The role of C₄ metabolism in the marine diatom Phaeodactylum tricornutum

The role of ( 4 metabolism in the marine diatom Phaeodactylum tricornutum

Maya Haimovich-Dayan

¹

*, Nitsan Garfinkel

¹

*, Daniela Ewe

²

*, Yehouda Marcus

^3,

Ansgar Gruber

^2,

Heiko Wagner

^4,

Peter G. Kroth

²

and Aaron Kaplan

^l

I Dcparrment ofPlaru and Environmental Sciences, Edmond j. Sarra Campus -Givat Ram, Hebrew University of Jerusalem, Jerusalem, 91904, Israel; 2Fachbercich Biologic, Univcrsitat Konstanz, Konstanz, 78457. Germany; ·"Department of Molecular Biolob'Y and Ecology ofPlanrs, Tel Aviv University, 'rei Aviv, 69978. Israel; "Institur fUr Biologic, Abrcilung Pfl3llzcnphysioiogic, Univcrsiriit Leipzig, Leipzig, 04103, Germany

Summary

Author for correspondence:

Aaron Kaplan Tel.: +972 2 6585234

Email: aaron.kaplan@mail.huii.ac.il

• Diatoms are

important

players

in

the global carbon cycle. Their apparent photosynthetic affinity for amb

ient CO2 i

s much higher than that of ribulose 1

,5

-bisphosphate carboxylase/

oxygenase (Rubisco),

indicating that a COrconcentrating mechanism (CCM) is functioning.

However, the nature of the CCM, a biophysical or a biochemical C4, remains elu sive. Although

14C labeling

experiments and presence of complete sets of genes for C4 metabolism

in

two diatoms supported the presence of C4, other

data

and predicted

localization

of the decarboxylating enzymes, away from Rubisco,

makes this unlikely.

•

We used

RNA-interference to silence the single gene encoding pyruvate-orthophosphate dikinase (PPDK) in

Phaeodactylum tricornutum,

essential for C4

metabolism, and examined the

photosynthetic characteristics.

Key words: (4

metabolism,

((M,

diatoms, photosynthesis, pyruvate-orthophosphate dikinase.

• The mutants possess much

lower ppdk

transcript a

nd

PPDK activity but the photosynthetic

1<1/2

(C0

_{2 )}

was

hardly affected, thus clearly indicating

that the C4 route

does

not serve the

purpose of raising the CO2

concentration in close proximity of Rubisco in

P. tricornutum. The photosynthetic Vmax

was slightly

reduced in

the

mutant,

possibly

refl

ecting a

metabolic

constraint that also resulted

in

a larger lipid accumulation.

•

We

propose that the C4

metabolism does not function in net CO2

fixation but he

lps the cells to

dissipate excess light energy and in pH homeostasis.

Introduction

Diatoms play an important role in the global carbon cycle and it is estimated that they perform c. 20% of global CO

2

fixation (Falkowski

&

Raven, 2007). Information on the uptake of inorganic carbon (Ci) and its fixation by diatoms is rather limited, as only a few model organisms such as Thalassiosira weissflogii, Thalassiosira pseudonana, Thalassiosira oceanica, and Phaeodactylum tricornutum have been examined

. Nevertheless, the

K

I/2

(C0

2)

of their ribulose-l ,5-bisphosphate carboxylase/oxygenase (Rubisco), c. 28-40

~M

(Badger etal., 1998; Whitney etaI., 2001), is c. 20- to 40-fold higher than the apparent photosynthetic K

I/2

(C0

2)

for ambient CO

₂

(Burkhardt et al., 2001) and c. three- to fourfold higher than th

e

CO

₂

concentration in the present-day marine environment (Riebesell etal., 1993). Measurements indicated that in most cases Ci uptake and carbonic anhydrase (CA) activity are strongly affected by the CO

₂

concentrations experienced by the cells during growth. They both increase significantly with a declining ambient CO

₂

from a high concentration (1

-

5% CO

₂

in air) to the concentration of CO

₂

in air or lower, and that some of the

·These amhors contributed equally to this work.

CA encoding genes are up-regulated when the cells are exposed to

low concentrations of

CO

₂

(Tortell et aI., 1997, 2008; Burkhardt et aI., 2001; Morel et aI., 2002; Cassar et aI., 2004; Harada et aI., 2005; Tanaka et aI., 2005; Trimborn et aI., 2008; Mab'erly et aI.,·

2009; Matsuda et al., 2011; Tachibana et aI., 2011). These findings have

led

to the recognition that, like many other phytoplankton species, the model diatoms must rely on CO

₂

concentrating mechanisms (CCMs) to perform significant rates of CO

₂

fixation under contemporaty CO

₂

concentrations. These data also sup- ported the notion that a CCM is activated in cells exposed to declining CO

₂

concentrations (see Kaplan

et

aI., 1991, 1994;

Kaplan & Reinhold, 1999; Badger etal., 2002; Giordano etaI., 2005; Price etaI., 2008; Fukuzawa etaI., 2011 for reviews).

The nature of the CCM operating in diatoms is not clear and the extent of its expression may be species-specific and strongly affected by growth conditions (Roberts et al., 2007a,b; Reinfelder, 2011).

Although suggestive, induction of genes and activiti es involved in Ci uptake such as CA cannot and must not be regarded as conclus

ive evidence for the operation of

a biophysical CCM. Here, the light energy is used to actively accumulate' Ci within the cells and thereby raise the CO

₂

concentrations in close proximity to Rubisco, the carboxylating enzyme that is mostly confined within

177

Ersch. in: New Phytologist ; 197 (2013), 1. - S. 177-185

http://dx.doi.org/10.1111/j.1469-8137.2012.04375.x

Konstanzer Online-Publikations-System (KOPS)

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-219675

the carboxysomes in cyanobacteria or pyrenoids

in

eukaryotes (Kaplan

&

Reinhold , 1999; G iordano

et ai. , 2005;

Espie

&

Kimber, 201l). The prese nce of pyrenoids and the localization of most of the plastidic Rubisco, together with a beta-CA (PtCA1) within th ese bodies Oenks

&

G ibbs, 2000; Roberts

etai., 2007b;

Hopkinson

etal.,

2011; M a

etai., 2011; Matsuda etai., 2011;

Reinf elder, 2011; Tachibana

etai.,

2011) , provide additional support to the notion that a biophysical CCM

i

s functioning in at

least some of the diato

ms.

On the other hand, the seminal studies ofRei

nfelder (Reinfelder,

2011 and ref erences therein) suggested the ex

istence of

a plant-rype biochemical C

₄

metabolism in

diatoms.

This was based on short-term

1

4C0

2 pulse-chase labeling

experiments, analyses of phospho enol pyruvate (PEP) carboxylase (PEPC) and PEP carboxykinase (PEPCK) activities, the effects of inhibitors on the photosynthetic rate, and the effect of CO

2

concentrations on gene expression (Reinfelder

etai., 2000;

Morel etai., 2002). However, there are opposing conclusions based on the slow rate of C

₄

acid accumul ation in some diatoms such as

T pseudonana and constit- u

tive expression of C

₄

-related genes, hardly affected by the CO

2

concentrations (Roberts

etal.,

2 007a, b). T

he evidence for

C

₄

metabolism

in

P.

tricornutum

rests mainly on th e

lower rate of

photosynthesis following inhibition ofPEPC and the constitutive

high transcrip

t abundance from genes encoding enzymes essential for this route (see Reinf elder, 2011 and references therein) .

The classical plant C

₄

metabolism rests on coo peration between two cell rypes, with the mesoph yll cell performing the first carboxylation using PEPC and HC03' followed by reduction. The C

₄

acid obtained is then transferred to the bundle sheath cells where decarboxylation takes place, leadin g to an elevated CO

2

concen- [I'ati on in close proximi ty to Rubisco confi

ned to these cells (Hatch, 1992). However, a C₄

metabolism functio

ning in a single plant cell

was already described in

Hydrilla verticillata

(Rao

et ai., 2002), Bienertia cycloptera

and

Borszczowia aralocaspica

(Edwards

et al.,

2004) and was recently proposed for

Ulva proliflra

(Xu

et ai.,

201 2).

A plant-like C

₄

activity in diatoms i

s further

suppo rted by the presence of a complete set of genes essenti al for this metabolic route in

T pseudonana and

P.

tricornutum, for which the entire genomi

c sequ ence

information

is available (Armbrust et ai., 2004; Bowler

etai., 2008).

However,

in silico

analyses of the subcellular

l

oca

lization

of the relevant enzymes predicted a scrambled C

₄

metabolism (Kroth

et ai., 2008),

possibly a consequence of the evolution al Y history of diato ms via secondary endosymbiosis (Armbrust

et ai.,

2004; Kroth

et ai.,

2008; Vardi

et ai., 2008).

Phosphoenol pyruvate carboxy

lase

I (PEPC

1)

is predicted to be

located either in

the endoplas mic reti culum or in the periplastidi c space between the second and the third membranes of the four- membrane-bound diatom plastid

,

representing the former cyto- plasm of the eukaryoti c endosymbiont. PEPC2, one pyruvate kinase (PK) isoform, PEPCK, malate dehydrogenase (MDH) , malic enzy me (ME) and pyruvate ca rboxylase (PYC1) are predicted to be targeted to the mitoch ondria, whereas pyruvate phosp hate d

ikinase

(PPDK), anoth er PK i soform, and PYC2 were predicted to be plas tid

-targeted

. As a res

ult of

th ese subcellular loca

lizations,

for a functional plant-like C

₄

metabolism to occur and in the

absence of a recogni zed plastidic deca rboxylase, the CO2 generated via deca rboxylation of th e C

4

acid

in

the mitochondria must cross six membranes in order to reach Rubisco in the plasti d (Kroth etal., 2008). Owing to the presence of multiple forms of CAs (nine and 13 CA sequences were d etected

in

the genomes of P.

tricornutum

and

T pseudonana, respectively)

and their localizati o n (Tachibana

etal., 2011)

, th e CO2 produced by deca rboxylation outside the plastid is most

likely

bein g converted back to H C 0

3'

. Conse- quently, a futile Ci cycling would be expected w

here

ATP is consumed during PEP form ation from pyruvate.

From measurem ents of Ci flu xes and comparisons of th e app arent photosynth etic affi

ni

ty to CO

2

with that of Rubisco, we conclude that a CCM is functioning in diatoms. However, we are unable to determine whether a biophysical or a biochemical CCM route is taking place. Therefore, we adopted a genetic approach using RNAi

technology to

down- regulate the sing

le

copy ge ne encoding PPDK in P.

tricornutum.

Regardless of whether the predicted localizations of the various enzymes poten tially involved in the C

₄

route (Kroth et al., 2008) are correct, PPDK that catalyzes the formation of PEP from pyruvate

using ATP performs an

essential step for net CO

2

fixation via a biochemical CCM (H atch

&

Kagawa,

1973); its down-regulation thus enables

us to assess the

role of

C

₄ in

P.

tricornutum.

Materials and Methods

Strains and media

Phaeodactylum tricornutum

Bohlin (Bacill ariophyceae) strain UTEX646 (available at UTEX Culture Collection of Algae, University of Texas, Austin, TX;

http

://www.bio.utexas.edu/

research/utex/) was grown in 22°C with continuous illumination at 75 Jlmol photons m

-2

s

- I in

seawater-enriched f/2 media (Guill ard & Ryther, 1962) and 2 mM Tr

i

s buffer, pH

=

8.0. Solid media co ntained 1.2% Bacto Agar (Difco, Becton Dickinson, France). Cell cultures were maintained in Erl enmeyer fl asks without any forced aeration or bubb

ling in

order to impose extreme CO

₂ limi

tations, conditions under which various ge nes essential for both types of CCM are

likely up

-regulated . All the experiments were perform ed in tripli cate,

using

at

least three

independent cultures of the wild type (WT) and transformed cell

lines.

Construction of plasmids and PCR

Standard cloning procedures were used for plasmid constructi ons

(Sambrook etai., 1989). PCR was perform ed with a conventional

thermocycler (GenePro

,

Bioer, Arlington, MA, USA) using a

PrimeSTARTM H S DNA polymerase (TaKaRa, Tokyo, Japan)

acco rding to the manufacturer's instructions. T

he transformation

vector pPh a-T

I

(Ge

nBank

accessio n AF219942.1) was

used

(Zaslavskaia

et ai.

2000).

It

co ntains a

she ble

gen e for Zeocin

resistance, thus allowing screening for positive coloni es of

P.

tricornutum and

an

amp

gene encodin g ampicillin resistance

for bacteri al selection and amplifi cat

i

o n. A sequence of 469 bp

,

sense PPDK and a sequ ence of

189 bp

, antisense PPDK were

amplified from P. tricornutum cDNA (Supporting Information, Fig. Sla) and cloned on each side of an 874-bp loop containing the eGFP gene into the pPha-TI vector, giving rise to pPha-T I- PPDK-RNAi (Fig. SIb).

Biolistic transformation

Cells were bombarded using the Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad) fitted with 1350 psi rupture discs as described in Kroth (2007). After transformation, cells were allowed to recover for 24 h before being plated onto an fl2 medium containing 75!-lg ml-

^I

Zeocin (Invitrogen) . The plates were incubated at 22°C under constant illumination (75 J..lmol pho- tons m -

² S-I).

Genomic DNA isolation

Cells were harvested by centrifugation (3000

g,

10 min) and resuspended in a lysis buffer containing 0.2 M Tris-HCI, pH = 9, 0.4 M LiCi and 25mM EDTA. Glass beads (diameter = 212- 300 J..lm; Sigma) were added to the cells before they were mechanically broken for 1 min in a bead beater. The cell lysate was centrifuged for 5 min at maximum speed and the supernatant was transferred to a clean Eppendorf tube containing isopropanol at equal volume, followed bya second centrifugation for 10 min at maximal speed. The DNA pellet was air-dried after an ethanol wash and resuspended in 30!-l1 double-distilled water (DDW). Primers, binding to the transformation vector, were used to screen for genomic integration of the RNAi construct in Zeocin-resistant colonies.

Isolation of RNA and cDNA synthesis

Fifty milliliters of logarithmic phase cells (c. 10

⁶

cells ml -

^I)

were harvested by centrifugation at 5500

g

for 3 min at room temper- ature. The pellet was resuspended in 1 ml phosphate-buffered saline (PBS) and centrifuged at 5500

g,

4°C, for 1 min. Cell pellets were resuspended in 1 ml RNAzol (Molecular Research Center, Inc., Cincinnati, OH, USA) and then homogenized by mechanical breakdown using acid-washed glass beads (diameter = 212- 300 J..lm) for 20 s in a bead-beater. Further RNA isolation steps were perform ed according to the RNAzol protocol. Contaminating DNA was digested with Turbo-DNase (Turbo DNA-freeTM;

Ambion) according to

tl~e

manufacturer's instructions. The RNA obtained was reverse transcrib ed using ImProm- IIT M Reverse Transcription System (Promega) according to the manufacturer's instructions. Complete removal of genomic DNA from RNA samples was verified after cDNA synthesis by quantitative PCR (qPCR) amplification of his ton H4 (M)gene.

Quantitative PCR assays

These were perform ed using a Rotor-GeneTM 6000 Thermal Cycler (Corbett Research, Brisbane, Australia) . T he primers Llsed are shown in Table 51. Five microliters of diluted cDNA, corresponding to lOng total RNA, were Llsed in the following program: DNA po lymerase activation at 95°C for 15 min,

followed by 40 cycles of denaturation at 95°C for l Os, annealing at 56°C for 15 s, product elongation at 72°C for 20 s, and signal acquiring at 79°C. Amplifications were carried out in a total volume of 15 !-ll using the Absolute Blue SYBR Green ROX Mix (Thermo Scientific, ABgene, Rockford, IL, USA) according to the manufacturer's instructions. Transcript abundances were exam- ined rel ative to the level of the gene transcript for histone 4

(h4)

(Siaut et at., 2007). All samples were analyzed in three replicates per experiment and each experiment was repeated independently at least three times.

PPDK activity assay

Pyruvate phosphate dikinase activity was measured essentially as described inAshton et al. (1990) butwithseveral modifications. The procedures developed for the higher plant enzyme were used as a starting point because of the presence of a plant-like regulatory ptotein of' PPDK (see the Results and Discussion section). Cells maintained in growth conditions or exposed to a higher light intensity, 300 !-lmol photons m

-2

s

- 1,

for 1 h were broken as described earlier in extraction medium containing 50 mM HE- PES-NaOH, pH 8.0,10 mM MgCl

2,

1 mM dithiothreitol(DTT), 10 mM EDTA, 1 % casein, 1% polyvinylpyrrolidone, 0.25 M mannitol, 0.05% Triton X- I00 and a mix of protease inhibitors 1:100 (Sigma) . The assay buff er contained 100 ruM H EPES- NaOH, pH 8.0,15 mM MgCh, 0.15 mM EDTA pH 8.0, 5 mM NaHC0

3,

5 mM NH

4

Cl, 2.5 mM K

2

HP0

4 ,

5 ruM DTT and 1 mM glucose 6-phosphate. Freshly prepared 0.3 mM NADH, 1.5 mM ATP, 10.5 U MDH, 1.25 mM pyruvate and 0.5 U of PEPC were added directly to the qu artz cuvette and the reaction was initiated by the addition of 20 J..ll of protein extract; the overall reaction volume was 1 ml. The changing absorption at 340 nm was recorded by the spectrophotometer (Cary 300 bio, Varian) and the results were normalized to the protein content in the reaction. An earlier published alternative protocol developed to assess PPDK activity in certain algae, including P. tricornutum, also used the coupling to PEPC reaction but relied on its intrinsic value (Mukerji, 1980), which might be rate-limiting for the coupled reaction.

CO r and light intenSity-dependent O

2

evolution

The rate of COr dependent O

2

evolution as a function of Ci concentrations was determined using a Clark type O

2

electrode (PS2108, PASPORT dissolved O

2

sensor; Pasco, Roseville, CA, USA) essentially as described in Kaplan etal. (1988). Approx. 10

⁸

cells were harvested by centrifugation for 10 min at 3000 g in a swinging-bucket rotor and resuspended in 0.5- 1 ml CO

₂

-free

f/2

medium containing 20 mM H epes. The pH was adjusted to 7.5 with saturated NaOH. Two hundred microli ters of CO r free cells were then diluted in 4 ml of th e same media and incubated in the O

₂

electrode chamber at 22°C and 1000 !-lmol photons m

-2

s

- I.

Cells were allowed to utilize the Ci in their medium until the CO

₂

compensation point was reached. Aliquots ofNaHC0

₃

of known

concentrations were injected to raise the Ci concentration by

known increments while measuring the resulting ri se in the rate of

O

2

concentration in the chamber. For light intensity-dependent O

2

evolution measurements, cells were incubated in darkness for at least 5 min followed by exposure to increasing light of known intensities

.

A saturating NaHC0

₃

(10 mM) concentration was added to ensure that the cells were not Ci-limited.

Photoinhibition experiments

The photosynthetic rate of cells exposed to 10 mM Ci and 500 ).lmol photons m

^-2

s

^{- I}

was measured as described earlier followed by exposure to excess light of2000 ).lmol photons m

-2 S- I

for 1 h. The photosynthetic O

₂

evolution was then measured after

lowering the light

intensity back to 500 ).lmol photons m

-2

s

- I.

To inhibit recovery of photosystem II during photoinhibition, the cells were exposed to 100 ).lgml-

^I

lincomycin for 15 min before exposure to excess

light.

Fluorescence measurements

Fluorescence emitted by photosystem II was measured by pulse amplitude-modulated (PAM) kinetics using a PAM-lOi (Walz, Eff'ertlich, Germany). The light intensity (measured at the surface of the chamber) of the modulated measuring beam (at 1.6 kHz frequency) was 0.1 ).lmol photons m

^-2

s

^-I.

White actinic light was delivered by a projector

lamp at

1000 ).lmol photons m

-2

s

- I.

Maximal fluorescence (Fm) was measured with saturating white light pulses of 4000 ).lmol photons m

-2

s

-I for

1 s, with 1 min intervals.

Chla measurement

Cells were centrifuged for 5 min at 12000 g. The pellet was resuspended in 100

~d

methanol followed by 900

~d

acetone and a short vortex for a final 90% acetone extraction. The extract was centrifuged again for 10 min at 12 000

g,

and the supernatant was measured in a spectrophotometer at 664, 639 and 750 nm. Ch la concentration was calculated according to Jeffrey

&

Humphrey (1975)

.

M easurement of lipids content using Nile red

To estimate lipid content, 200 ).ll of logarithmic-phase cells were

loaded

in triplicate on

a 96-well

plate and the O.D. 750 nm was measured before sta

ining. Ten microliters of Nile red (500 ).lg

of 9-diethylamino-5Hbenzo[exJphenoxazine-5-one per 1 ml acetone (Sigma)), a fluorescent probe of intracellular lipids and hydrophobic domains of proteins were added to the cells. Fluorometric ana

lysis

occurred 10 min after staining using a Sequoia-Turner Model 450 Digital Fluorometer with a 485 nm narrow-band excitation filter and a 590 nm narrow-band

emission filter.

With this technique, cellu

lar storage or neutral lipids display yellow-golden fluorescence

(Greenspan et at., 1985; McGinnis et at.,

1997).

Fourier transform infrared (FTIR) spectroscopy and , macromolecular composition

For FTIR spectroscopy analysis, cells were harvested by centrifu- gation at 4000 g for 1 min (Centrifuge 5415D; Eppendorf,

Hamburg, Germany) and washed with distilled water. After centrifugation, the pellet was freeze-dried (Christ Alpha 1-4, B.

Braun Biotech International, Allentown, PA, USA) and stored at

-

20°C before measurement. Cells were resuspended in c. 10).l1 distilled water to obtain a final cell density of 1.8 x 10

⁶

cells ).ll

-I.

A volume of 2 ).ll of this cell suspension was placed on a 384-well silicon microplate (with n> 5) and dried in a cabinet: dryer at 40°C for at least 10 min. FTIR spectra were measured usi ng a HTS-XT microtiterplate module (Bruker, Berlin, Germany) with a DTGS detector as described in Wagner et al. (2010)

. Transmission spectra

were recorded in the range between 4000 and

7

00 cm

-

I with 32 scans per sample and at a resolution of 4 cm

- I.

The spectra were analyzed using OPUS Lab Software (version 5.6, Bruker), corrected to the background spectra and baseline-corrected by the rubber band method. The carbohydrate,

lipid and

protein contents were calculated from the spectra according to Wagner

etal. (2010).

Level of Rubisco active sites

The amount of Rubisco was assessed both by western analyses and by the amount ofRuBP binding sites using 14Ccarboxypentitol- bisphosphate (CPBP) binding as described in Marcus et at.

(2005).

CPBP is an analog of the intermediate Rubisco's substrate ribulose 1,5- bisphosphate.

Results and Discussion

Silencing of PPDK in P. tricornutum

As indicated, we took a genetic approach to assess whether C

₄

metabolism plays an important role in the growth and photosyn- thetic performance of low-C0

2-grown

P tricornutum. Recent developments of gene silencing in P tricornutum using constructs that express either antisense or inverted repeat RNAs (De Riso et al., 2009) enable the application of this approach for down-regulation of genes in this organism. We applied RNAi technology ro silence the single ppdk gene copy (Phatr v2.0 Protein 10: 21988) that encodes PPDK (Fig. S1). This enzyme is predicted to be located within the plastid (Kroth et at., 2008) where it converts pyruvate to PEP at the expense of ATP, an essential step in net CO

₂

fixation via the C

4

route. To silence the ppdk gene in P tricornutum, we constructed a transformation vector containing sense and antisense fragments of this gene (see the Materials and Methods section for a detailed description of the plasmid construction)

and biolistically

transformed P tricornutum with this construct. ,Colonies growing on selective plates containing Zeocin were collected for further

analysis and screened for

ppdk down-regulation.

Transcript abundance of ppdk and PPDK activity

Two of the

Zeocin-resistant cell lines were selected

for further

analyses and had a significantly (70-

80%) lower abundance of the

ppdk transcript, as determined by qPCR. Data from one sPPDK

cell line are presented as an

example (Fig. 1

a). In all the aspects

examined here, the two mutants we investigated in parallel showed

(a)1.2 (b)0.4

3 e;

1.0 ^~

,

~ ^..c c 0.3

c _'Q;

~ 0.8

!!

.a a

_.;: c ^0.6

^~

^{Q, 0.2}

u :z: 0

~ 0.4 ^4:

z

C! ~

^0.1

~

..

0.2 ::I.

Q.

Q.

0

.0

WT 0.0

WT

Fig. 1 The transcript abundance of ppdk (a) and PPDK activity (b) in Phaeodactylum tricornutum and the sPPDK transformed cell line thereof. Abundance of ppdk was measured using quantitative PCR and set to 1.0 relative to the level of the gene transcript for histone 4 (h4); ppdk transcript abundance in the transformed cell line was calculated relatively to the wildtype. Pyruvate-orthophosphate dikinase (PPDK) activity was calculated from the change in absorbance at 340 nm resulting from the forward direction by coupling the production of phosphoenol pyruvate (PEP) to NADH oxidation via PEP carboxylase and malate dehydrogenase (Ashton et al., 1990; Marshall et al., 2001). Note that the activity is provided per total soluble extracted proteins, whereas the rate of photosynthesis (Fig. 2) is shown per Chla. The ratio of Chla to soluble protein in P. tricornutum is c.15.

similar responses. Although various approaches were applied, we could not raise useful antisera against the

P. tricornutum

PPDKand thus were unable to examine whether the PPDK level is lower in the silenced transformant strains. However, as the purpose of the gene silencing was to reduce PPDK activity,' we focused on this parameter, because it was more important for the sake of this study than the enzyme level. Our analysis clearly shows that the PPDK activity was much lower in the sPPDK mutants (Fig. 1 b), which was the goal of our genetic transformation efforts.

In C4 plants, theactivityofPPDK is strongly affected by reversible phosphorylation of a threonine residue in the active site by the bifunctional protein kinase/phosphatase PPDK regulatory (PR) protein (see Chastain

etaL.,

2011 for a recent review and references therein). The phosphorylation of this threonine blocks PPDK activity in the dark, thereby avoiding futile ATP consumption in darkness. To the bestofour knowledge, analyses ofPPDK activation and/or involvement of a regulatory protein were not examined in diatoms. Analysis of the

P tricornutumgenome

database identified a single gene

OGI

Pror-lD 49027), bearing a plastid targeting sequence, that shows significant amino acid homology (38%

(a) 300

~ 250

1:

101 200 :s

i 150-

0 r

¹⁰⁰

I

⁵⁰

O~--~----.---~----.---~

50 100 150 pM HCO.-

identity) to the plant type enzyme and contains the typical DUF299 domain of the bacterial PPDK-PR protein (Chastain

et at., 2011) .

On the other hand, we could not detect a PPDK-PR encoding gene in the

T pseudonana

genome, although other genes essential for the C₄ metabolism were found. This is consistent with the suggestion that

T pseudonanadoes

not perform a biochemical CCM (Roberts

et ai. ,

2007b). Clearly, ifan active PPDK is present in

T pseudonana,

it would be important to down-regulate its activity in the dark to avoid futileATP consum ption. We did not perform a detailed study on the activation ofPPDK in

P tricornutum,

but the presence of the plant type PPDK regulatory protein may suggest that PPDK activation in

P tricornutum

is similar to that observed in the plant type protein.

Photos ynthetic and growth characteristics

Since PPDK is a key player in net CO2 fixation in organisms that perform a C₄ metabolism (Matsuoka

etai.,

2001; Edwards

etaL .,

2004), decreasing its cellular activity by silencing the gene would be expected to lower the carbon assimilation abilities, particularly

(b) 300

"i 250

.I:

101

:s200 i 150

0 r

¹⁰⁰

I

⁵⁰

O __ ---r---.---.---~

o

200 400 600 800

IAmol photons m-¹ s-'

Fig.2 COrdependent (a) and light-dependent (b) O2 evolution in wildtype (WT) Phaeodactylum tricornutum (red squares) and the sPPDK transformed cell line (blue diamonds). (a) P. lricornulum cells at the CO2 compensation point were exposed to increasing NaHC03 concentration and O2 evolution was measured correspondingly. (b) Dark-adapted cells were exposed to increasing light intensity at saturating inorganic carbon (Ci) and O2 evolution was measured correspondingly. For both cases, gross rate of O2 evolution is provided after correcting forthe rate of O2 consumption in the dark, which was similar in the WT and sPPDK mutant.

under low C i concentrations. COTdependent O

₂

evolution was measured as an indicator of CO

₂

assimil ation (Fig. 2a) and to assess the apparent photosynthetic affinity to C i. In both the sPPDK transformants and the WT, the calculated K

_II2(C0₂)

was c. 0.6 11M CO

_{2 ,}

This value was calculated from the

K_II2(Ci)

obtained in Fig. 2, based on the assumption that the Ci species in the medium are at chemical equilibrium accord ing to the experimental pH 7.5.

This value is within the range reported previously (Reinfelder, 20 11). The rise in photosynthetic performance wi'th increasing light intensity was also simi lar in the WT and sPPDK transformed cell line (Fig. 2b). The simil ar photosynthetic performance (Fig. 2) in the mutants, despite the much lower PPDK activity (Fig. 1 b) , provides strong evidence that photosynthetic net CO

₂

fixation in P. tricornutumdoes not take the C

₄

route. The cultures were grown under limiting Ci level and thus must have exploited a biophysical CCM to perform efficient photosYIHhesis where the apparent photosynthetic affinity to extracellular CO

₂

is c. 50- to 70-fo ld higher than that of Rubis co. These data are consistent with the fact that the growth rates of the WT and sPPDK transformants were identical, even when grown without forced aeration where the sole supply of CO

2

is via diffusion from the atmosp here into the flasks (Fig. 52).

The photosynthetic V;"ax (at saturating CO

₂

concentration and light intensity) was somewhat lower in the transformants but the reasons are not clear. We did not derect significant differences in the abundance of rbel (encoding the large subunit of Rubisco) transcripts between the WT and the sPPDK mutants (not shown) apd in the amount of Rubisco. T he latter was measured by CPBP binding (Fig. 53). CPBP is an analog of the intermediate Rubisco's substrate, ribulose 1 ,5-bisp hosphate; it binds essentially irreversibly to the reaction center, thereby allowing very accurate (far better than western blots) assessment of Rubisco binding site concentra- tion (Marcus et al., 2005). A metabolic imbalance as a result of reduced consumption of pyruvate in the sPPDK mutant, for instance, could lead to lower photosynthetic V;nax' This may be reflected in the change in cell constituen ts, a ri se in carbo hydrates and lipid levels at the expense of proteins in thesPPDK transformed

(a) ^1.2

r+-

^{5 0.9 (b)}

~ + ^g

1: t

^0.6

i

^0.8 _Is

I 11

⁰⁰⁽

::; 0.4 0.3

0.0 0.0

WT sPPDK

cell line (Figs 3, 54), presumably because of the higher pyruvate availab ility.

The photosynthetic V;"ax in the WT cells is

c.

250 Ilmol O

₂

evolved mg

- I

ChI a h

- I

(Fig. 2b), whereas that of PPDK activity (Fig. 1 b) is significan tly (c. 40-fold) lower. Please note that PPDK activity is provided per protein and that photosynthesis activity is per Ch la; the ratio of ChI alto tal soluble protein, measured during the enzyme assays, is close to 15 in P. tricornutum. We do not present all the various modifications exam ined to get maximal PPDK activity (the focal point of this study was the role of the C

4

, not optimization ofPPDK). One of the modifications applied was exposure to various light intensities before rapidly breaking the cells (see the Materials and Methods section). Of the 25 conditions performed on independent cultures, the PPDK activity increased in some (up to c. 10% of the photosynthetic V;naJ after pretreatments with 300 Ilmol photons m-

²

s- 1 for I h. However, this activity was only a small fraction of the photosynthetic v;:"ax and the ratios of PPDK activities in the WT and mutant were not affected by the light treatment. However, we cannot rule out the possibility that the PPDK activity observed here is below the maximal rate in situ.

Most importantly, an identical procedure was applied to both the WT and the transformed cell lines, strengthening our conclusion that PPDK activity in the latter is considerably reduced.

The role of the C

4

route in P . tricornutum

The results presented here clearly indicate that the C

₄

route does not playa significaIH part in ca rbon acquisition in P. tricornutum.

T he possibility that a biophysical CCM compensates for the reduced ppdk expression in the sPPDK mutant, minimizing the effect of the gene silencing on the CO

₂

response curves (Fig. 2), should be considered. However, in view of the very low PPDK activity, it is unlikely that a C

₄

mechanism plays an important part in net CO

₂

fixation even in the WT. Nevertheless, a significant amount of 1 4C fixation into C

₄

acids has been observed in several diatoms, and inhibition of PEPC lowered the rate of photo- synthesis in P. tricomutum (see Reinfelder, 2011 and references

1700 1500 1300 1100 900

Wavenumber (em-I,

Fig.3 Lipid content in the sPPDK transformed Phaeodaclylum Iricornulum cell line compared with the wild type (WT). (a) Lipid content was measured using the dye Nile red, which produces a golden fluorescence in a hydrophobic environment. Fluorescence was calibrated to 0.0. 750; data are provided in relative units compared with the WT. (b) Fourier transform infrared (FTIR) spectra of the sPPDK transformed cell line (blue line) compared with the WT (red line).

Mean spectra (n > 9) have been baseline-corrected and normalized to the amide I band. Dotted lines indicating the specific peaks of the vibrational bands for lipids (C=O; 1740 cm-'), proteins (amide I band c=o of peptids; 1655 cm-') and carbohydrates (C-O-C; 1200-900 cm-'). The amounts of proteins, carbohydrates and lipids, calculated as a percentage of total biomass from FTIR spectra according to Wagner et al. (2010), are provided in Fig. 54.

therein) . This raises the question of the biological role of the C

4

route in P. tricornutum, if any. In view of the subcellular loca lization of the proteins involved (Kroth et aI., 2008), it is possible that the C

4

route is a futile cycle dissipating excess light energy and ATP (see the Introduction) . To examine possible involvement in protection against photo inhibition, P. tricornutum WT and sPPDK transformed cells were exposed to 2000 )..lmol photons m

-2

s

-I

for 1 h (c. threefold higher than required to saturate the photosynthetic rate). This was followed by measure- ments of O

2

evolution at saturating Ci concentrations. As a result of this treatment, the rate of photosynthesis declined to 85 and 82%

(from the initial rate) in the WT and sPPDK mutants, respectively.

Treatments with 100)..lg ml -

^I

lincomycin, which inhibits protein synthesis in plastids and thereby replacement of damaged D 1 protein in the photosynthetic reaction center II (the main reason for photoinhibition), caused a faster decline of the photosynthetic rate.

It reached 40 and 30% in the WT and the sPPDK transformant, respectively, within 1 h. Clearly, the sPPDK mutant is somewhat more sensitive to excess light than the WT, but the differences are very small.

It should be noted that diatoms possess an array of mechanisms that enable them to cope with varying abiotic factors such as excess light or fast-changing light intensities in the water body (Lavaud etal., 2007; Eisenstadt etal., 2008; Wu etal., 2011; Lepetit etal., 2012). Our measurements (Fig. 4) showed that the rate of fluorescence quenching (as indicated by the slope of the fluores- cence decline from the maximal obtained after turning on the actinic light) is faster ill the sPPDK murant than in the WT, presumably through nonphotochemical quenching (NPQ). This may indicate that the transformed cell lines are able to compensate for the reduced ability to dissipate excess energy by the PPDK- dependent C

₄

route. Such mechanisms may involve the 'activation of the xanthophyll cycle and reaction center events, both leading to the apparent rise in NPQ (Eisenstadt et aI., 2008; Lepetit et al., 2012) and possibly minimizing the expected photoinhibitoty

120

20'+---r---~~----~---_r----~

5 10 15 20 25

Time (min)

Fig.4 The fluorescence yield emitted from wildtype (WT) Phaeodactylum tricornutum (red line) and sPPDK mutants (blue line). Fluorescence was measured using a PAM-1 01. Actinic light intensity was 1500 ~Lmol. photons m-² s-' at 22°e. Saturating pulses (4000 ~Lmol photons m-² s-') were given for 1 s every min. Cells were dark-acclimated for 5 min before turning on actinic light. The arrow pointing up represents the light being switched on; the arrow pointing down represents the light being switched off.

damage in the transformed cell line. Alternatively, the cells may use the PEPCK route of C

_4,

whereby decarboxylation of oxaloacetate leads to the formation of PEP with little, if any, net CO

₂

fixation but significant energy dissipation.

The extent of up- or down-regulation of genes presumably involved in one of the CCM modes following a shift in CO

₂

concentration was used as supporting criteria for the occurrence of C

₄

in diatoms (see Reinfelder, 2011 and references therein), but this may not be the case. One example is the significant rise in the PEPC transcript abundance shortly after an upshift of the pH in the culture media of T. pseudonana from 7.6 to 8.5, in an attempt to lower the concentration of dissolved CO

2 ,

Whether these data demonstrate a rapid up-regulation of PEPC by t he lower CO

₂

concentration, as proposed by Reinfelder (2011), remains to be elucidated. One possibility is that the rise in PEPC tra nscript and in the activity of the C

4

route is the result of the increasing ambient pH. Formation and conversion of dicarboxylic acids could serve as important means of pH homeostasis (Raven et aI., 1990). This may be particularly necessary under conditions where the cells are intensively cycling Ci species between the cytoplasm and the surrounding media (Tchernov etal., 1997,2003). The extent of this cycling is strongly affected by the ambient conditions and by the extent to which they were acclimated to low concentrations of CO

2

(and induced the Ci transport capabilities; Fukuzawa et al., 2011). As an example, Ci cycling rises significantly with light intensity and may reach values much higher than the photosyn- thetic rate and thereby causing a very large load on pH homeostasis (Tchernov etal., 1997,2003).

Acknowledgements

This study was supported by the German-Israel Science Founda- tion (GIF; A.K. and P.G.K.), the Deutsche Forschungsgemeins- chaft (German Research Foundation, DFG) trilateral program (A.K.), the Israel Science Foundation (ISF; A.K.), and by DFG grant number INST 268/149-1. The authors further thank Doris Ballert for help in biolistic transformation and the Universitat Konstanz for financial support.

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Fig. 51 Schemes of the PpDK gene (Prot- ID 21988) (a) and the pPh-TI-PPDK-RNAi construct (b) .

Fig. 52 Growth of the WT and sPPDK transformed cell line under the growth conditions used here.

Fig. 53 The amount of Rubisco's catalytic units.

Fig. 54 The amounts of proteins, carbohydrates and lipids, calculated as percentage of total biomass from FTIR spectra such as presented in Fig. 3(b).

Table 51 Primers used for qPCR