrogen photo-production under sulfur deprivation was studied in the Chlamydomonas reinhardtii pgr5 pgrl1 double mutant and respective single mutants. Under medium light conditions, the pgr5 exhibited the highest performance and produced about eight times more hydrogen than the wild type, making pgr5 one of the most efficient hydrogen producer reported so far. The pgr5 pgrl1 double mutant showed an increased hydrogen burst at the beginning of sulfur deprivation under high light conditions, but in this case the overall amount of hydrogen produced by pgr5 pgrl1 as well as pgr5 was diminished due to photo-inhibition and increased degradation of PSI. In contrast, the pgrl1 was effective in hydrogen production in both high and low light. Blocking photosynthetic electron transfer by DCMU stopped hydrogen production almost completely in the mutant strains, indicating that the main pathway of electrons toward enhanced hydrogen production is via linear electron transport. Indeed, PSII remained more active and stable in the pgr mutant strains as compared to the wild type. Since transition to anaerobiosis was faster and could be maintained due to an increased oxygen consumption capacity, this likely preserves PSII from photo-oxidative damage in the pgr mutants. Hence, we conclude that increased hydrogen production under sulfur deprivation in the pgr5 and pgrl1 mutants is caused by an increased stability of PSII permitting sustainable light-driven hydrogen production in Chlamydomonas reinhardtii.

본문

_Edited by:_

_Flavia Vischi Winck,_

_Brazilian Center for Research_

_in Energy_ _and Materials, Brazil_

_Reviewed by:_

_Mikko Tikkanen,_

_University_ _of Turku,_ _Finland_

_Toshiharu_ _Shikanai,_

_Kyoto_ _University, Japan_

_Giuseppe_ _Torzillo,_

_Consiglio Nazionale_ _delle Ricerche,_

_Italy_

_*Correspondence:_

_Michael Hippler_

_mhippler@uni-muenster.de_

_†Present address:_

_Leonardo_ _Magneschi,_

_CNRS, UMR 5168,_ _Laboratoire_ _de_

_Physiologie Cellulaire et Végétale,_

_Commissariat à l’Energie_ _Atomique_ _et_

_aux Energies_ _Alternatives – Institut_

_National Recherche_ _Agronomique,_

_Institut de Recherche_ _en Sciences_ _et_

_Technologies_ _pour le Vivant,_

_Université_ _Grenoble_ _Alpes, Grenoble,_

_France_

_Specialty section:_

_This article was submitted to_

_Plant Biotechnology,_

_a section_ _of the journal_

_Frontiers_ _in Plant Science_

_Received:_ _31 July 2015_

_Accepted: 07 October_ _2015_

_Published: 27 October_ _2015_

_Citation:_

_Steinbeck J, Nikolova D,_

_Weingarten_ _R, Johnson_ _X, Richaud_ _P,_

_Peltier G, Hermann M, Magneschi_ _L_

_and Hippler_ _M (2015)_ _Deletion_

_of Proton_ _Gradient_ _Regulation_ _5_

_(PGR5) and PGR5-Like 1 (PGRL1)_

_proteins_ _promote_ _sustainable_

_light-driven_ _hydrogen_ _production_

_in Chlamydomonas_ _reinhardtii_ _due_

_to increased_ _PSII activity under_ _sulfur_

_deprivation._ _Front._ _Plant Sci. 6:892._

_[doi: 10.3389/fpls.2015.00892](http://dx.doi.org/10.3389/fpls.2015.00892)_

[ORIGINAL RESEARCH](http://www.frontiersin.org/Plant_Science/editorialboard)
published: [27 October](http://www.frontiersin.org/Plant_Science/editorialboard) 2015
[doi: 10.3389/fpls.2015.00892](http://dx.doi.org/10.3389/fpls.2015.00892)

# Deletion of Proton Gradient Regulation 5 (PGR5) and PGR5-Like 1 (PGRL1) proteins promote sustainable light-driven hydrogen production in Chlamydomonas reinhardtii due to increased PSII activity under sulfur deprivation

_[Janina Steinbeck](http://loop.frontiersin.org/people/262114/overview)_ _1_ _[, Denitsa Nikolova](http://loop.frontiersin.org/people/262533/overview)_ _1_ _[, Robert Weingarten](http://loop.frontiersin.org/people/264520/overview)_ _1_ _[, Xenie Johnson](http://loop.frontiersin.org/people/261107/overview)_ _2,3,4_ _,_
_[Pierre Richaud](http://loop.frontiersin.org/people/266044/overview)_ _2,3,4_ _, Gilles Peltier_ _2,3,4_ _[, Marita Hermann](http://loop.frontiersin.org/people/284345/overview)_ _1_ _[, Leonardo Magneschi](http://loop.frontiersin.org/people/86170/overview)_ _1†_ _and_
_[Michael Hippler](http://loop.frontiersin.org/people/26501/overview)_ _1_ _*_

_1 Institute of Plant Biology and Biotechnology,_ _University of Münster, Münster, Germany,_ _2 Laboratoire_ _de Bioénergétique_ _et_

_Biotechnologie_ _des Bactéries et Microalgues,_ _Institut_ _de Biologie Environnementale_ _et de Biotechnologie,_ _Direction_ _des_

_Sciences du Vivant, Commissariat à l’Energie_ _Atomique et aux Energies_ _Alternatives,_ _Saint-Paul-lez-Durance, France,_

_3 CNRS, UMR 7265,_ _Biologie Végétale et Microbiologie_ _Environnementale,_ _Saint-Paul-lez-Durance,_ _France,_ _4 Laboratoire_ _de_

_Bioénergétique_ _et Biotechnologie_ _des Bactéries et Microalgues,_ _Aix Marseille Université, Saint-Paul-lez-Durance,_ _France_

Continuous hydrogen photo-production under sulfur deprivation was studied in the

_Chlamydomonas_ _reinhardtii_ _pgr5_ _pgrl1_ double mutant and respective single mutants.

Under medium light conditions, the _pgr5_ exhibited the highest performance and

produced about eight times more hydrogen than the wild type, making _pgr5_ one of

the most efficient hydrogen producer reported so far. The _pgr5_ _pgrl1_ double mutant

showed an increased hydrogen burst at the beginning of sulfur deprivation under high

light conditions, but in this case the overall amount of hydrogen produced by _pgr5 pgrl1_

as well as _pgr5_ was diminished due to photo-inhibition and increased degradation of

PSI. In contrast, the _pgrl1_ was effective in hydrogen production in both high and low

light. Blocking photosynthetic electron transfer by DCMU stopped hydrogen production

almost completely in the mutant strains, indicating that the main pathway of electrons

toward enhanced hydrogen production is via linear electron transport. Indeed, PSII

remained more active and stable in the _pgr_ mutant strains as compared to the wild type.

Since transition to anaerobiosis was faster and could be maintained due to an increased

oxygen consumption capacity, this likely preserves PSII from photo-oxidative damage in

the _pgr_ mutants. Hence, we conclude that increased hydrogen production under sulfur

deprivation in the _pgr5_ and _pgrl1_ mutants is caused by an increased stability of PSII

permitting sustainable light-driven hydrogen production in _Chlamydomonas reinhardtii_ .

Keywords: PGR5, PGRL1, hydrogen production, PSII stability, sulfur deprivation

INTRODUCTION

Solar fuels are an important motive for the development of
future renewable energy systems with zero CO2 emission. This
development is necessary to meet one of the most urgent
challenges of our society today, to counter the problems of
global warming, fossil fuel depletion, concurrent increasing
energy demand, and consequently the maintenance of economic
and political stability (Organisation for Economic Co-operation
and Development (OECD)/International Energy Agency (IEA),
2011). Among other fuels, hydrogen is considered to be one
of the most effective and clean fuels (Hankamer et al., 2007).
Solar-driven H2 production by photosynthetic microorganisms,
particularly cyanobacteria and microalgae, is a promising
complement to clean and sustainable technologies of hydrogen
production beside chemical techniques.

Photobiological hydrogen production was first discovered by
Gaffron and Rubin, 1942. In this process, electrons and protons
from water splitting are directed via photosynthesis toward
specific H2-evolving enzymes, the hydrogenases. The algal FeFe hydrogenase is very efficient compared to other hydrogenases
(turnover rate in thousands per second, 100-fold higher than
other hydrogenases; Volgusheva et al., 2013; Lubitz et al., 2014).
However, direct light-to-hydrogen conversion efficiency is very
low, because hydrogenase activity is extremely sensitive to oxygen
(Ghirardi et al., 1997; Rupprecht et al., 2006; Stripp et al., 2009),
thus oxygenic photosynthesis cannot easily be directly coupled
to hydrogen production in green algae. Therefore, hydrogen
production is a transient phenomenon in nature and stops after a
few minutes of illumination (Bishop and Gaffron, 1963).

Melis et al. (2000) proposed an experimental protocol
for prolong H2 evolution based on sulfur deprivation that
circumvents this limitation. This method allows the separation
of photosynthetic oxygen evolution and hydrogen production by
a two stage process: In the first phase of cell cultivation, oxygenic
photosynthesis drives production of biomass and carbohydrate
stores in the presence of acetate as an additional carbon source.
Shifting cells to sulfur depleted medium in sealed flasks induces
the switch to the second anaerobic stage, inducing hydrogenase
expression and sustainable H2 production for several days.
During acclimation to this nutrient stress, cells stop dividing and
undergo morphological changes (Zhang et al., 2002). Both light
and dark reactions of photosynthesis are down-regulated, with
the amounts of Rubisco being substantially reduced within the
first 24 h of sulfur starvation (Zhang et al., 2002). Photosystem
II activity drops gradually, attributed to an impaired PSII repair
cycle due to the restricted _de_ _novo_ synthesis of the D1 reaction
center protein (which contains methionines and cysteines) by
S-limiting conditions, driving down O2 evolution (Wykoff et al.,
1998). When O2 consumption overtakes O2 evolution, anaerobic
conditions are established. However, active PSII in the first hours
of S-depletion was shown to be essential for H2 generation, as no
hydrogen evolution could be observed when the PSII inhibitor
DCMU was added directly after transfer to S-free medium
(Fouchard et al., 2005; Hemschemeier et al., 2008). Indeed,
PSII activity was reported to contribute a substantial amount of
electrons from water oxidation at PSII to hydrogen production

(Antal et al., 2003; Kosourov et al., 2003). Beside this direct
PSII-dependent pathway, electrons toward the hydrogenase can
also derive from an indirect pathway that relies on nonphotochemical reduction of PQs from metabolites such as starch
(Fouchard et al., 2005; Jans et al., 2008; Chochois et al., 2009).
Starch is massively accumulated during the first hours of –S
conditions and is subsequently degraded (Zhang et al., 2002).
The direct sunlight to hydrogen pathway has nevertheless the
potential for higher energy conversion efficiency.

The improvement in hydrogen production by the method
of sulfur deprivation implicated the highest efficiency for
photobiological systems reported by then (Rupprecht et al.,
2006). Yet, this H2 production efficiency has still to be
advanced to reach economic profitability. Optimization of
the electron supply to the hydrogenase appears to be a
critical issue. Since the majority of electrons toward the
hydrogenase have been shown to derive from PSII activity,
but O2 evolution by PSII has to be prevented not to inhibit
hydrogenase activity, possible solution scenarios involve either
a more O2 tolerant hydrogenase or a more efficient oxygen
scavenging system. Engineering resulting in a decrease in
the O2 sensitivity of the Fe–Fe hydrogenase has not been
reported yet. Among some other _Chlamydomonas_ _reinhardtii_
mutant strains, the _state_ _transition_ _6_ ( _stm6_ ) mutant lacking
the mitochondrial respiratory chain assembly factor _moc1_,
and the _proton_ _gradient_ _regulation_ _like_ _1_ ( _pgrl1_ ) mutant
displayed the highest improved hydrogen production rates
(Kruse et al., 2005; Tolleter et al., 2011). The PGRL1 protein
was first discovered in _Arabidopsis_ (DalCorso et al., 2008)
as an essential component of the PGR5 (proton gradient
regulation 5) dependent cyclic electron flow (CEF) pathway
(Munekage et al., 2002). Moreover, PGRL1 has been suggested
to operate as the elusive ferredoxin-plastoquinone reductase
(Hertle et al., 2013). In _Chlamydomonas_, PGRL1 is also required
for effective CEF (Petroutsos et al., 2009; Tolleter et al.,
2011) and suggested to be a functional component of a CEFsupercomplex formed under CEF promoting conditions (Iwai
et al., 2010; Terashima et al., 2012). A _pgr5_ mutant has recently
been characterized in _Chlamydomonas_ (Johnson et al., 2014),
which revealed that PGR5 deficiency leads to a diminished
proton gradient across the thylakoid membrane accompanied
with less effective CEF capacity. The proton gradient across
the thylakoid membrane was shown to restrict electron flow
toward the hydrogenase in _pgrl1_, and also proton uncouplers like
carbonyl cyanide- _p_ -trifluoromethoxyphenylhydrazone (FCCP)
and nigericin increased hydrogen evolution (Antal et al., 2009;
Tolleter et al., 2011). Interestingly, both _stm6_ and _pgrl1_ share
the characteristic of an increased respiration rate (Kruse et al.,
2005; Petroutsos et al., 2009; Dang et al., 2014). Enhanced
H2 production in _stm6_ was recently linked to prolonged PSII
activity under sulfur starvation (Volgusheva et al., 2013). In
this study, we investigated hydrogen production under similar
conditions in the PGR5-deficient mutant and a _pgr5 pgrl1_ double
mutant in comparison to the _pgrl1_ mutant. These mutants also
exhibit enhanced hydrogen production and higher PSII stability
under –S conditions. Since blocking PSII activity by DCMU
abolished hydrogen production almost completely, we conclude

that electrons for enhanced hydrogen production mainly derive
from PSII. Anaerobiosis and therefore hydrogenase activity can
still be maintained due to a higher oxygen consumption capacity
in the mutants.

MATERIALS AND METHODS

Strains and Growth Conditions
The _Chlamydomonas_ _reinhardtii_ _pgrl1_ (Tolleter et al., 2011)
mutant was back-crossed four times with the wild type (wt)
strain CC124 (137c, nit2 −, mt − ) and then mated to _pgr5_ (137c
background; Johnson et al., 2014) in order to obtain _pgr5_
_pgrl1_ double mutants in the 137c background. Zygotes were
germinated on paromomycin plates (10 mg mL −1 ) and progeny
was screened for both insertion of the _AphVIII_ resistance marker
within the _PGRL1_ coding sequence (primers AphVIII_Fw1,
5 ′ -TCGGGCCGGAGTGTTCC-3 ′ and PGRL1_Rev, 5 ′ -TTACGC
AGCGGCCTTAGCC-3 ′ ) and deletion of the PGR5 locus
(primers PGR5 FW2, 5 ′ -CTACTCGCAGCCAAAACACA-3 ′

and PGR5 REV2, 5 ′ -GGAAACCAGTGTGCAAGTCA-3 ′ ).
Mating type of the isolated clones was assessed with primers
MID_Fw (5 ′ -ATGGCCTGTTTCTTAGC-3 ′ ), MID_Rev (5 ′ -CTA
CATGTGTTTCTTGACG-3 ′ ), FUS1_Fw (5 ′ -ATGCCTATCTT
TCTCATTCT-3 ′ ) and FUS1_Rev (5 ′ -GCAAAATACACGTCT
GGAAG-3 ′ ).

Wild-type, _pgrl1_, _pgr5,_ and the _pgr5_ _pgrl1_ double mutants
were maintained on TAP medium (Harris, 1989), pH 7.0,
solidified with 1.2% (w/v) agar at 25 ◦ C and 50 µE m −2 s −1

photosynthetically active, constant irradiance, and in case of
the mutants supplemented with paromomycin (5 µg mL −1 ).
Strains were cultured in standard TAP medium at 25 ◦ C under
continuous light of 50 µmol photons m −2 s −1 on a rotary shaker
(120 rpm).

Long-term Hydrogen Production
Long-term hydrogen production was measured under conditions
of sulfur deficiency (Melis et al., 2000). Cultures grown in
TAP medium to a chlorophyll concentration of 20–25 mg L −1

were washed twice and resuspended (2500 × _g_, 5 min) in
sulfur-deprived medium. Sealed in 250-ml glass flasks (Schott,
conventional measuring system) or 1000-ml flasks (BlueSens gas
measuring system) at a final chlorophyll concentration of 14–
15 mg L −1, cells were incubated at room temperature, constant
stirring and under continuous one-side illumination at medium
light (60 µE m −2 s −1 ) or high light (200 µE m −2 s −1 ). If
indicated, DCMU (10 µM final concentration) or Lincomycin
(2 mM final conc.) were added 48 h after sealing. For the
conventional measuring system, the gas phase was removed
daily, with H2 and O2 concentrations analyzed from a
0.2 ml gas sample each day by gas chromatography (GC2010, Shimadzu). Liquid samples (1 ml) were taken for
immunoblot and pulse-amplitude modulated (PAM) chlorophyll
fluorometry analysis. For the BlueSens gas measuring system,
the gas phase was constantly removed and analyzed every
20 s with online gas sensors (BCP, BlueSens gas sensor
GmbH).

Chlorophyll Fluorescence Measurements
Fluorescence was measured at room temperature using a MaxiImaging PAM chlorophyll fluorometer (Heinz Walz GmbH).
Samples were dark adapted for 20 min to maximally oxidize QA
before each measurement. The effective photochemical quantum
yield of PSII was measured as PSII yield [Y(II) = (Fm ′ - F)/Fm ′ ].

Mass Inlet Membrane Spectroscopy
(MIMS) Analysis of O2 Exchange
Mass Inlet Membrane Spectroscopy (MIMS) analysis was
performed as described in Johnson et al. (2014) and a detailed
description of the protocols for analyzing MIMS data are
provided in Cournac et al. (2002). For O2 exchange experiments,
cultures were grown in TAP at 10 µmol photons m −2 s −1

illumination. They were then centrifuged and resuspended in
Minimal media and incubated for 24 h in 120 µmol photons
m −2 s −1 of light with 2% CO2 in an INFORS. Cells were then
centrifuged and resuspended in fresh minimal medium without
the addition of HCO3 − to a concentration of 10 mg mL −1

chlorophyll and left in the dark for 30 min before performing the
experiments; 1.5 mL of the concentrated culture was added to the
MIMS cuvette. Cells were incubated in the dark in the cuvette
until the oxygen isotopes were in equilibrium and then exposed
to a single light intensity at a time for around 10 min.

RESULTS

The depletion and/or absence of the PGRL1 induced increased
hydrogen production under sulfur-deficiency in _Chlamydomonas_
_reinhardtii_ (Tolleter et al., 2011). Here, we investigate hydrogen
production under similar conditions in a PGR5-deficient mutant
and a _pgr5 pgrl1_ double mutant.

Generation of the _pgr5 pgrl1_ Double
Mutant
Both _pgr5_ (Johnson et al., 2014) and _pgrl1_ (Tolleter et al.,
2011) were obtained by insertional mutagenesis with the _AphVIII_
cassette, conferring resistance to the antibiotic paromomycin.
In _pgr5_, integration of this cassette resulted in a complete
deletion of the PGR5 locus, a phenomenon already observed
in other insertional mutants (Peers et al., 2009; Johnson et al.,
2014). In _pgrl1,_ on the other hand, insertion of the _AphVIII_
cassette occurred at the level of the first exon, resulting in

Immunoblot Analysis
Whole cell samples (50 µg total protein, measured by Pierce

⃝R

BCA TM Protein Assay Kit) were analyzed by discontinuous
13% SDS-PAGE according to Laemmli (1970) and transferred
to nitrocellulose membrane (Hybond ECL membrane, GE
Healthcare), which was incubated with antibodies against PSAD
(1:1000, Naumann et al., 2005), ATPB CF1 (1:10 000, Agrisera)
and PSBA D1 (1:2500, Agrisera) as described (Hippler et al.,
2001; Naumann et al., 2005). Secondary antibody was anti-rabbit
(Invitrogen) and signal detection was by enhanced chemical
luminescence (ECL).

lack of accumulation of the PGRL1 protein (Tolleter et al.,
2011; Kukuczka et al., 2014). We took advantage of these
genetic differences to screen for _pgr5_ _pgrl1_ double mutants.
Primers spanning the first 500 bp of the _PGR5_ locus were used
to differentiate wt-like (band) from _pgr5_ (no band) progeny
(Supplementary Figure S1). To select for absence of the PGRL1
protein in this _pgr5_ background, we assessed integration of the
resistance cassette inside the first exon of the _PGRL1_ gene by
_AphVIII_ - and _PGRL1_ -specific primers. Successful amplification
of a ∼800 bp fragment represents the signature of the _pgrl1_
mutant background (Supplementary Figure S1). From this
screening, three independent _pgr5_ _pgrl1_ double mutants were
isolated, two in the mating type minus (mt–) and one in the
mating type plus (mt+). The mt– _pgr5_ _pgrl1_ double mutant
#15 was further characterized at the physiological level, since it
revealed the highest H2 production rates in a pre-screening test.

Enhanced Long-term Hydrogen
Production in the _pgr_ Mutants under
Different Light Conditions
Continuous hydrogen production under sulfur deprivation was
measured with two independent measuring systems over a period
of up to 14 days ( Figures 1A and 2 ). The first approach
used sealed flasks with a gas collection apparatus similar to
that previously reported (Tolleter et al., 2011; Figure 1 ) from
which gas was removed on a daily basis and analyzed using gas
chromatography. Second, a continuous gas measuring system
(BCP, BlueSens gas sensors GmbH) attached to the culture
flasks was tested ( Figure 2 ). Data collected with the BlueSens
gas sensor system agreed well with data collected using the
conventional apparatus, with a slight decrease in hydrogen
production volumes ( Figures 1A and 2B ). Continuous gas
measurements have the ability to dissect more accurately the
different phases of sulfur deprivation: (i) the O2 evolution phase,
(ii) the O2 consumption phase, (iii) the anaerobic phase, and (iv)
the H2 production phase. All four phases could be observed with
the continuous BlueSens gas measuring system in the wild type,
with a short transition from anaerobiosis to hydrogen production
( Figure 2C ), while the transition from oxygen consumption to
complete anaerobiosis toward the onset of hydrogen production
was less discrete using the conventional apparatus ( Figure 1D ).
Both experimental setups, however, independently showed that
oxygen consumption capacity was increased in the _pgr_ mutants
( Figures 1D and 2C ). Therefore, they reached anaerobiosis faster
and started producing hydrogen earlier than the wild type, and
stayed anaerobic during the remaining time of sulfur deprivation
( Figures 1A,D and 2B,C ). This was especially pronounced in the
_pgr5_ mutant under medium light conditions.

The _pgr_ mutants showed enhanced hydrogen production with
four times higher maximal production rates than the wild type
(7 ml L −1 culture per hour, Figures 1A and 2 ). The _pgr5_ mutant
produced 7–10 times more hydrogen than the wild type in
continuous medium light ( Figures 1A and 2B ). With an overall
volume of more than 800 ml hydrogen per liter (at 15 µg ml −1

chlorophyll, Figure 1A ), _pgr5_ exceeded the hydrogen production
of the _pgrl1_ mutant by about 1.5 times ( Figure 1A ). Total

FIGURE 1 | Continued

Continues hydrogen production is enhanced in _pgr5_, _pgrl1_, _pgr5 pgrl1_
compared to the wild type. (A) Long-term hydrogen production of _pgr5_,
_pgrl1_, _pgr5_ _pgrl1_ and wild type under sulfur deprivation illuminated at 60 µmol
photons m −2 s −1 ( _n_ = 4). (B) Total hydrogen volumes produced by _pgr_
mutants and the wild type after 9 days of sulfur deprivation in the presence of
DCMU (10 µM final conc.) or lincomycin (2 mM final conc.) compared to the
control ( _n_ = 2). (C) Fv/Fm values measured after dark acclimation for 20 min
of 200 µL aliquots from the S-deprived cultures ( _n_ = 4). (D) O2 (left axis) and
H2 (right axis) concentrations of the gas phase of the cultures measured daily
by gas chromatography. Concentrations remained constant after cells
reached complete anaerobiosis on day 4 ( _n_ = 3). Data show mean ± SD.

hydrogen volumes by _pgrl1_ of 600 ml L −1 culture correspond
to ∼1.6 µmol H2 µg −1 chlorophyll and were therefore in good
agreement with previous published results (Tolleter et al., 2011).
The _pgr5_ _pgrl1_ double mutant was as effective in hydrogen
production as the _pgrl1_ mutant under medium light.

Under elevated light conditions, oxygen evolution in _pgr5_ and
_pgrl1_ was increased compared to medium light ( Figures 2C,E ),
leading to a longer oxygen evolution phase. In contrast,

the double mutant reached the anaerobic phase even faster
( Figure 2E ) and showed an increased hydrogen burst at the
beginning of sulfur deprivation ( Figure 2D ). However, it stopped
producing hydrogen much earlier compared to medium light
conditions. Also _pgr5_ produced less hydrogen when exposed
to high light, while _pgrl1_ remained effective in H2 production
in both light settings ( Figure 2, Supplementary Figure S2).
Fractionation of whole cells by SDS-PAGE and immunoblotting
using anti-PSAD and anti-PSBA antibodies revealed differences
in the stability of the two photosystems ( Figure 3 ). After 5 days
of high light exposure under sulfur deprivation, the PSI complex
in pgr5 was severely degraded as indicated by the strong decrease
in the PSAD immunoblot signal (∼70%). In the absence of both
PGR5 and PGRL1, this effect was even greater, since by day
4, PSI levels were already low and the double mutant stopped
hydrogen production completely by day 5. At the same time,
levels of PSII, which is known to be prone to degradation under
sulfur deprivation (Melis et al., 2000), remained comparably
stable up to day 5. Thus the observed decrease in hydrogen
production in _pgr5_ and the double mutant can be attributed
to increased photosensitivity of PSI in the absence of PGR5.

FIGURE 2 | Long-term hydrogen production under sulfur deprivation at different light conditions acquired with the continuous BluSens gas
measuring system. (A) Picture of the BlueSens 1 L-photobioreactor (PBR) gas measuring system. (B,D) represent continuous hydrogen production of _pgr5_, _pgrl1_,
_pgr5_ _pgrl1_ and the wild type at medium light (60 µmol photons m −2 s −1, _n_ = 1) and high light (200 µmol photons m −2 s −1, _n_ = 1, 2 for _pgr5_ _pgrl1_ ), respectively.
(C,E) show the corresponding O2 (left axis, dotted line) and H2 (right axis, solid line) concentrations of the gas phase of the cultures measured every 20 s for medium
and high light, respectively. Since gas concentrations remained constant after day 8, data are only shown till day 9 here ( _n_ = 1).

FIGURE 3 | Immunoblot analysis of the PSII-subunit PSBA D1 and the PSI-subunit PSAD during 5 days of sulfur deprivation in the wild type, _pgrl1_,
_pgr5,_ and _pgr5 pgrl1_ mutant strains under high light conditions reveal that PSAD is severely degraded in _pgr5 pgrl1_ and _pgr5_ . ATPase subunit CF1
served as loading control. Samples represent 50 µg total protein extract from high light exposed sulfur deprived cells. The corresponding hydrogen production and
PSII maximum efficiency measurements are shown in Supplementary Figure S2.

Moreover, the augmented decrease in PSI in the absence of PGR5
and PGRL1 indicate an additive effect between these two gene
products, which are known to interact physically (DalCorso et al.,
2008).

The _pgr_ Mutants show Prolonged PSII
Activity and Increased Oxygen
Consumption Capacity under Sulfur
Deprivation Leading to Increased
Hydrogen Production
Sustainable hydrogen production under continuous light
requires low oxygen concentrations due to the extremely oxygen
sensitive Fe–Fe hydrogenases. Sulfur deprivation was reported
to achieve anaerobiosis by down-regulating the activity of PSII
(Melis et al., 2000). However, the maximum efficiency of PSII
photochemistry, measured as the Fv/Fm values after 20 min of
dark adaption (Baker, 2008), remained significantly higher in
the _pgr_ mutants under such sulfur limiting conditions compared
to the control (wild type –S, Figure 1C ). Despite this, _pgr_ cells
became anaerobic after 2 days of S deprivation, two times faster
than the wild type ( Figures 1D and 2D ). This suggests that
in the _pgr_ mutants, the driving force behind the more rapid
attainment of anaerobiosis is not primarily the loss of PSII
activity but instead is mediated by higher rates of respiration
and light-dependent oxygen uptake, as demonstrated for _pgrl1_
by Petroutsos et al. (2009), Tolleter et al. (2011), and Dang et al.
(2014). This leads to a greater capacity to consume oxygen,
even though some loss of PSII activity does occur. To test
whether oxygen uptake is also increased in the _pgr5_ mutant,
we applied a MIMS technique that uses isotopic oxygen to
differentiate between the rates of oxygen uptake in the light
and photosynthetic oxygen evolution. Oxygen evolution (E) at
different light intensities is similar between the wild type and
_pgr5_ showing that photosynthetic rates are not affected in _pgr5_
under the conditions used ( Figure 4, dotted lines). In contrast,
oxygen uptake (U) rates in the light are higher at all light
intensities in the _pgr5_ strain compared to the wild type ( Figure 4,
solid lines). This is clearly demonstrated by the oxygen uptake
in the light plotted as a percentage of total oxygen exchange
(U/E + U = U/T): while wild type U/T is around 40%, the
_pgr5_ mutant is around 60%, with the highest tendency at the

lowest light intensity. This supports the idea that respiratory
and/or light induced oxygen photo-reduction pathways are
considerably more active in the _pgr5_ mutant than in the wild
type.

The decline in Fv/Fm is similar between _pgr_ mutants and wild
type up to 1.5 days of sulfur deprivation. By the time the wild type
reached complete anaerobiosis after 3.5 days of sulfur starvation,
PSII activity already dropped to 10% ( Figures 1C,D and 2D ).
In contrast, PSII activity in the _pgr_ mutants remained at ∼60–
85% of the starting cultures by the time they reached anaerobiosis
and remained higher compared to the wild type throughout the
entire period of sulfur deprivation ( Figure 1C ). Thus, higher
oxygen consumption capacities in the _pgr_ mutants lead to faster
induction and maintenance of anaerobiosis, thereby preserving
PSII activity under –S conditions.

To test whether the electrons for increased hydrogen evolution
could be derived from residual PSII activity in the _pgr_ mutants,

FIGURE 4 | Gas exchange analysis of competing O2 flows in the light
comparing the wt and _pgr5_ strains. The left _y_ -axis shows the rate of O2
Evolution (E) or O2 Uptake in the Light (U), normalized to the chlorophyll
content of the cells. The right _y_ -axis shows a ratio (U/T) between O2 Uptake in
the light (U) and Total O2 exchange (U + E = T):. The _x_ -axis shows the actinic
light intensities used to excite photosynthesis in the cuvette. Data show
mean ± SD ( _n_ = 3).

the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
was added to the cultures after 2 days of sulfur starvation to
block linear electron transfer at the level of the QB side of PSII.
This time point was chosen to be able to distinguish between the
contribution of the PSII-direct and indirect pathway of electrons
toward the hydrogenase (Fouchard et al., 2005; Hemschemeier
et al., 2008). The link between photosynthetic water oxidation
and hydrogen production, and the mechanism behind it,
is important for understanding photobiological hydrogen
production and its further development (Volgusheva et al.,
2013). Indeed, addition of DCMU blocked hydrogen production
immediately ( Figure 1B ). Hydrogen volumes displayed in
Figure 1B were mainly produced prior to addition of DCMU,
with a residual amount of only 1% produced after adding
DCMU. This indicates that electrons from linear photosynthetic
electron transfer driven by the residual PSII activity contribute
to hydrogenase-mediated H2-production, with elevated oxygen
consumption being responsible for driving down O2 levels to
achieve rapid anaerobiosis.

While the chlorophyll content remained stable in all strains,
the chlorophyll _a/b_ ratio of the wild type increased by 20%
from 2.3 to 2.8 within 5 days of sulfur starvation. In contrast,
the chlorophyll _a/b_ ratio remained constant at 2.35 in all _pgr_
mutants, an indication that PSII could be less degraded. In
agreement, the PSBA amounts as revealed by the immunoblots
analyses of sulfur-deprived cells showed a higher stability than
the wt at certain time points in the _pgr_ mutants ( Figure 3 ).
To further investigate whether PSII is more stable in the
thylakoid membranes of the _pgr_ mutants, the effect of the
prokaryotic translation inhibitor lincomycin on PSII activity and
hydrogen production was investigated ( Figures 1B,C ). Inhibiting
chloroplast translation at day 2 of sulfur starvation reduced
the PSII activity of the mutants as well as the wild type in
a similar manner with 50% decreased Fv/Fm levels by day 5
compared to the sulfur deprived control samples ( Figure 1C ).
The decrease in hydrogen production by 33% ± 10% was also
similar between strains ( Figure 1B ). However, Fv/Fm levels
and hydrogen production rates still remained higher in the _pgr_
mutants compared to the wild type. This supports the conclusion
that a substantial amount of electrons for hydrogen production
in the _pgr_ mutants derived from PSII. Moreover, as lincomycin
had a similar effect on wild type as well as the _pgr_ mutants,
it can be assumed that the larger residual PSII activity under

sulfur-deficiency is sustained due to the absence of PGR5 or
PGRL1. However, it cannot be distinguished whether this is due
to an altered repair cycle or a higher stability of PSII in the
membrane.

DISCUSSION

In the present study, we investigated _pgr5_ and _pgr5 pgrl1_ double
mutants in comparison to a PGRL1 deficient mutant and wild
type in their capacity of light-driven hydrogen production. As
already reported earlier for PGRL1 deficiency in _C._ _reinhardtii_
(Tolleter et al., 2011), the absence of the _pgr5_ gene product also
increased the capacity of hydrogen production as compared to
wild type significantly. Importantly, the _pgr5_ mutant is, with
hydrogen volumes of up to 10 times higher than the wild type,
one of the highest hydrogen producers reported to date. The
maximum amounts of 850 ml L −1 culture (at 15 µg ml −1

chlorophyll referring to 2.4 µmol H2 µg −1 chlorophyll) exceed
reported volumes of _pgrl1_ and other high hydrogen producers
like _stm6_, _stm6glc4_, _LO1_, or the PSII D1 mutant _L159I-N230Y_
(see Table 1, Kruse et al., 2005; Doebbe et al., 2007; Torzillo
et al., 2009; Oey et al., 2013). Our data indicate that (i) the
ability to maintain linear photosynthetic electron flow rates
under S deplete conditions in conjunction with (ii) an increased
oxygen consumption capacity explains the increased hydrogen
production in the _pgr_ mutants.

Elevated Hydrogen Production in the pgr
Mutants: Interplay between Capacities of
Photosynthetic Electron Transfer and
Oxygen Consumption
It is known that the hydrogenase is extremely sensitive to
oxygen (Ghirardi et al., 1997; Stripp et al., 2009). On the other
hand, linear electron transfer will produce oxygen. Compared to
maximal wild type rates, the hydrogen amounts produced per
hour were four times higher in the _pgr_ mutants with maximal
rates of 7 ml L −1 culture per hour over a period of 2–3 days
( Figure 1A ). The wild type stopped producing hydrogen slightly
earlier than the mutants ( Figures 1A and 2B ). After 2 days of
sulfur deprivation (time point of DCMU addition, Figure 1B ),
the wild type produced already 46% vs. only 12% of hydrogen
produced by the _pgr_ mutants compared to the total hydrogen

TABLE 1 | Comparison of hydrogen amounts produced by different _Chlamydomonas_ mutants.

Mutant H2 volume (ml/L culture) Chlorophyll ( µ g/ml) H2 volume ( µ mol H2/ µ g chl) PBR size (ml, ø in cm) Reference

_pgr5_ 850 15 2.4 250, 6.5

_pgrl1_ 580 15 1.6 250, 6.5 Also Tolleter et al., 2011

_pgr5_ _pgrl1_ 610 15 1.7 250, 6.5

_stm6_ 540 26 0.9 500, 8.0 Kruse et al., 2005

_stm6 glc4_ 150% of _stm6_ 26 1.3 500, 8.0 Doebbe et al., 2007

_LO1_ 400 15 1.1 500, 8.0 Oey et al., 2013

_L159I-N230Y_ 504 12 1.75 1000, 5.0 Torzillo et al., 2009

_Absolute_ _H_ 2 _volumes are taken from specified publications._ _For a better_ _comparison,_ _H_ 2 _amounts are equalized_ _to chlorophyll_ _content_ _(column 4). Photobioreactor_ _(PBR)_
_sizes are given as one_ _possible factor_ _introducing_ _some variance_ _between_ _studies due_ _to differences_ _in experimental set-ups._

amounts produced over 9 days. This time point correlates with
the drop in PSII activity in the wild type ( Figure 1C ). Thus,
PSII activity of the _pgr_ mutants remained higher until the late
phase of sulfur deprivation ( Figures 1C and 3 ) suggesting that
linear electron flow from PSII is responsible for the prolonged
and significantly higher hydrogen production rates in the _pgr_
mutants.

Why the hydrogenase is not inhibited by elevated PSII
activity in the _pgr_ mutants? This is because anaerobiosis can
be maintained in the _pgr_ mutants due to an increased oxygen
consumption rate, as oxygen is removed much faster from the
measuring system compared to wild type ( Figures 1D and 2C,E ).
This is further supported by the fact that light-induced oxygen
uptake is increased in the _pgr5_ mutant ( Figure 4 ). Increased
respiration rates were already reported for the _pgrl1_ _ko_ and _kd_
lines (Petroutsos et al., 2009; Tolleter et al., 2011). Recently,
Dang et al. (2014) reported increased mitochondrial cooperation
and increased O2 photo-reduction in the _pgrl1_ mutant and also
in _Arabidopsis_ _pgr5_, this cooperation was enhanced (Yoshida
et al., 2007). Metabolic shuttles such as the malate-oxaloacetate
shuttle can export reducing power from the stroma to the
mitochondria (Scheibe, 2004; Shen et al., 2006). This cooperation
is particularly enhanced under stress conditions, where ATP
demand is increased (Lemaire et al., 1988). Mitochondrial
inhibitors induced a drop in the PSII yield in _pgrl1_ (Dang
et al., 2014). Also light-dependent O2 uptake and the abundance
of flavodiiron proteins were higher in the mutant, indicating
an increased capacity of Mehler-like reactions and possibly
photorespiration. High antioxidant capacity was reported for
_Arabidopsis pgr5_ (Suorsa et al., 2012). A recent study showed an
up-regulation of those proteins in the early acclimation phase
of sulfur deprivation, suggesting an involvement of flavodiiron
proteins in acclimation to anoxia during hydrogen production
(Jokel et al., 2015). In addition to the very similar phenotype
of the _pgrl1_ and _pgr5_ mutants in regard to CEF impairment,
NPQ reduction and PSI photo-inhibition (Tolleter et al., 2011;
Johnson et al., 2014; Kukuczka et al., 2014), O2 consuming
mechanisms are also up-regulated under S-deplete mixotrophic
conditions in _pgr5_ ( Figures 1D, 2D,E and 4 ) and participate in
a faster anaerobic induction at the onset of sulfur deprivation
( Figures 1D and 2D,E ). This is in line with the conclusion, that
increased oxygen consumption rates allow higher PSII activity
under anaerobic –S conditions leading to increased hydrogen
production in the _pgr_ mutants. Interestingly, in a recent study
using the _stm6_ and PSII-D1 _L159I-N230Y_ mutant (see also
Table 1 ), the authors reported very similar phenotypes under
sulfur deprivation as is shown here for the _pgr_ mutants: the
mutants reached anaerobiosis faster and increased hydrogen
production was mainly achieved by an increased electron supply
from PSII toward the hydrogenase due to an increased respiration
rate (Torzillo et al., 2009; Scoma et al., 2012; Volgusheva et al.,
2013).
The contribution of the indirect pathway of electrons from
starch via NAD2 toward the hydrogenase to H2 production
has been reported to be variable, depending on experimental
conditions and particularly on the phase of the sulfur
deprivation process (Kosourov et al., 2003; Fouchard et al.,

2005; Hemschemeier et al., 2008; Jans et al., 2008). It might
also contribute to increased hydrogen production in the _pgr_
mutants. Starch breakdown was reported to be faster in the
_pgrl1_ mutant (Tolleter et al., 2011) and from a study with a
starch deficient mutant and the inhibitor DCMU and proton
uncoupler FCCP, it was concluded that the proton gradient
generated by cyclic electron flow around PSI inhibits the indirect
pathway of electrons from starch to the hydrogenase (Chochois
et al., 2009). Tolleter et al. (2011) reported hydrogen production
rates in _pgrl1_ are enhanced compared to the wild type, and
to a similar extent independent of the presence of DCMU.
However, H2 production was 10 times lower than in the
absence of DCMU (Tolleter et al., 2011). In our hands, the
hydrogen production in the presence of DCMU was even lower
( Figure 1B ), possibly because DCMU was added with a 1-day
delay (48 h vs. 24 h of sulfur deprivation). Therefore, since
blocking the PSII-dependent direct pathway by DCMU had such
a dramatic effect on hydrogen production, the contribution of
the indirect pathway to hydrogen production in the _pgr_ mutants
should be relatively small compared to the PSII-dependent
pathway.

Deletion of PGRL1 and PGR5 Sustains
PSII Activity under –S Conditions
The mechanisms contributing to the inhibition of photosynthetic
O2 evolution in S depleted conditions and their relative
importance are still a matter of debate (Ghysels and Franck,
2010). Sulfur deprivation has a general effect on the transcription
of the chloroplast (Irihimovitch and Stern, 2006; Irihimovitch
and Yehudai-Resheff, 2008). It is well described that acclimation
to sulfur deficiency is highly controlled and induces a downregulation of photosynthesis, particularly of PSII (Davies et al.,
1996). The absence of down-regulation of photosynthetic
activity, as observed in the _sac1_ mutant (Davies et al., 1996),
is deleterious. It has been suggested that sulfur limitation
will result in a general decrease in protein synthesis, which
in turn would impact the capacity of D1 turn-over and
repair cycle (Wykoff et al., 1998; Zhang et al., 2002). Hence,
under low sulfur and in the absence of efficient PSII-repair,
high activity of photosynthetic electron transfer would likely
result in damage due to the production of reactive oxygen
species, which could not be sustained by D1-repair. In the
_pgr_ mutants, however, PSII photochemistry efficiency remained
high ( Figure 1C ) and amounts of the D1 protein in the
mutants showed some stability compared to the wild type
during sulfur starvation ( Figure 3 ), at which PSII activity in
the wild type had already dropped to 10% ( Figure 1C ). The
inhibitory effect of chloroplastida translation by lincomycin
on PSII activity and H2 production further indicates that a
substantial amount of PSII remains functional under sulfur
deprivation ( Figures 1B,C ). Thus, the absence of PGRL1 and
PGR5 sustained PSII activity under sulfur deprivation. How
could this be explained?

The increased oxygen consumption capacity in the mutants,
and the faster transition to anaerobiosis is, in its own
right, sufficient to explain PSII preservation, via at least two

mechanisms. First, rapid attainment of anaerobiosis leads to
early induction and high activity of the hydrogenase, the final
electron acceptor of linear electron flow in –S conditions. This
is important because CO2 fixation is lost during S deprivation,
which normally limits LEF by slowing down the reoxidation
of its main final electron acceptor NADP + : Rubisco is affected
even earlier than D1 and declines by about 80% in the first
24 h of S deprivation, becoming undetectable after 60 h
of starvation (Zhang et al., 2002). Loss of CO2-fixation has
been reported to slow down the D1 repair cycle and was
therefore predicted to stimulate the loss of PSII activity during
S deprivation (Takahashi and Murata, 2005, 2006). In summary,
early anaerobiosis releases acceptor side limitation of LEF faster
due to an earlier activation of the hydrogenase ( Figures 1D and
2D ). Second, faster induction of anaerobiosis at the onset of
sulfur starvation and induction of hydrogenase activity preserves
existing PSII from photo-oxidative damage, as it reduces the
amount of oxygen available to cause potential damage at PSII.
All strains show an initial decline in Fv/Fm, which slows
after the system becomes anaerobic, but as this occurs much
later in the wt, the corresponding damage to PSII is much
worse.

Under High Light Exposure, _pgr5 pgrl1_
shows an Increased Hydrogen Burst at
the Beginning of Sulfur Depletion and
PSI becomes Photo-inhibited in the _pgr5_
and _pgr5 pgrl1_ Mutants
In this study, we report the generation of a _pgr5_ _pgrl1_ double
mutant. This double mutant displays a similar phenotype
compared to its respective single mutants, confirming that PGR5
and PGRL1 both act on the same pathway as earlier proposed
(DalCorso et al., 2008; Johnson et al., 2014). However, _pgrl1_
and _pgr5_ differ in the severity of one particular phenotype:
While PSII remained stable in sulfur limiting conditions, PSI
was degraded much faster in the _pgr5_ and _pgr5_ _pgrl1_ mutants
when they were exposed to additional stress of high light
( Figure 3 ), reducing the amount of hydrogen produced ( Figure 2 ;
Supplementary Figure S2). In contrast, _pgrl1_ remained effective
in hydrogen production also in high light and PSI was not
degraded at the same rate ( Figures 2 and 3 ). Although PGR5
was previously not detectable by western blot analysis in the
_pgrl1_ mutant (Johnson et al., 2014), the different phenotype of
the _pgr5_ _pgrl1_ and _pgr5_ mutant compared to the _pgrl1_ single
mutant suggests that the PGR5 protein is present in the _pgrl1_
mutant, though below the limit of detection of immunoblot
analysis, which could detect about 50% of PGR5 wild type
protein amounts (Johnson et al., 2014). High light sensitivity
has already been reported for _pgr5_ under photoautotrophic
conditions (Johnson et al., 2014). In _pgrl1_, PSI becomes photoinhibited under high light exposure (Kukuczka et al., 2014),
although not degraded as severely as in _pgr5_ . In the absence
of both PGR5 and PGRL1, PSI becomes even more affected,
indicating an additive role of these two proteins in terms of PSI
protection and suggesting that both proteins operate in the same
pathway.

PSI photo-inhibition in high light was also described in
a _Chlamydomonas_ _stt7-1_ mutant locked in state 1 (Bergner
et al., 2015). However, this phenotype was less severe than
in the _pgr5_ and _pgrl1_ mutants (Johnson et al., 2014; Bergner
et al., 2015). Furthermore, the _stt7-1_ mutant displayed high
CEF rates and enhanced formation of a CEF-supercomplex
(Bergner et al., 2015). A non-successful acclimation to high
light was recently also linked to altered dephosphorylation
patterns of LHCII proteins in _Arabidopsis_ _pgr5_ (Mekala
et al., 2015). These findings indicate a regulatory link
between the PGR5/PGRL1 pathway, phosphorylation and
dephosphorylation dynamics and the susceptibility of PSI to
photo-inhibition.

In high light, the thylakoid lumen becomes acidified, which
down-regulates linear electron flow and induces dissipation
of excess energy at PSII in the form of heat. This so called
non-photochemical quenching (NPQ) requires the induction
of the LHCSR3 protein in algae (Peers et al., 2009) and
acidification of the thylakoid lumen (Petroutsos et al., 2011).
Both _pgr5_ and _pgrl1_ have reduced levels of NPQ due to a
reduced proton gradient across the membrane, while LHCSR3
levels are unaltered in the mutants (Tolleter et al., 2011;
Johnson et al., 2014). Without a sufficient proton gradient,
linear electron flow is not down-regulated and the acceptor side
of PSI becomes over-reduced. An indication that an increased
electron drive toward the hydrogenase occurs under elevated
light conditions is the enhanced hydrogen burst at the beginning
of the hydrogen production phase in the _pgr5_ _pgrl1_ mutant
( Figure 2C ). Hydrogen production at PSI was proposed to
act as a safety valve to protect the photosynthetic electron
transport chain from over-reduction under natural conditions
by safely disposing of excess electrons from PSI (Melis and
Happe, 2001; Happe et al., 2002; Hemschemeier et al., 2009).
Now, the deletion of PGR5 and/or PGRL1 is deleterious for PSI
under high light conditions. Thus, while PSII stability under
sulfur deficiency is more affected in wild type, PSI integrity is
most affected in the absence of PGR5 and/or PGRL1; a topic
that certainly requires additional work for a more in-depth
understanding.

CONCLUSION

Enhanced hydrogen production rates in the _pgrl1,_ _pgr5,_ and
the _pgr5_ _pgrl1_ double mutant under S deplete conditions
lead to the highest continuous photobiological produced
hydrogen amounts of eukaryotic cells reported so far. These
rates are achieved by a prolonged residual PSII activity
providing an increased electron supply toward the hydrogenase.
PSII activity can be maintained in the mutants without
inhibiting the oxygen sensitive hydrogenase, because the
oxygen consumption capacity is increased. Our results suggest
that respiration and light-dependent O2-uptake rates are
higher in the _pgr_ mutants, and that this is responsible
for the faster transition to anaerobiosis, especially when the
greater residual PSII activity of the mutants is taken into
account.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Ian Ross for critical proofreading of the manuscript and productive discussion. They
would like to thank Dr. Dimitris Petroutsos for assistance at
the start of the project. LM acknowledges support from the
Alexander von Humboldt Stiftung/Foundation during his stay
at the Institute of Plant Biology and Biotechnology (University
of Münster, Germany). MH acknowledges support from the

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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.

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