Microbiology Papers in Press. Published May 1, 2015 as doi:10.1099/mic.0.000101

Microbiology Deciphering the mechanisms against oxidative stress in developing and mature akinetes of the cyanobacterium Aphanizomenon ovalisporum --Manuscript Draft-Manuscript Number:

MIC-D-15-00048R2

Full Title:

Deciphering the mechanisms against oxidative stress in developing and mature akinetes of the cyanobacterium Aphanizomenon ovalisporum

Short Title:

Potassium deficiency, oxidative stress and akinetes formation in cyanobacteria

Article Type:

Standard

Section/Category:

Physiology and metabolism

Corresponding Author:

Assaf Sukenik Israel Oceanographic & Limnological Research Mogdal, ISRAEL

First Author:

Ruth N. Kaplan-Levy, PhD

Order of Authors:

Ruth N. Kaplan-Levy, PhD Ora Hadas Assaf Sukenik

Abstract:

Cells of filamentous cyanobacteria of the orders Nostocales and Stigonematales can differentiate into dormant forms called akinetes. Akinetes play a key role in the survival, abundance and distribution of the species, contributing an inoculum for their perennial blooms. In the cyanobacterium Aphanizomenon ovalisporum, potassium deficiency triggers the formation of akinetes. Here we present experimental evidence for the production of ROS during akinetes development in response to potassium deficiency. The function of ROS as a primer signal for akinetes differentiation was negated. Nevertheless, akinetes acquired protective mechanisms against oxidative damage during their differentiation and maintained them as they mature, giving akinetes advantages to survive harsh conditions.

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Deciphering the mechanisms against oxidative stress in developing and mature akinetes of the cyanobacterium Aphanizomenon ovalisporum

Ruth N. Kaplan-Levy, Ora Hadas and Assaf Sukenik The Yigal Allon Kinneret Limnological Laboratory, Israel Oceanographic & Limnological Research P.O. Box 447 Migdal 14950, Israel

Corresponding author: Assaf Sukenik; The Yigal Allon Kinneret Limnological Laboratory, Israel Oceanographic & Limnological Research P.O. Box 447 Migdal 14950, Israel. Tel: +972 4 6721444 ext. 205 Fax: +972 4 6724627 e-mail: [email protected]

Running title: ROS and akinetes differentiation in Nostocales

Key words: akinetes/ cyanobacteria/ dormancy/ reactive oxygen species Word count: Summary – 105

Main text - 5520

Number of figures: 7 Number of Tables: 1 Number of supplements: 2

1

1

Summary

2

Cells of filamentous cyanobacteria of the orders Nostocales and Stigonematales can

3

differentiate into dormant forms called akinetes. Akinetes play a key role in the survival,

4

abundance and distribution of the species, contributing an inoculum for their perennial

5

blooms. In the cyanobacterium Aphanizomenon ovalisporum, potassium deficiency

6

triggers the formation of akinetes. Here we present experimental evidence for the

7

production of ROS during akinete development in response to potassium deficiency. The

8

function of ROS as a primer signal for akinete differentiation was negated. Nevertheless,

9

akinetes acquired protective mechanisms against oxidative damage during their

10

differentiation and maintained them as they mature, giving akinetes advantages to survive

11

harsh conditions.

12

2

13

Introduction

14

Cyanobacteria species of the orders Nostocales and Stigonematales, (sections IV and V of

15

the five taxonomic sections, Rippka et al. 1979) exert cell differentiation. The

16

morphological structures emerge from photosynthetic vegetative cells and may form

17

dormant akinetes, nitrogen fixing heterocysts or small filaments of motile cells known as

18

hormogonia (Maldener et al. 2014). These specialized cells contribute to the species

19

adaptability and survival. In that context, akinetes are resting cells that preserve genotypes

20

while surviving harsh conditions.

21

A variable number of vegetative cells along the filament differentiate to reach mature

22

akinetes when conditions are unfavorable for growth. These akinetes detach from the

23

trichome providing an inoculum for the subsequent seasonal recruitment to produce a new

24

population when environmental conditions improve (Hense and Beckmann 2006).

25

Akinetes are larger than vegetative cells, surrounded by a thickened cell wall and a

26

multilayer extracellular envelope (Adams and Duggan 1999) and are resistant to low

27

temperatures and desiccation. A wide range of environmental factors was reported to

28

trigger akinetes differentiation, such as light, temperature and diverse nutrient limitations

29

(Kaplan-Levy et al. 2010). In A. ovalisporum depletion of potassium from the growth

30

medium triggered the formation of akinetes (Sukenik et al. 2013). During differentiation,

31

akinetes accumulate glycogen, nitrogen in the form of cyanophycin granules, and nucleic

32

acids (Simon 1987; Sukenik et al. 2012). Through the maturation process, a decrease in

33

akinete metabolic activity and an increase in biomass were observed (Fay 1969). Akinetes

34

reached 10 fold higher biovolumes than adjacent vegetative cells (Sukenik et al. 2013). It

35

was demonstrated that akinetes of Aphanizomenon ovalisporum (Nostocales) lost their

36

phycobilisome antenna, as they matured (Sukenik et al. 2007). The phosphate storage

37

found in vegetative cells in the form of polyphosphate bodies was used for the benefit of

38

genome replication in akinetes, reaching 10 to 15 times more genome copies.

39

Furthermore, akinetes contain more ribosomes than vegetative cells (Sukenik et al. 2012).

40

The cessation of vegetative growth due to harsh conditions implies enhancement of

41

oxidative stress, i.e. accumulation of reactive oxygen species (ROS). ROS act as

42

molecular messengers in a wide range of biological processes and they are involved in the

43

development of dormancy in microbial eukaryotes (Aguirre et al. 2005) and plants

44

(Leymarie et al. 2012; Mittler et al. 2011). However, at high levels, ROS can cause

45

oxidative damage such as lipid peroxidation, protein carbonylation and DNA damage

46

(Cabiscol et al. 2000). 3

47

Oxidative stress can ultimately lead to cell death, therefore living organisms evolved

48

various defense mechanisms to protect themselves from ROS damage and keep the

49

cellular ROS homeostasis (D'Autréaux and Toledano 2007). Cyanobacteria, as any other

50

oxygenic phototrophic organisms, are exposed to oxidative damage due to respiration, and

51

during photosynthesis. Singlet oxygen (1O2) and hydrogen peroxide (H2O2) are formed

52

due to the activity of Photosystem II (PSII), superoxide anions are by product in

53

photosystem I (PSI) and hydrogen peroxide is created by photorespiration (Latifi et al.

54

2009). Furthermore, hydrogen peroxide can impair the electron transfer chain and oxygen

55

evolution in the cell (Samuilov et al. 2004). In cyanobacteria, the non-enzymatic

56

antioxidant protection involves carotenoids and α-tocopherol (Kesheri et al. 2011; Latifi et

57

al. 2009), while their enzymatic antioxidative machinery resembles that of other bacteria.

58

The enzymatic defense system includes superoxide dismutase (SOD) and peroxidases. The

59

SOD enzyme catalyzes the dismutation of O2− into oxygen and hydrogen peroxide.

60

Catalases or Peroxiredoxins (Prxs) then convert the hydrogen peroxide into water and

61

oxygen. Prxs belong to a family of enzymes that react with diverse peroxide substrates

62

sharing a similar catalytic mechanism of a redox-active cysteine in their active site. The

63

cysteine is oxidized to a sulfenic acid by the peroxide substrate. Subsequently, the sulfenic

64

acid is reduced back to a thiol by several proteins such as glutaredoxin or thioredoxin,

65

allowing the Prx to proceed to the next catalytic cycle. The Prx family can be divided into

66

4 classes: a) 1-Cys Prxs, b) 2-Cys Prxs, c) Type II Cys Prxs and d) PrxQ, all found in

67

cyanobacteria (Bernroitner et al. 2009; Dietz 2011). In bacteria, the Prxs are responsible

68

for the removal of most of the formed hydrogen peroxide (Perelman et al. 2003; Seaver

69

and Imlay 2001).

70

In this study, we examined the cellular ROS accumulation caused by potassium depletion,

71

conditions that impose the formation of akinetes in A. ovalisporum. The possible

72

involvement of ROS in the regulation of akinete differentiation process was examined.

73

Finally, the cellular response to the oxidative stress, through changes in transcript levels

74

and enzymatic activities were evaluated.

75

Experimental Procedures

76

Culture maintenance, akinetes induction and experimental setup

77

Stock and experimental cultures of Aphanizomenon ovalisporum strain ILC-164 from Lake

78

Kinneret, Israel (Banker et al. 1997) were grown batch wise in liquid media. Cultures were

79

transferred bi-weekly into freshly prepared BG11 medium in a 10-fold dilution (Stanier et

80

al. 1971) grown in 500-1000 ml flat tissue-culture flasks at 20±1 ºC with aeration by a 4

81

regular aquarium air pump (HAILEA, ACO-2203) under continuous illumination at 10

82

mol quantam-2s-1. The basic experimental setup was comprised of control cultures

83

maintained in full BG11 medium (hereafter BG) and of experimental cultures maintained

84

in BG11 medium depleted of potassium (hereafter BG/-K), which imposes akinete

85

differentiation (Sukenik et al 2013). Cultures were grown at 20±1 ºC with moderate

86

shaking under continuous light with diverse intensities, high light (HL) 50 mol quanta m-2

87

s-1, medium light (ML) 25 mol quanta m-2 s-1, and low light (LL) 4 mol quanta m-2 s-1.

88

At day 21 the number of filaments and akinetes in the cultures were assessed and akinetes

89

were isolated as described in Sukenik et al (2007).

90

Filament and akinete count

91

Cultures were sampled at time intervals (at day 0, 5, 7, 14 and 21) starting upon their

92

transfer to a BG/-K medium. Samples were collected after vigorous shaking, to assure

93

homogeneous suspension of filaments and akinetes. Sample aliquots were preserved in

94

Lugol’s solution, and filaments and akinetes were counted in an Utermöhl sedimentation

95

chamber using an inverted microscope (Lund et al., 1958, Utermöhl 1958). Two categories

96

of akinetes were defined, filament-attached akinetes and mature free akinetes. The

97

trichome vegetative cells (named vegetative cells hereafter) had an average wet weight

98

biomass of 0.05 ng (w.w.), whereas the size of filament-attached akinetes increased as

99

their differentiation proceeded and they exceeded a biomass of 0.4 ng (Sukenik et al.,

100

2013). During their differentiation, akinetes were characterized by a thickening of the cell

101

wall and accumulation of apparently cyanophycin granules. The average biomass of young

102

akinetes was 0.1 ng. At later stages of their development, akinetes reached an average

103

biomass of 0.4 ng and eventually detached from the trichome. These free akinetes were

104

defined as mature.

105

ROS in situ staining

106

Samples for ROS detection were collected from BG and BG/-K cultures at days 0, 2, 5, 7,

107

9, 14, 17 and 21, and stained with a fluorescent probe 2’,7’-dichlorodihydrofluorescein

108

diacetate (DCFH-DA; Sigma, cat. D6883, Rastogi et al. 2010). The probe was used at a

109

final concentration of 10 µM in 1 ml culture following incubation at 28°C for 45 min in

110

the dark. The cells were washed with BG/-K medium and dispensed in a 96 well plate in

111

triplicates, 200 µl cell suspension per well, next to non-stained cells serving as controls.

112

Fluorescence was measured in a Fluoroskan ascent (Thermo scientific) plate reader with

5

113

excitation at 485 nm and emission of 538 nm. Absorbance at 630 nm of the same plate

114

was read in a µQuant spectrophotometer (BIO-TEK Instrument Inc.).

115

To observe the intracellular ROS generation during akinete formation 1 ml samples were

116

stained with dihydrorhodamine 123 (Molecular Probes®, cat. D-632), to a final

117

concentration of 200 µM. The samples were incubated at room temperature for 20 min in

118

the dark, washed with BG/-K medium and observed under a fluorescence microscope

119

(Zeiss Axioskop) with GFP filter. The images were analyzed using ImageJ software

120

(http://imagej.nih.gov/ij/index.html). The cellular ROS (relative units) was determined

121

from the integral density measured from individual cells or akinetes normalized to the

122

background intensity and further corrected by subtracting the time zero relative units.

123

RNA extraction, RT-PCR and qPCR conditions

124

RNA extraction - Samples collected at 0, 5, 9 and 14 days from all cultures and from

125

isolated akinetes were frozen in liquid nitrogen and stored at -80°C until processing. Total

126

RNA extraction from A. ovalisporum was conducted using RNeasy Plant Mini Kit

127

(QIAGEN, cat. 74904), according to manufacturer’s protocol. To improve cell lysis prior

128

to the extraction, glass beads (Sigma, 425-600 μm cat. G8772 and ≤106 μm cat. G4649)

129

were added to the RLT lysis buffer and vigorously vortexed for 1 min. The extraction

130

included the use of an on-column DNase step with RNase-Free DNase Set (QIAGEN, cat.

131

79254). Total RNA was eluted and an additional step of DNase was conducted to remove

132

any traces of genomic DNA contamination with DNA-free™ DNase treatment (Ambion,

133

cat. AM1906). The RNA concentration in each sample was estimated using Nanodrop

134

2000C (Thermo Scientific).

135

Reverse Transcription (RT) – all RT reactions in this study were done using Bio-RT kit

136

(Bio-Lab Ltd, cat. 9597 58027300) with 1µg total RNA as template, and 0.5 µg of random

137

primers (Promega, cat. C1181). Following RT, samples were treated with RNase H (NEB,

138

cat. M0297S) according to the suppliers protocol, in a total volume of 50 µl.

139

Polymerase Chain Reaction – The PCR reaction was conducted in a volume of 25 µl with

140

0.5 µl of 1st strand cDNA, 400 nM dNTPs, 120 nM of each primer according to Table S1,

141

2.5 µl of 10x Taq Buffer and 0.5 µl Taq polymerase (Pluthero 1993). The amplification

142

program for all the tested genes in this study was as follows: a hot start of 3 min at 95°C,

143

following 95°C for 45 sec, annealing for 30 sec and extension at 72°C for 40 sec, with a

144

final step at 16°C. The number of cycles and annealing temperatures for each gene are

145

detailed in Table S1. Three types of controls were done for each gene: negative control of 6

146

total RNA as template to verify the absence of genomic DNA in the sample, no template

147

control and A. ovalisporum genomic DNA.

148

qPCR – Transcript levels were assessed using Rotor-Gene 6000 (Corbett Research

149

Mortlake, Australia). The PCR reaction was performed in a final volume of 25 µl with 2

150

µl of 1st strand cDNA template, 400 nM dNTPs, 72 nM of each primer according to Table

151

S1, 1:30,000 final dilution of SYBR® Green I (Invitrogen S7563), 2.5 µl of 10x Taq

152

Buffer and 0.5 µl Taq polymerase (Pluthero 1993). For all the genes tested in this study,

153

the amplification program was as follows: a hot start of 3 min at 95°C, following cycles of

154

95°C for 10 sec, annealing at 58°C for 15 sec and extension at 72°C for 20 sec. Each time

155

point was done in triplicates with the corresponding controls: 1) no template control, 2)

156

total RNA as a template and 3) genomic DNA as positive control. To establish the

157

transcript levels in a sample a standard curve was done using genomic DNA as template

158

for each gene. The data was processed using the Rotor-GeneTM 6000 real-time PCR

159

analyzer software, version 1.7 (Qiagen Inc, CA USA). Transcript levels were normalized

160

to 16S rRNA and calculated relative to their expression at time zero of the induction.

161

In gel enzyme activity assays

162

Samples collected at 0, 7, 14, 21 days from all cultures and from isolated akinetes were

163

collected by centrifugation using a swinging bucket rotor at 2700 g at room temperature

164

for 10 min. The pellet was immediately frozen with liquid nitrogen and stored at -80°C

165

until processing. Prior to the protein assay, samples were thawed in 300 µl breaking buffer

166

(50 mM PBS pH 6.8, 5 mM EDTA pH 8 and 1 mM phenylmethylsulfonyl fluoride). Total

167

protein was extracted by sonication with 12 cycles of a 30 sec pulse followed by a 10 sec

168

pause. Sonication intensity of 0.3 Kw was used for cultures and 0.6 Kw for isolated

169

akinetes. Following sonication, the samples were centrifuged at 12,000 g for 3 min and the

170

supernatant collected. Total protein concentrations were assessed using Bradford stain

171

(Sigma B6916). In-gel enzyme activities were performed onto a 10% acrylamide native

172

gel (Sambrook et al. 2001), using 7 µg total protein. SOD activity on native gel was

173

conducted according to Beauchamp and Fridovich (1971). Achromatic bands were formed

174

where SOD isoforms were present and had catalyzed the removal of superoxide radicals

175

produced by riboflavin. The metal ion in the active centers of the enzymes were identified

176

by adding an inhibitor to the staining solution, 2 mM H2O2 to inhibit Fe-SOD (Weissman

177

et al. 2005). Peroxidase activity on native gel was conducted according to Goldberg and

178

Hochman (1989). Achromatic bands resulted from the enzymatically removal of hydrogen

179

peroxide indicate the presence of peroxidase isoforms. 7

180

Results

181

ROS accumulation under potassium deficiency

182

Cultures of A. ovalisporum grown in BG11 medium with (BG) and without potassium

183

(BG/-K) at 20°C, gradually accumulated ROS when continuous light was provided at

184

medium or high intensity (ML - 25 or HL - 50 mol quanta m-2 s-1 respectively, Fig. 1).

185

BG/-K medium enhanced ROS accumulation also under low light (LL - 4 mol quanta m-2

186

s-1), unlike the cultures growing on BG (Fig.1C). Under high light conditions ROS levels

187

in BG/-K cultures were significantly higher (Student’s t-test p≤ 0.05) than in BG cultures

188

from day 5 on, although temporal fluctuations in ROS levels were recorded in both

189

cultures (Fig. 1A). Under ML no significant differences in ROS levels were found

190

between the potassium deprived and the full media cultures (Student’s t-test p>0.5) until

191

day 21. As in HL, also in ML temporal fluctuations in ROS intensities in both cultures

192

were recorded (Fig. 1B).

193

After 21 days, akinetes were observed in all BG/-K cultures, with high abundance under

194

HL or ML conditions and low abundance under LL conditions (Fig. 2). Substantially low

195

concentrations of akinetes were detected in the BG cultures under ML, whereas no

196

akinetes were recorded in LL and HL controls (Fig. 2). The highest concentration of

197

akinetes was observed in BG/-K cultures maintained under HL, in accordance with our

198

previous report (Sukenik et al. 2013).

199

Enhancement of oxidative stress and akinete formation

200

Since potassium deficiency triggers ROS accumulation and akinete formation, the effect

201

of oxidation agents on the differentiation of akinetes was tested to examine a possible link

202

between these two pathways. The experiments took place under low light conditions to

203

neutralize the effect of light intensity on ROS production and akinete differentiation. First,

204

the effect of various hydrogen peroxide concentrations (0, 0.01, 0.05, 0.1, 0.5, 1 and 5

205

mM) on growth and akinete formation was evaluated. Low H2O2 concentrations (0.01 and

206

0.05 mM) showed no difference in growth rate as compared to a non-treated culture.

207

Treatment with 0.1 mM H2O2 caused minor growth inhibition whereas the high

208

concentrations (0.5, 1 and 5 mM) resulted in the collapse of the culture. Nevertheless, no

209

akinetes were formed in any of these treatments (Table 1). Finally, to attain a mild

210

oxidative stress in A. ovalisporum cultures, ascorbate was added to final concentrations

211

ranging between 0.1 and 1 mM (Table 1) under low light conditions. Ascorbate at

212

concentrations higher than 1mM imposed culture collapse (data not shown). A mild 8

213

cellular ROS pool was generated upon the addition of 0.7, 0.8 or 0.9 mM ascorbate (Fig.

214

3A, B). A. ovalisporum cultures grown on BG media responded to the addition of

215

ascorbate on day 5, with a prompt peak of ROS that was practically quenched on day 7

216

(Fig. 3A). Unlike BG cultures, the addition of ascorbate to BG/-K grown cultures resulted

217

with biphasic mode of ROS generation. The first ROS peak was observed immediately

218

after the addition of ascorbate, as in the BG cultures, and a second higher peak, 4 days

219

later. From that point on, ROS level decreased to the level found in the ascorbate free

220

BG/-K cultures (Fig 3B). The higher the ascorbate concentration added, higher ROS levels

221

were observed during the second phase (Fig. 3B). While the addition of ascorbate imposed

222

the elevation of ROS level under all culture conditions, akinetes were formed mainly in K+

223

deprived cultures (BG/-K ), with highest akinete numbers formed in response to the

224

addition of 0.7 mM ascorbate (Fig. 3C). Nonetheless, the synergistic effect of K+ depletion

225

and 0.7 mM ascorbate on akinete formation was minor as compared to the number of

226

akinetes formed in BG/-K medium under high light conditions (Fig. 2). These results do

227

not support the assumption that oxidative stress is linked with akinetes’ differentiation,

228

and/or served as a direct developmental signal in this process.

229

Intracellular ROS generation and akinete differentiation

230

To follow variations in ROS at the cellular level, we applied dihydrorhodamine (D-632) to

231

cultures deprived from potassium, during cell differentiation, and to isolated akinetes

232

suspension, and observed ROS accumulation by fluorescence microscopy (Fig. 4 & 5).

233

Intracellular ROS accumulation was observed 5 days post potassium deficiency in both

234

vegetative cells and in developing akinetes, with similar fluorescence intensity in both cell

235

types (Fig 4 & 5). By day 9 in BG/-K medium, vegetative cells showed homogeneous

236

ROS accumulation, whereas variable fluorescence intensity was observed in akinetes (Fig.

237

4 red and blue arrows and Fig. 5). This variability can be attributed to akinete age, as

238

akinetes differentiate at an unsynchronized mode (not all cells initiate their differentiation

239

at the same time). While young differentiating akinetes (average w.w. of 0.1 ng) showed

240

higher ROS intensities, akinetes at later developmental stages (biomass, w.w. ranged

241

between 0.1of 0.4 ng) showed significantly lower ROS levels than the vegetative cells

242

(Fig. 5). At day 14, a decrease in ROS pools in all cell types was observed, followed by

243

variable and reduced levels of ROS in mature isolated akinetes (Fig. 4 & 5).

244

The expression pattern of the antioxidative machinery during akinete differentiation

245

A survey for genes encoding proteins putatively associated with antioxidative stress was

246

conducted, using a translated BLASTN: tblastn (search of translated nucleotide database 9

247

using a protein query) protocol on the draft genome of A. ovalisporum. In total, 14 open

248

reading frames (ORF) encoding putative antioxidative enzymes were found (Table S2). In

249

agreement with other cyanobacteria, no ascorbate peroxidase encoding ORF was

250

identified. As in other Nostocales, A. ovalisporum genome contains two ORFs encoding

251

putative superoxide dismutase (SOD): 1) a predicted membrane bound SOD (SodA) with

252

77% similarity to Ava_1445 protein sequence; and 2) a soluble Fe-SOD (SodB) with 91%

253

similarity to Ava_0963 amino acids sequence. Two open reading frames code for

254

glutathione reductase (GR1 and GR2) and one ORF encoding glutaredoxin were detected.

255

A gene encoding a putative manganese catalase (MnCAT) with a similarity of 95% to the

256

Anabaena variabilis MnCAT Ava_3821 was found. Other peroxidase (Prx) encoding

257

ORFs were identified in A. ovalisporum genome, as detailed in Table S2. An ORF

258

encoding for vanadium-dependent haloperoxidase (vdPrx) was found in addition to two

259

ORFs that putatively encode 2-cysteine Prx (Prx2a and Prx2b). These A. ovalisporum

260

ORFs are located at consecutive loci as found for Anabaena variabilis and other

261

Nostocales. Additionally, a single ORF that codes for type II peroxiredoxin (t2Prx) was

262

identified in A. ovalisporum. It is located upstream to the gr1 locus. Only 3 putative ORFs

263

encoding peroxiredoxin Q (PrxQ) were found in A. ovalisporum genome, while other

264

Nostocales contain 4 different PrxQ encoding genes (Bernroitner et al. 2009). Based on

265

earlier observations that the phycobilisome antenna is degraded during akinete maturation

266

to reduce photo-oxidative damage (Sukenik et al. 2007), we identified an ORF with a

267

similarity of 100% to the NblA amino acid sequence in Anabaena variabilis. Finally, as a

268

reference to akinete differentiation, an ORF encoding the akinete marker AvaK from A.

269

ovalisporum was identified, with a 70% similarity to the A. variabilis Ava_1657 and 66%

270

similarity to the protein sequence from Anabaena sp. 90 ANA_C12209 (Zhou and Wolk

271

2002).

272

A semi-quantitative RT-PCR approach was applied to study variations in antioxidative

273

gene expression during akinete differentiation in akinete-inducing (BG/-K, HL) versus

274

non-inducing conditions (BG, LL) (Fig. 6A). In the non-inducing conditions, in 8 out of

275

the 14 antioxidative genes tested, a low level of constitutive expression was observed.

276

These results suggest that those genes are involved in the cell redox homeostasis and not

277

necessarily transcribed only under oxidative stress conditions. In addition, nblA was

278

expressed in low levels under both, inducing and non-inducing conditions, probably as

279

part of the phycobilisome antenna turnover process. The putative MnCAT transcript was

280

not detected either in the non-induced or in the akinete-induced cultures. Subsequently, the

281

expression patterns of the putative antioxidative and other akinete related genes were 10

282

tested by RT-qPCR during akinete differentiation (Fig. 6B). Transcript levels were

283

normalized to 16S rRNA and then standardized relatively to the expression at time zero

284

(upon imposing potassium deprivation and HL conditions). In order to maintain clear

285

visibility at different scales, the data was grouped into four panels due to large variations

286

in expression levels of the tested genes. Overall, most of the antioxidative genes

287

expression, including nblA and the akinete marker- avaK, coincided with high levels of

288

ROS in the cultures, with a peak by day 9 and a fall by day 14-post induction (Fig. 1C and

289

Fig. 6B). Moreover, all genes showed a higher expression in isolated akinetes as compared

290

to that of the 14 days old culture, ranging from 1.9 for gr2 to 47.7 folds for prxQ1 (Fig.

291

6B).

292

The expression patterns of both SOD encoding genes differed from each other. sodB was

293

transcribed in both akinete-inducing (BG/-K, HL) and non-inducing conditions, whereas

294

sodA was expressed mainly during akinete-inducing conditions (Fig. 6B panels 3 and 4).

295

The main difference in the expression levels of these two genes was observed on the 5th

296

day upon akinete-inducing condition, as sodB transcription levels dropped almost 3 folds,

297

while sodA transcript levels increased by 20 fold. Furthermore, sodA was the only gene

298

that its expression level at the peak of the ROS burst (9th day) was twice higher than in

299

isolated akinetes. A difference in the transcription patterns of the two genes encoding for

300

glutathione reductase was also observed. gr2 was expressed only under akinete-inducing

301

conditions with a peak of 10 fold expression by day 9 (Fig. 6B panels 2 and 4). gr1 was

302

expressed both, in non-inducing and in akinete-inducing conditions, and unlike the other

303

genes tested, its expression increased gradually through akinete differentiation, to reach

304

almost 20 times higher levels by day 14 and an elevation of 4.2 fold in isolated akinetes.

305

The examination of the Prx encoding genes showed that the majority of them were

306

expressed during the vegetative growth of the non-induced culture, with the exception of

307

t2Prx and PrxQ1. In isolated akinetes, the transcripts of these genes increased 200 and 20

308

fold, respectively. The expression pattern of the 2-cys Prx a and b was similar, with a

309

down regulation of more than 1.5 fold (5th day), following an increase in transcription at

310

day 9, and a decline in transcript levels at day 14. Isolated akinetes showed elevated

311

expression of both genes (Fig. 6B panel 2). Unlike the transcription of the other genes

312

studied, PrxQ3 transcription was upregulated 3 fold after 5 days at BG/-K and HL

313

conditions, remained stable at day 9 and decreased at day 14, however, the gene

314

transcription was ~8 fold higher as compared to day zero in isolated akinetes (Fig. 6B

315

panel 1). The remaining Prxs, including - vdPrx, Prx1 and PrxQ2, showed similar

11

316

patterns, with high expression levels at day 9, following a decrease at day 14 and the peak

317

of expression levels in the isolated akinetes.

318

The activity of SOD and peroxidase

319

The gene expression analysis was completed by measuring variations in the activity of

320

SOD and peroxidase by native gel activity (Fig. 7). SOD activity assay resulted in one or

321

more achromatic bands, formed where SOD native proteins were present and catalyzed the

322

removal of superoxide radicals produced by riboflavin (labeled with arrows and roman

323

numbers in Fig. 7A). In the BG culture, a decrease in SOD activity was observed after 7

324

days following a low level of activity until day 21 (isoform IV). The BG/-K culture

325

showed an increase in activity of isoform V after 7 days, followed by the appearance of

326

additional enzyme isoforms. The highest number of SOD isoforms was detected in

327

isolated akinetes. SOD isoforms II and V appeared by day 7, whereas isoform III was

328

detected only in the isolated akinetes (Fig. 7A). Running the in-gel activity assay in the

329

presence of 2 mM H2O2 confirmed that Fe is the metal ion active in the SOD reaction

330

center of the observed isoforms (data not shown).

331

The peroxidase in-gel activity assay demonstrated several achromatic bands resulting from

332

the enzymatic removal of hydrogen peroxide (Fig 7B). In the non-induced culture,

333

peroxidase activity was observed as a major achromatic band (labeled IV), 14 days post

334

inoculation with increasing intensities on day 21, as the culture aged (Fig. 7B). Two other

335

minor bands (I & II) were detected at day 14 and 21. In the akinete induced culture, a

336

different pattern of peroxidase activity was observed with 4 isoforms detected 14 days

337

post inoculation. While in the non-induced culture the two peroxidase isoforms activity

338

increase at day 21, in the akinete induced culture a decrease in activity was recorded in all

339

isoforms. Peroxidase isoform III with high activity appeared on day 14 post inoculation in

340

the akinete-induced culture, following a decrease at day 21, and seems to be absent from

341

isolated akinetes. A higher activity of isoform I was detected in isolated akinetes.

342

Discussion

343

Reactive oxygen species are associated with various stress conditions but also linked with

344

cellular differentiation and developmental signaling pathways in diverse organisms

345

(Aguirre et al. 2005; Bailly et al. 2008; Gapper and Dolan 2006). In this study, we

346

examined the presence of ROS under akinete formation conditions, investigated their role

347

in cell differentiation in cyanobacteria and showed the cellular response to oxidative stress

348

during akinetes’ development. 12

349

ROS generation was observed in response to potassium deficiency under all light

350

intensities. This phenomena is consistent with findings in mitochondria and chloroplasts,

351

where potassium channels present in the same membrane of the electron transfer chain are

352

involved in the maintenance of membrane potential (Checchetto et al. 2012; Malinska et

353

al. 2010). Ion fluxes across the thylakoid membrane contribute to the regulation of the

354

osmotic (ΔpH) and electric (ΔΨ) constituents of the proton motive force (Checchetto et al.

355

2012; Kramer et al. 2003). Potassium deficiency may also affect photosynthesis, since this

356

process is balanced by K+ efflux from the thylakoid lumen (Chow et al. 1976; Dilley and

357

Vernon 1965). The efflux of positive charge out of the lumen balances the proton flow,

358

resulting in the dissipation of the trans-thylakoid membrane potential and establishing a

359

significant ΔpH for ATP synthesis (Checchetto et al. 2013; Pfeil et al. 2013). Therefore,

360

lack of K+ ions might lead to a low ΔpH resulting in photoinhibition and ROS production

361

(Joliot and Johnson 2011). As potassium deficiency induces pleiotropic effects in

362

cyanobacteria (Alahari and Apte 1998), ROS formation and akinete differentiation in A.

363

ovalisporum seem to be part of these multiple phenotypic expression. Does the ROS

364

accumulation influence cellular differentiation in cyanobacteria? Does it play a role in

365

akinetes’ differentiation signaling?

366

In many organisms, ROS serve as signaling molecules for various processes including

367

cellular differentiation and development (Mittler et al. 2011). In the filamentous fungi

368

Neurospora crassa, a hyper-oxidative state is developed at the initiation of the three

369

morphogenic transitions of the asexual reproduction stage from spores, named conidiation

370

(Hansberg et al. 2008). The transcription of Mn-sod gene is induced during spore

371

development in fungi, as shown in Colletotrichum graminicola (Fang et al. 2002) and

372

catalase A (catA) is highly expressed during sporulation in Aspergillus nidulans (Navarro

373

et al. 1996), emphasizing the potential role of ROS burst in developmental processes. In

374

plants, biotic and/or abiotic stress conditions stimulate a burst of ROS that plays an

375

integral role as signaling molecules in the regulation of numerous biological processes.

376

Stress responses in plants are mediated by a temporal–spatial coordination between ROS

377

and other signals that rely on production of stress-specific chemicals, compounds, and

378

hormones (Baxter et al. 2014). Zhao et al (2007) reported that hydrogen peroxide

379

accumulates in heterocysts, probably as a result of respiration and nitrogenase activity but

380

no signaling role has been ascribed to this ROS formation. Claessen et al. (2014)

381

postulated that in cyanobacteria from the order Nostocales ROS mediate programmed cell

382

death, leading to the formation and release of hormogonia. Here we challenged the role of

383

ROS accumulation as an intracellular signal for akinete differentiation by implementing 13

384

environmental stress conditions such as H2O2 or ascorbate. The addition of ascorbate

385

(between 0.7 and 0.9 mM) succeeded to mimic the ROS levels generated under akinete-

386

inducing conditions (potassium deficiency and ML or HL conditions), but yielded only a

387

limited number of akinetes.

388

Ascorbate is known as an antioxidant but may also reduce redox-active metals such as

389

copper and iron, thereby increasing the pro-oxidant chemistry of these metals and

390

resulting in hydroxyl radicals through the Fenton reaction (Buettner and Jurkiewicz 1996;

391

Halliwell 1996). This may explain the burst of ROS observed post ascorbate addition. The

392

biphasic ROS production in A. ovalisporum cultures growing in BG/-K (Fig. 3) can be

393

attributed to the additional stress elicited by the lack of potassium. The addition of

394

ascorbate induced akinete formation under LL conditions, but at a significantly lower

395

abundance than under akinete inducing conditions (potassium deficiency and ML or HL

396

conditions – Fig. 2). Therefore, we postulate that in A. ovalisporum, ROS accumulation

397

under potassium deficiency and the concomitant akinetes development are not necessarily

398

linked.

399

Akinete differentiating cells in potassium deficient cultures under high light intensities

400

need to cope with oxidation stress prior the formation of a viable akinete, and maintain the

401

capacity to survive an extended period of dormancy. Cells committed to form akinetes

402

seemed to have higher ROS levels than their adjacent vegetative cells at day 9 post

403

inoculation on akinete inducing medium. However, as the akinete development proceed, a

404

decrease in ROS levels (Fig. 4 & 5), concomitant with an up-regulation of the

405

antioxidative machinery was observed, both, at the gene and protein levels (Fig. 5-7).

406

Although a decrease in ROS levels was observed in all cell types, by day 14th post

407

inoculation, mature akinetes maintained their antioxidative defense (Fig. 5-7).

408

Nevertheless, some of the genes tested were also expressed in the BG11 cultures, as being

409

involved both, in the antioxidative mechanism and in the cellular redox homeostasis

410

(Bernroitner et al. 2009). An additional strategy of cyanobacteria to decrease ROS

411

generation upon nutrient deficiency is by phycobilisome degradation to reduce light

412

energy input by decreasing light absorption capabilities (Schwarz and Forchhammer

413

2005). In isolated akinetes, the transcript abundance of nblA was 7 fold higher than that of

414

the whole culture, concomitant with the depletion of phycobilisome proteins reported for

415

A. ovalisporum akinetes (Sukenik et al. 2007).

416

In this study we reveal that environmental conditions that trigger akinete formation (BG/-

417

K, HL) also induce ROS accumulation in the cyanobacterium A. ovalisporum. However, 14

418

the differentiating cells manage to overcome the oxidative burst by degradation of the

419

phycobilisome and up-regulation of the antioxidative machinery. Although the ROS level

420

decreases in mature akinetes, the antioxidative machinery is maintained. This

421

developmental process resembles the one occurring in developing plant seeds, where ROS

422

metabolism is important during the three typical developmental stages – embryogenesis,

423

reserve accumulation and maturation/ drying (Bailly 2004). ROS and the accumulation of

424

the antioxidative machinery, in particular the Prx pathway, are involved in the acquisition

425

of desiccation tolerance in developing plant seeds (Bailly et al. 2001). This information

426

leads us to speculate that in cyanobacteria the ROS burst in developing akinetes is part of

427

the differentiation process and not the trigger itself. Furthermore, this process enables the

428

differentiating cell to activate the antioxidative machinery that in turn, like in plant seeds,

429

may confer the desiccation tolerance known to exist in cyanobacteria akinetes. Mature

430

akinetes should survive an extended period of dormancy. Therefore the protection of their

431

polyploid genome (Sukenik et al. 2012) and other essential cellular components are crucial

432

to assure a successful germination on due time and conditions. The antioxidative

433

machinery found in isolated mature akinetes certainly contributes to the persistence of

434

akinetes. It would be interesting to investigate the antioxidative capabilities of an akinete

435

after a prolonged period of dormancy in laboratory cultures, as well as in lake bottom

436

sediment, and to elucidate if this machinery serves as a defense strategy through

437

dormancy, or/and plays a role in the germination process.

438

Acknowledgement

439

The contribution of Drs. Michael Kube and Richard Reinhardt (Max Plank Institution,

440

Berlin) to the sequencing of A. ovalisporum genome is acknowledged. We thank Dr Esther

441

Lubzens for her constructive comments on the manuscript. This study was funded by the

442

Israel Ministry of Science and Technology (MOST), Levy Eshkol Fellowship (No. 3-

443

9476) to RNKL and by the Israel Science Foundation (Grant No. 319/12) to AS. We thank

444

the European Cooperation in Science and Technology COST Action ES 1105

445

CYANOCOST for networking and knowledge-transfer support.

446

Conflict of interest statement - there is no conflict of interest in connection with this

447

study and its publication.

448 449 450 15

451 452

References

453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500

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595 596 597 598 599 600 601 18

602 603

Table 1: ROS induction by diverse treatments in Aphanizomenon ovalisporum cultures

604

under low light conditions. The different treatments employed to induce ROS generation

605

in A. ovalisporum cultures are listed below including the cultures behavior and the

606

concentration of akinetes detected in the culture after 14 days. (–) No akinetes observed;

607

(SO) Akinetes seldom observed.

608

Treatment BG/-K HL (Reference) BG BG/-K BG + H2O2

BG + Ascorbate

BG/-K + Ascorbate

Final Concentration [mM] 0.01 0.05 0.1 0.5 1 5 0.1 0.25 0.3 0.5 0.6 0.75 0.8 0.9 1 0.1 0.3 0.5 0.6 0.7 0.8 0.9

Culture growth

Akinetes/ml

Growth inhibition by day 7 Normal growth Similar to BG/-K HL Normal growth Normal growth Growth inhibition Death Death Death Normal growth Normal growth Normal growth Normal growth Normal growth Normal growth Normal growth Normal growth Death Similar to BG/-K HL Similar to BG/-K HL Similar to BG/-K HL Similar to BG/-K HL Similar to BG/-K HL Similar to BG/-K HL Similar to BG/-K HL

~ 4-8x105 SO ~ 4x103 SO SO SO ~ 1x103 ~ 1x103 ~ 1x103 ~ 3x103 ~ 1x104 ~ 3x103 ~ 3x103

609 610 611 612 613 614 615 19

616 617

Figure Legends:

618

Figure 1: Levels of reactive oxygen species (ROS) during Aphanizomenon

619

ovalisporum growth in BG/-K vs BG media. ROS levels were measured with the probe

620

DCFH-DA and fluorescence intensities were normalized to the optical densities of the

621

samples and presented as arbitrary units. All cultures were grown at 20°C under

622

continuous light for 21 days. (A) ROS level at light intensity of 50 mol quanta m-2 s-1

623

(high light, HL); (B) ROS level at light intensity of 25 mol quanta m-2 s-1 (medium light,

624

ML); (C) ROS level at light intensity of 4 mol quanta m-2 s-1 (low light, LL). Data points

625

present average and standard deviation (n=3)

626 627

Figure 2: Akinetes concentration in BG/-K and BG cultures under three different

628

light conditions as specified in Fig. 1. Akinete concentrations f A. ovalisporum cultures

629

were counted at day 21. Both attached to trichomes and free akinetes were counted. Bars

630

present average and standard error (n=3).

631 632

Figure 3: The effect of ascorbate on ROS generation and akinete formation during

633

Aphanizomenon ovalisporum growth in BG/-K and BG media. ROS levels were

634

measured with the probe 2,7-DCFH-DA and fluorescence intensities were normalized to

635

the optical densities of the samples. BG (A) and BG/-K (B) cultures were grown at 20°C

636

under continuous light for 21 days at light intensity of 4 mol quanta m-2 s-1 (low light,

637

LL). Arrow marks the time of ascorbate addition to the culture and the concentration

638

[mM] of ascorbate added to the culture are noted. (C) Number of akinetes attached and

639

free per ml at 21 days post inoculation. Data points present average and standard deviation

640

(n=3).

641 642

Figure 4: Cellular ROS generation during akinete differentiation. Filaments were

643

stained with dihydrorhodamine (D-632) to detect cellular ROS pools by epifluorescence

644

microscopy. Samples were collected from BG/-K, HL cultures at day 0, 5, 9 and 14-post

645

inoculation. Akinetes were isolated on day 14. Left – Bright field; Right – Fluorescence

646

observed with GFP filter set. Red arrows indicate early stages of akinete differentiation,

647

Blue arrows show later stages of akinete maturation; Scale bars - 10µm.

648

Figure 5: Quantitation of the cellular ROS pools during akinete differentiation. ROS

649

intensities per cell were extrapolated for each time point from the pictures using imageJ. 20

650

The units are normalized to time zero of inoculation. The different developmental stages

651

presented are: Vegetative cells - with an average biomass of 0.05ng (w.w.); Young

652

akinetes – differentiating cells at early stages of development with an average biomass of

653

~ 0.1 ng (w.w.); Akinete later stage – akinetes that reached an average biomass of ~ 0.4 ng

654

(w.w.), but are still attached to the trichome. Mature free akinetes – akinetes that detached

655

from the trichome. Isolated akinetes – Akinetes were isolated following the procedure

656

described in Sukenik et al., 2007. Bars present average and standard deviation. For each

657

time point and cell type, more than 10 cells were analyzed.

658

Figure 6: Expression pattern of putative antioxidative genes during akinete

659

differentiation. (A) Expression pattern of putative antioxidative genes were tested by

660

semi quantitative-RT-PCR during akinete formation in induced cultures (BG/-K, HL) vs.

661

non-induced culture (BG, LL). Samples were collected on day 0, 5, 9 and 14 since

662

induction. Akinetes were isolated on day 14 (AK). Three controls were made per gene: A

663

negative control of total RNA as template to verify the absence of genomic DNA in the

664

sample, no template control and A. ovalisporum genomic DNA. Results are representative

665

of three independent biological replicas. (B) Quantitative analysis of gene expression

666

during akinete differentiation was tested in BG/-K cultures by RT-qPCR. Transcript levels

667

are normalized to 16 rRNA and relative to T0. In order to maintain clear visibility at

668

different scales, the data was grouped into four panels. RU –relative units. Bars represent

669

average of three technical replicas.

670

Figure 7: On gel activity of antioxidative enzymes during Aphanizomenon

671

ovalisporum growth in akinete induced vs. non-induced media. Samples were collected

672

on day 0, 7, 14, 21 since induction. Akinetes were isolated on day 21 (AK). Total protein

673

extracts were run on native gels and stained for Superoxide dismutase activity (A) and

674

Peroxidase activity (B). The various activity bands showing enzyme isoforms are labeled

675

with arrows and roman numbers. These analyses were run with two independent

676

biological replicates. Here, representative results are shown.

21

Figure 1 Click here to download Figure: Fig1 AS.tif

Figure 2 Click here to download Figure: Fig2 AS.tif

Figure 3 Click here to download Figure: Fig3_Asc AS.tif

Figure 4 Click here to download Figure: Fig4_ROScells.tif

Figure 5 Click here to download Figure: Fig5 AS revised.tif

Figure 6 Click here to download Figure: Fig6-HD.tif

Figure 7 Click here to download Figure: Fig7_proteins-rev.tif

Click here to download Supplementary Material Files: Table S1 S2 V1 200415.pdf