Effect of fulvic acid induction on the physiology, metabolism, and lipid biosynthesis-related gene transcription of Monoraphidium sp. FXY-10

Accepted Manuscript Effect of fulvic acid induction on the physiology, metabolism, and lipid bi osynthesis-r elated gene tr anscr iption of Monoraphidium sp. FXY-10 Raoqiong Che, Li Huang, Jun-Wei Xu, Peng Zhao, Tao Li, Huixian Ma, Xuya Yu PII: DOI: Reference:

S0960-8524(16)31668-6 http://dx.doi.org/10.1016/j.biortech.2016.12.017 BITE 17393

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

15 October 2016 2 December 2016 3 December 2016

Please cite this article as: Che, R., Huang, L., Xu, J-W., Zhao, P., Li, T., Ma, H., Yu, X., Effect of fulvic acid induction on the physiology, metabolism, and lipid biosynthesis-r elated gene tr anscr iption of Monoraphidium sp. FXY-10, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech. 2016.12.017

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1

Effect of fulvic acid induction on the physiology, metabolism, and

2

lipid biosynthesis-related gene transcription of Monoraphidium sp.

3

FXY-10

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Raoqiong Chea1, Li Huangb1, Jun-Wei Xua, Peng Zhaoa, Tao Lia, Huixian Mac, Xuya Yu a* a

Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China b Institute of Chemical Industry, Kunming Metallurgy College, Kunming, China c School of Foreign Languages, Kunming University, Kunming 650200, China *Corresponding Author: [email protected] Tel: +086-0871-65920548. 1These authors contributed equally to this work

1

20 21

Abstract

22

Fulvic acid (FA) triggers lipid accumulation in Monoraphidium sp. FXY-10, which

23

can produce biofuels. Therefore, the metabolism shift and gene expression changes

24

influenced by fulvic acid should be investigated. In this study, lipid and protein

25

contents increased rapidly from 44.6% to 54.3% and from 31.4% to 39.7% under FA

26

treatment, respectively. By contrast, carbohydrate content sharply declined from 49.5%

27

to 32.5%. The correlation between lipid content and gene expression was also

28

analyzed. Results revealed that accD, ME, and GPAT genes were significantly

29

correlated with lipid accumulation. These genes could likely influence lipid

30

accumulation and could be selected as modification candidates. These results

31

demonstrated that that FA significantly increased microalgal lipid accumulation by

32

changing the intracellular reactive oxygen species, gene expression, and enzyme

33

activities of acetyl-CoA carboxylase, malic enzyme, and phosphoenolpyruvate

34

carboxylase.

35

Keywords: Monoraphidium sp. FXY-10; fulvic acid; lipid; gene expression; enzyme

36

activity

37

2

38

1. Introduction

39 40

An increasing global demand for a renewable and sustainable energy sources has

41

emerged because of the exhausted usage of fossil fuels. Attempts have been made to

42

use various organisms as feedstock of fatty acid-derived biofuels and chemicals

43

(Chisti, 2007). Biodiesel has also been extensively explored as a promising alternative

44

to petroleum-based fuels because of its environmental adaptability, high oxygen ratio,

45

and excellent ignition properties. Microalgae have been regarded as the most potential

46

biodiesel source because of their fast growth rate, high lipid content, high

47

photosynthesis efficiency, and slight competition with crops for arable land (Chisti,

48

2007; Yu et al., 2012).

49

Among oleaginous microalgae, Monoraphidium species have been widely

50

investigated and have evolved three nutrition modes for growth, namely,

51

photoautotrophy, heterotrophy, and mixotrophy. Moreover, Monoraphidium species

52

can switch their nutritional mode according to their environment. Monoraphidium

53

species can accumulate large quantities of oils, which are ideal precursors for

54

biodiesel production, under certain culture conditions (Yu et al., 2012). Furthermore,

55

the whole genome sequence of Monoraphidium neglectum has been obtained, and a

56

well-developed gene toolbox has been available (Bogen et al., 2013), and these

57

techniques have facilitated studies on lipid biosynthesis at molecular levels. Thus,

58

Monoraphidium species have been considered new oleaginous microalgal models for

59

oil biosynthesis and promising biodiesel feedstock. 3

60

The growth and lipid accumulation of Monoraphidium vary greatly depending

61

on growth conditions, irradiance, and nutrient stress. (Yu et al., 2012; Huang et al.,

62

2014; Zhao et al., 2016). The manipulation of growth conditions can also influence

63

lipid and triacylglycerol (TAG) accumulation in microalgae. Several chemical and

64

physical stimuli, such as light (He et al., 2015; Liu et al., 2012), salinity (Zhao et al.,

65

2016; Yang et al., 2014), temperature (Converti et al., 2009), and nutrient (Hang et al.,

66

2014), have been extensively used to produce microalgae with desirable lipid

67

accumulation. Besides these stimuli, microalgae growth and lipid accumulation are

68

also influenced by plant hormones (Tarakhovskaya et al., 2007). However, the effects

69

of plant hormones on microalgae at a gene level have been poorly described. Studying

70

lipid synthesis from the gene level will be helpful in understanding the molecular

71

mechanism of storage lipid accumulation in hormone-induced conditions of

72

microalgae. In addition, based on varying lipid content, the key genes responsible for

73

the storage lipid biosynthesis can be identified.

74

Fulvic acid (FA) as a plant growth regulator is involved in increasing cell

75

membrane permeability, photosynthesis, oxygen uptake, respiration, and phosphate

76

uptake; FA also controls hormone levels and enhances secondary metabolites (Çimrin

77

et al., 2001). Heil et al. (2004) reported that FA additions resulted in high stimulation,

78

with more than a doubling of growth rate and a five-fold increase in maximum cell

79

yields of the dinoflagellate Prorocentrum minimum. A total of 20 mg L−1 of

80

Microcystis aeruginosa K-5 yield was observed at 20 mg L−1 FA, which is

81

significantly higher than that of control (Ohkubo et al., 2010). FA can significantly 4

82

promote biomass and astaxanthin accumulation in Haematococcus pluvialis (Zhao et

83

al., 2015). FA plays a key role in plant signal transduction, but the function of this

84

plant growth regulator in the molecular mechanism of storage lipid biosynthesis in

85

oleaginous microalgae has yet to be fully described. Microalgal lipid metabolism at

86

biochemical and molecular levels have been comprehensively described, but studies

87

have mostly focused on nutrient stress and several model species, such as

88

Chlamydomonas reinhardtii and Dunaliella salina (Boyle et al., 2012; Ramanan et al.,

89

2013). Key genes regulating lipid biosynthesis in Monoraphidium sp. FXY-10 under

90 91

plant growth regulator-inducing conditions have been rarely identified. Therefore, this

92

study described the time-course changes in the growth and chemical composition of

93

Monoraphidium sp. FXY-10 under FA-related. In addition to, the expression of

94

several lipid biosynthesis-related genes was also examined upon exogenous FA

95

application, and correlation analysis was performed to determine the relationship

96

between gene expression and lipid accumulation in Monoraphidium sp. FXY-10 and

97

to obtain further information about physiological and molecular changes triggered by

98

FA.

99 100

2. Materials and methods

101

2.1 Microalgae and culture conditions

102 103

Monoraphidium sp. FXY-10 (provided by the Biorefinery Laboratory of the Kunming University of Science and Technology) was used in this study (Yu et al., 5

104

2012). The alga was first cultured heterotrophically in 500 mL flasks containing 250

105

mL of sterilized Kuh1 medium. The seed cultured heterotrophically was placed on a

106

reciprocating shaker (150 rpm) and maintained at 25 °C ± 1 °C in the dark for 8 days

107

to deplete the organic carbon source. The heterotrophic cells were collected through

108

centrifugation and re-suspended at a density of about 7 × 107 cells mL−1 in Kuh1

109

medium with 0 and 25 mg L−1 FA for photoautotrophic cultivation. Microalgae were

110

cultured and placed in a shaker (150 rpm) maintained at 25 °C ± 1 °C under

111

continuous fluorescent illumination at 3500 lx for 10 days.

112

2.2 Determination of cell dry weight and lipid content

113

The microalgal biomass was quantified in terms of dry cell weight (DCW) by

114

regulating the relationship between absorbance optical density (OD) 750 nm and

115

DCW, as described previously (Che et al., 2016).

116

Cell biomass was harvested through centrifugation at 12000 × g for 5 min. The

117

wet cell mass was frozen overnight at −70 °C and freeze-dried at −80 °C under

118

vacuum conditions. Total lipids were extracted from lyophilized algal cells, as

119

described by Yu et al. (2012). In brief, freeze-dried microalgal biomass (300 mg to

120

500 mg) was ground into fine powder. The powder was rinsed with 3 mL of

121

chloroform:methanol solution (2:1, v/v). The mixture was incubated at room

122

temperature for 20 min in a shaker (150 rpm) and centrifuged at 8000 × g for 5 min.

123

The supernatant was then transferred to a new tube. This extraction procedure was

124

repeated twice, and all of the resulting supernatants were pooled together. The

125

supernatants were dried at 40 °C in a vacuum oven until constant weight. The total 6

126

lipid content was calculated as the percentage of the total biomass (in % dry weight).

127

2.3 Determination of reactive oxygen species

128

To evaluate the oxidative stress caused by FA addition, the intracellular ROS

129

levels were monitored. ROS were determined using the cell-permeable fluorescent

130

probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, China)

131

described by Soo-Jin Heo (Heo et al., 2008) with minor modifications. In brief, 5 mL

132

of the fresh culture was harvested by centrifugation (12000 × g, 3 min). The cells

133

were re-suspended by using 1 mL of DCFH-DA (10 mM) and incubated at room

134

temperature for 30 min in a shaker (150 rpm) under dark condition. The suspension

135

was centrifuged at 12000 g for 5 min at 4 °C and washed twice with the 0.5 M

136

phosphate buffer solution (PBS) to remove the excess fluorescent probe. The average

137

fluorescence density of intracellular cells was measured to index the ROS level. The

138

fluorescence of the samples was determined by using a spectrofluorophotometer

139

(RF-540) with an excitation wavelength of 488 nm and emission band between 500

140

and 600 nm.

141

2.4 Determination of chlorophyll a, protein and carbohydrate contents

142

Chlorophyll a was extracted with dimethyl sulfoxide (DMSO) and measured by

143

UV-visible spectroscopy in accordance with a previously described method (Wellburn,

144

1994). In the control treatment, no FA was added.

145

FA-induced fresh culture were harvested by centrifugation at 12000 × g for 5 min.

146

The cell pellet was washed twice with distilled water and re-suspended in 1 mL of

147

DMSO. Cellular debris was pelleted (12000 × g, 5 min) following resuspension, and 7

In brief, 2 mL of the control and

148

the absorbance spectrum of the supernatant was measured from 600 nm to 700 nm,

149

DMSO as the reference. Chlorophyll a levels were measured in triplicate, and its

150

concentrations (in µg mL−1) were calculated using the following equation described

151

by Wellburn (1994): ሾChlሿ = ൫12.47ｘ‫ݏܾܣ‬665.1൯ − (3.62ｘ‫ݏܾܣ‬649.1)

152

Lyophilized algal powder (10 mg) from the control and FA-induced condition

153

were used for analysis of total carbohydrate content (Ma et al., 2016). The algal

154

powder was incubated in 0.5 mL acetic acid at 80 °C for 20 min and added with 10

155

mL acetone then centrifuged at 3500 g for 10 min. The supernatant was discarded,

156

and the pellet was re-suspended in 2.5 mL 4 M trifluoroacetic acid (TFA) then boiled

157

for 4 h. The suspension was cooled and centrifuged at 10000 g for 3 min. Then, 20 µL

158

of the supernatant was mixed with 900 µL sulfuric acid (15 mL):H2O (7.5 mL):phenol

159

(0.15 g) and boiled for 20 min prior to reading the optical density at 490 nm (OD490).

160

Glucose was used to establish the standard curve and to quantify the total

161

carbohydrate content.

162

Total protein content was extracted and determined as previously described

163

(Berges et al., 1993). Freeze-dried algal powder (10 mg) from the control and

164

FA-induced condition were ground into fine powder, hydrolyzed in 100 µL of 1 M

165

sodium hydroxide, and then incubated in water bath at 80 °C for 10 min.

166

Subsequently, 900 µL of H2O was added to the hydrolysate to obtain the final volume

167

of 1 mL. The mixture was centrifuged at 12000 × g for 30 min, and the supernatant

168

was transferred to a new tube. This extraction procedure was repeated twice, and the 8

169

resulting supernatants were pooled together. The protein concentration was measured

170

with Bradford assay, and bovine serum albumin (BSA) was used as standard.

171

2.5 Cloning and sequencing of lipid biosynthesis-related gene in Monoraphidium sp.

172

FXY-10

173

The nucleotide sequences of seven genes, such as NADP-dependent malic enzyme

174

(ME), acetyl-CoA carboxylase beta subunit (accD), β-ketoacyl-ACP synthase III

175

(KAS III), glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol

176

acyltransferase (DGAT1), ribulose 1,5-bisphosphate carboxylase/oxygenase

177

(RuBisCO) large subunit (rbcL), and phosphoenolpyruvate carboxylase (PEPC), of

178

different microalgal and plant species were retrieved from NCBI

179

(http://www.ncbi.nlm.nih.gov) and aligned using ClustalW to find the conserved

180

regions. Primers were designed from the conserved regions by using Primer 5.0 and

181

amplified on the cDNA of Monoraphidium sp. FXY-10. For amplification, PCR

182

reactions were performed in a final volume of 20 µL containing 10 µL of 2× Taq PCR

183

masterMix (Tiangen, China), 1 µL of each primer (10 µM), 2 µL of cDNA, and 6 µL

184

of distilled water. Amplification programs included 94 °C for 5 min, 35 cycles of

185

94 °C for 30s, annealing temperature (50 °C to 61 °C) for 30 s, 72 °C for 3 min and a

186

final extension of 7 min at 72 °C. Then, 6 µL of each PCR product was verified

187

through 1% to 2% agarose gel electrophoresis and anthocyanin staining. The PCR

188

products were recycles with a universal DNA purification kit (Tiangen, China) if the

189

size of the PCR product was right. The recycled products were inserted into T-vector

190

pMD19 (TaKaRa, Japan), cloned in DHAα competent cell (Tiangen, China), and 9

191

sequenced by Sangon Biotech (Shanghai, China). BLAST

192

(http://www.ncbi.nlm.nih.gov/ Blast) was used to calculate the identities of sequence

193

similarities. The partial coding sequences of the selected genes isolated from

194

Monoraphidium sp. FXY-10 are listed in Supplemental Data S1.

195

2.6 cDNA synthesis and Real Time-PCR analysis

196

RNA extractions were performed at 2-day intervals on FA-induced cells 10 days

197

from the beginning of induction. RNA extracted from the cultures without FA in the

198

same time period was used as the control. Total RNA was extracted using TriZol

199

reagent (Invitrogen) according to the manufacturer’s instructions, and the

200

concentration was determined using a Nanodrop 2000 (Thermo). All of the RNA

201

samples were digested with RNAase-free DNAase I (Fermentas) to remove DNA

202

contamination, and cDNA was synthesized using a Superscript RNAse H- first-strand

203

synthesis kit (Invitrogen) according to the manufacturer’s recommendations. The

204

cDNAs from three independent cultures for each time and treatment were subjected to

205

quantitative PCR analysis. Real-time PCR was performed in a 7900HT Fast real-time

206

PCR system (Applied Biosystems, Spain) by using SYBR® Green real-time PCR

207

Master Mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s

208

instructions: 1 cycle of 95 °C for 30 s, 40 cycles of 95 °C for 5 s each, 57 °C for 30 s,

209

and 72 °C for 30 s. The 18S rRNA genes were used as internal references to

210

normalize the expression data and to calculate the transcript abundances. The

211

candidate reference genes were constantly expressed under all experimental

212

conditions. Relative fold changes were analyzed using the 2 -∆∆Ct method (Schmittgen 10

213 214

et al., 2008). The primers used for real-time PCR are shown in Table 1. These primers were

215

designed by the gene sequences obtained from Monoraphidium sp. FXY-10 in this

216

research. The real-time PCR primers were designed using Primer 5.0 and synthesized

217

by Sangon Biotech (Shanghai, China).

218

2.7 Determination of enzymatic activities

219

Centrifugation (12000g, 5 min at 4 °C) was done to harvest 20 mL of the fresh

220

culture. The cell pellet was washed twice, frozen with liquid nitrogen, and pulverized

221

with pestle and mortar. Enzyme activity was determined subsequently. ME,

222

acetyl-CoA carboxylase (ACCase), and DGAT activity of microalgae were analyzed

223

with colorimetric quantitative detection kit (Ke Ming Co., Suzhou, China) according

224

to manufacturer's instructions. One unit (U) of enzyme activity is defined as the

225

amount of enzyme catalyzing the formation/ consumption of 1 nmol of each

226

enzymatic reaction product/substrate per minute or hour under the aforementioned

227

conditions (Xue et al., 2015).

228

2.8 Statistical analysis

229

Results were shown as the means of the three biological replicates, and the error

230

bars indicated the standard deviation. The statistical significance of the results was

231

evaluated using Student's t-test. Correlation analysis was performed using the

232

Spearman correlation analysis (SPSS19.0). A P-value < 0.05 was considered

233

statistically significant for all of the analysis.

234 11

235 236

3. Results and discussion

237

3.1 Effect of FA on the physiological and biochemical changes of Monoraphidium sp.

238

FXY-10

239

The physiological and biochemical changes underlying FA induction in

240

Monoraphidium sp. FXY-10. The heterotrophic cells were transferred as seed to

241

photoautotrophy and induced with 25 mg L-1 FA. The cell density and intracellular

242

component characteristics were recorded on two separate days and over the

243

cultivation period. The time-course of Monoraphidium sp. FXY-10 cell growth in

244

response to FA addition was examined (Fig. 1a). Figure 1a shows that FA addition

245

had an insignificant (p >0.05) effect on biomass concentration compared with the

246

control. This result is in agreement with the previous study. The study showed that

247

when the heterotrophic cells were transferred into photoautotrophic culture and

248

treated with different chemical elicitors, chemical elicitors addition had an

249

insignificant effect (p >0.05) on biomass concentration compared with that of control

250

(Zhao et al., 2016). Cells grown under all the culture conditions displayed continuous

251

growth throughout the culture period. The cell density ranged from 1.83 g L-1 to 2.29

252

g L-1, and 1.83 g L-1 to 2.28 g L-1 on the two different test days, respectively.

253

Photosynthetically fixed carbon can be diverted into multiple pathways for

254

synthesis of major macromolecules, such as carbohydrates, lipids, and proteins. The

255

protein content increased continuously both in the FA induction and control samples

256

during the whole culture period (Fig. 1b). Monoraphidium sp. FXY-10 rapidly 12

257

synthesized protein in the presence of FA, and protein content increased from 31.4%

258

to 39.7%. Moreover, the protein content increased from 28.8% to 38.9% in the control

259

sample. Figure 1b illustrates the significant (p < 0.05) production of protein observed

260

on the 2nd, 4th, 6th, and 8th induction days with percentage contents of 31.4%, 32.7%,

261

33.7%, and 38.8% for FA-treated cells relative to the control sample. This result is in

262

agreement with that of several studies, which reported that protein content rapidly

263

increases for several hours and then gradually increases until 55% cell dry weight is

264

reached in cells transferred from a heterotrophic mode to a photoautotrophic mode

265

and subjected to illumination (Fan et al., 2012). Previous research has shown that low

266

concentrations of 2,4-Dichlorophenoxyacetic acid can stimulate the increase of

267

chlorophyll and protein in Chlorella vulgaris and Spirulina platensis, whereas high

268

concentrations of 2,4-Dichlorophenoxyacetic acid has a reverse effect. The study

269

indicate that the low concentrations of 2,4-Dichlorophenoxyacetic acid can promote

270

the photosynthetic efficiency of algae, whereas high concentrations of

271

2,4-Dichlorophenoxyacetic acid can inhibit algae photosynthetic efficiency

272

(Saygideger et al., 2008). The increasing rate of protein content was related to light

273

intensity and carbon reallocation of carbohydrates, lipids, and proteins in response to

274

FA induction.

275

Biochemical analyses showed that cell chlorophyll content had a very similar

276

trend to that of protein. Chlorophyll content rapidly increased to 21.46 µg mL-1 during

277

the first 6 days after FA treatment then kept a steady state (approximately 23.06 µg

278

mL-1) until the end of the culture period (Fig. 1c). The cellular chlorophyll content 13

279

increased from 5.25 µg mL−1 to 21.66 µg mL-1 during the first 6 days and steadily

280

increased to 23.88% on day 10 without FA treatment. The chlorophyll synthesis was

281

initially fast then steadied when the seed cultured heterotrophically was transferred to

282

photoautotrophic culture. Fan et al. (2012) indicated that the chlorophyll content in

283

cell initially increased rapidly and then slightly when the cells were briefly transferred

284

into photo-induced phase, reaching 35–40 mg/g DCW after 48-h photoinduction,

285

which is in line with the results of the present study. This may be attributed to the

286

improved photosynthetic efficiency of the cells. The chlorophyll content of

287

FA-treated cells was almost comparable to that of control cells (Fig. 1c) throughout

288

the cultivation period. This phenomenon is different from nitrogen starvation wherein

289

the nitrogen defects result in a sharp reduction in pigments, and this phenomenon is a

290

widespread response to nitrogen depletion in microalgae explained by the nutrient

291

recycling from degradation of structural cell components, such as chloroplast (Fan et

292

al., 2014b). Under FA-treated condition, the cell photosynthetic efficiency was

293

unaffected, and this condition was better than nitrogen stress.

294

Figure 1d illustrates the carbohydrate content variations of the Monoraphidium

295

sp. FXY-10 in FA and control treatments. Under FA-treated condition, the cellular

296

carbohydrate content rapidly increased from 42.65% to 49.54% in the first 4 days,

297

decreased to 33.51% on the 6th day, and remained unchanged thereafter. The cellular

298

carbohydrate content variation trend of the Monoraphidium sp. FXY-10 in the control

299

treatment showed slight differences compared with the FA treatment. Carbohydrates

300

continuously decreased throughout most of the time-course, but maintained levels 14

301

between 39.26% and 34.38% of dry weight (Fig. 1d). Figure 1d illustrates the

302

significant (p < 0.01) production of carbohydrates only on the first 4 days for the

303

FA-treated cells relative to the control cells. Compared with that of the control, the

304

stimulatory effect of FA on the carbohydrate content was recorded on the 6th and 8th

305

days, with approximately 3.1% and 3.8% reduction, respectively. Previous study

306

reported a transient increase of starch, followed by a lipid increment accompanied

307

with starch reduction (Fernandes et al., 2013). The carbohydrate content significantly

308

decreased during the lipid increase phase in the present study, which is in line with

309

results reported by Ikaran et al. (2015).

310

Intracellular ROS were detected by DCFH-DA, a fluorescent probe for hydrogen

311

peroxide, and the results were presented as fluorescence intensity in the same number

312

of cells (1ｘ106 number). FA treatment could significantly (p < 0.01) increase the

313

ROS production (Fig. 1e). The ROS production levels upon exposure to FA treatment

314

on the 6th, 8th, and 10th days were 1.46, 1.47, and 1.38 times of the control value,

315

respectively (Fig. 1e). ROS are formed by the inevitable leakage of electrons onto

316

molecular oxygen from the electron transport activities of chloroplasts, mitochondria,

317

and plasma membrane in the plant. ROS production has been found to be stimulated

318

by various environmental stresses, such as exposure to high levels of light, drought,

319

heavy metals, high salt concentration, extremes temperature, UV irradiation,

320

mechanical stress, and physical stress, and also in response to biotic stresses such as

321

invasion of various pathogens (Mallick et al., 2000). As reported by Liu et al. (2012),

322

hypo-osmotic stress (5‰ and 10‰) can significantly increase ROS production, and 15

323

ROS production was increased by 2-fold and 1.7-fold in salinities of 5‰ and 10‰

324

compared with the control cells. Zhao et al. (2016) also reported that the level of ROS

325

increased in cells exposed to light stress with 5 mM glycine betaine, and the ROS

326

content was increased by 29.74% compared to the control on day 1. Similar to glycine

327

betaine, FA is a plant growth regulator which that may enhance the absorption of

328

metal trace elements including iron in microalgae to provoke oxidative stress via the

329

iron-catalyzed Haber–Weiss reaction (Hong et al., 2015).

330

3.2 Effects of FA-induced changes on total lipid accumulation

331

Figure 2 shows the total lipid content variations in FA-induced Monoraphidium

332

sp. FXY-10. The total lipid content increased from 44.3% to 48.4% over a 10-day

333

period, with the maximum lipid level observed on the 6th day. However, in

334

FA-induced condition, a lipid level of 54.3% was obtained on the 6th day, which was

335

a 1.1-fold increase compared with the corresponding day equivalent in the control.

336

Protein, chlorophyll, lipid, and carbohydrate are the main algal cell components, and

337

their contents may be changed and converted during stress. The increase of oil in the

338

case of FA induction might be caused by the degradation of carbohydrates and its

339

conversion into oil (Fig. 1d). Similar variations in biochemical composition attributed

340

to FA induction had also been studied by Han et al. (2012). In their study, the

341

carbohydrate content decreased from 55.3% to 32.8%, whereas the lipid content

342

increased from 9.10% to 28.9%, protein content increased from 32.2% to 34.8%, and

343

chlorophyll increased from 1.22% to 2.26%. Moreover, lipid production was

344

enhanced, possibly because of excessive ROS (Zhao et al., 2016). As shown in Fig. 1e, 16

345

a significant (p < 0.01) ROS production was observed in the FA-induced condition

346

compared with the control condition.

347

3.3. Lipid biosynthesis-related gene expression associated with FA treatment in

348

Monoraphidium sp. FXY-10

349

The changes in gene expression induced by FA for seven lipid

350

biosynthesis-related genes (ME, KAS III, GPAT, DGAT1, accD, rbcL, and PEPC)

351

were quantified through real-time PCR to evaluate lipid regulation as mediated by FA.

352

Gene expression analysis showed that FA induced changes in the expression of rbcL

353

gene from 2 days to 8 days, and the expression was enhanced 2.5 times at the end of

354

two days. This upregulation is consistent with the increment of carbohydrates

355

observed for FA-treated cells during the early period of this experimental (Figs. 1d

356

and 1c). The rbcL gene encodes the catalytic large subunit of RuBisCO, which is a

357

required enzyme that catalyzes initial carbon fixation in the first reaction of the Calvin

358

cycle and is an overall rate-limiting step in photosynthesis (Ikaran et al., 2015).

359

During the photosynthetic process, atmospheric carbon dioxide is fixated by RuBisCO

360

and used to synthesize more energy-dense molecules such as sucrose and lipids (Wan

361

et al., 2011). Carbon dioxide is the exclusive carbon source in photoautotrophy, hence

362

this reaction is the dedicated biosynthetic step to form lipids from carbon dioxide.

363

Thus, the rbcL gene was used to examine the photosynthetic rate in the present study.

364

Wan et al. (2011) reported that the rbcL showed up-expression in both logarithmic

365

and stationary phases of photoautotrophic culture and indicated a higher

366

photosynthetic rate in the logarithmic and stationary phases of photoautotrophic 17

367

culture. Compared with control, FA treatment led to a significant up-regulation of

368

rbcL by 2.5-fold and approximately 1.1-fold to 1.6-fold after 2 days and 6 days of

369

induction, respectively. The present study showed that FA treatment can promote

370

carbon dioxide fixation and improve the photosynthetic efficiency of Monoraphidium

371

sp. FXY-10. This can provide more carbon precursor, which is used in the

372

biosynthesis of the multiple forms of storage compounds, such as water-soluble

373

polysaccharides, starch, and TAG. Therefore, the carbohydrate content increased at in

374

the early stage of FA induction.

375

The expression levels of ME in the FA-treated cells is illustrated in Fig. 3B. The

376

transcript abundance of ME increased significantly in the FA-treated cells compared

377

with that of the control cells, and a 1.3-fold increase was observed on the 4th

378

induction day. ME catalyzes the irreversible oxidative decarboxylation of malate to

379

pyruvate, to produce pyruvate, NADH, and CO2 (Xue et al., 2015). NADH production

380

is vital for fatty acid biosynthesis, which provides the necessary reducing power for

381

cell metabolism. ME has been reported to be involved in diverse metabolic pathways,

382

including lipogenesis, energy metabolism, and photosynthesis. Furthermore, ME is

383

the rate-limiting step in fatty acid biosynthesis in oleaginous fungi (Zhang et al.,

384

2007). The increased expression of ME gene leads to both increased biosynthesis of

385

fatty acids and formation of unsaturated fatty acids (Zhang et al., 2007). Xue et al.

386

(2015) revealed that the overexpression of ME significantly increases the total lipid

387

content by 2.5-fold and reaches 57.8% of dry cell weight. In nitrogen-starved cultures,

388

ME is significantly upregulated during the experiment and closely matches with total 18

389

lipid increase (Ikaran et al., 2015). The stimulatory effect of FA on ME expression

390

observed in the present study implies that ME was significantly up-regulated on the

391

4th day. Consequently, oil rapidly accumulated on the 6th day.

392

The first committed step in fatty acid synthesis, which is the synthesis of

393

malonyl-CoA from acetyl-CoA, is catalyzed by ACCase. Two forms of ACCase are

394

found in plants: the heteromeric form, which plays an exclusive role in de novo fatty

395

acid synthesis in plastids; the homomeric form, which is located in the cytosol. The

396

heteromeric ACCase is composed of four subunits: nuclear-encoded biotin

397

carboxy-carrier, alpha-carboxyltransferase, biotin carboxylase subunits, and

398

beta-carboxyltransferase which is encoded in the plastid genome. The

399

beta-carboxyltransferase (accD) appears to be a good indicator of lipid content

400

(Sasaki et al., 2004). Thus, only the expression pattern of the nuclear-encoded gene

401

for the beta-carboxyltransferase of the heteromeric form (accD) was analyzed in this

402

study. The expression of this gene was affected by FA. Compared with the control,

403

FA treatment significantly up-regulated accD by 1.1- and 1.6-fold in the first 4 days

404

(Fig. 3C). A positive response in the expression of accD genes in relation to lipid

405

accumulation triggered by different induction conditions has been reported in

406

microalgae. In a previous study, N, P, and Fe deficiency can also trigger an increase

407

in accD expression levels (Fan et al., 2014b). Wan et al. (2011) indicated that the

408

increased expression levels of accD reflects the increased lipid content in stationary

409

phase of mixotrophic growth. The results of these studies agree with the present

410

results and suggest a positive correlation between accD gene expression and lipid 19

411

content in Monoraphidium sp. FXY-10 (Fig. 2a).

412

The synthesis of short to long saturated acyl chains (C4–C18) involves the

413

condensation of C2 units from malonyl-acyl carrier protein to acyl chains and can be

414

achieved by enzyme complex, β-ketoacyl ACP synthase (KAS I, KAS II, and KAS III)

415

(Fofana et al., 2004). KAS III initiates the fatty acid synthesis in plants by catalyzing

416

the condensation of acetyl-CoA and malonyl-ACP to form 3-ketobutyryl-ACP. In the

417

present study, the expression profile of KAS III gene showed a pattern quite similar to

418

that of ME, with 1.9-fold increase during the first 4 days after FA treatment and being

419

kept under low levels until the end of the culture period (Fig. 3D). This result suggests

420

that FA could induce KAS III gene expression. Sharma et al. (2015) reported that KAS

421

III exhibited upregulated expression in Scenedesmus species (including SD12 and

422

SQ19) under stress conditions and showed higher significance for high lipid content.

423

GPAT is the first enzyme that catalyzes the acylation of glycerol 3-phosphate

424

(G3P) resulting in lysophosphatidic acid (LPA), which is the precursor for the

425

biosynthesis of phosphatidic acid, diacylglycerol (DAG), TAG (Niu et al., 2016).

426

Compared with control, a significant upregulation of GPAT was obtained, with

427

1.2-fold and 1.5-fold increase during the first 4 days of induction (Fig. 3E). GPAT

428

isoforms have been identified in varied species, such as mammals, humans, plants,

429

microalgae, etc. (Niu et al., 2016).

430

Yokoi et al. (1998) reported that expression of Arabidopsis GPAT resulted in

431

increased content of unsaturated fatty acids in transgenic rice compared to wild type.

432

Expression of Brassica napus GPAT resulted in increased oil content in transgenic 20

433

tobacco (Liu et al., 2015). Fatty acid content was increased in the transgenic green

434

microalga, Chlamydomonas reinhardtii, expressing Lobosphaera incisa GPAT, and

435

resulted in increased TAG accumulation (Iskandarov et al., 2016). Similarly, induced

436

expression of Helianthus annuus GPAT in transgenic Escherichia coli resulted in

437

increased unsaturated fatty acid content (Payá-Milans et al., 2015). Niu et al. reported

438

that the number of oil bodies is similar in both transgenic and wild type cells, whereas

439

the volume of oil bodies in transgenic cells is considerably increased and larger than

440

in wild type (Niu et al., 2016). The present result is in agreement with the results of

441

the aforementioned studies, that is, upregulation GPAT is consistent with the increase

442

of lipid content in Monoraphidium sp. FXY-10 during the early FA induction period

443

(2 days to 4 days).

444

The final step in de novo TAG biosynthesis is catalyzed by DGAT that transfers

445

the third fatty acid to position 3 of DAG, resulting in the production of triacylglycerol.

446

DGATs has been identified as one of the rate limiting enzymes for TAG accumulation

447

in some oil-producing plants. In algae, DGATs have at least two major families,

448

namely, type 1 and type 2. Type 1 and type 2 DGATs do not share any significant

449

similarities in amino acid sequence although both catalyze the same enzymatic

450

reaction. DGAT1 and DGAT2 also have differences in functionality and temporal and

451

spatial expression profiles (Chen et al., 2012). In the present study, the expression of

452

DGAT1 gene insignificantly increased under FA-induced conditions (Fig. 3F).

453

DGAT1 gene in Chlamydomonas was expressed at relatively low levels and was not

454

affected by nitrogen depletion (Msanne et al., 2012). On the contrary, DGAT1 gene 21

455

showed a significant up-regulation at 72 h, just before the observed TAG rise (Ikaran

456

et al., 2015). The up-regulation of DGAT1 gene linked to TAG accumulation has also

457

been described in C. reinhardtii and C. vulgaris and the diatom Phaeodactylum

458

tricornutum (Miller et al., 2010; Ikaran et al., 2015). DGAT1 expression is different in

459

diverse microalgae and appears to be species specific. Additionally, DGAT1

460

expression was also affected by N, Fe, and P starvation, illumination, and training

461

phase (Fan et al. 2014a; Fan et al. 2014b).

462

PEPCase is a ubiquitous cytoplasmic enzyme present in a wide spectrum of

463

organisms, including archaea, bacteria, unicellular green algae, and vascular plants

464

(Ikaran et al., 2015; Fan et al., 2014a). This enzyme catalyzes the irreversible

465

carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate (OAA) that feeds the

466

Krebs cycle, providing the precursors and energy needed for several metabolic

467

pathways including protein synthesis (Ikaran et al., 2015). There are evidences

468

supporting that PEPCase activity is related to carbon flux into lipids (Ikaran et al.,

469

2015). Chen et al. (1998) reported that oil content increased by 6.4% to 18% in

470

transgenic rapeseed lines, in which the expression of PEPC was blocked by antisense

471

RNA. Fan et al. (2014a) demonstrated that the expression of pepc g6833 increases by

472

3.1 times at the end of the growth stage but decreases rapidly in the oil-accumulation

473

stage. The expression levels of PEPC in the FA-treated cells are illustrated in Fig. 3G.

474

The expression of PEPC increased by 1.14 and 1.71 times on the 4th and 6th days of

475

FA treatment but decreased rapidly during late induction. This result is in agreement

476

with that of several studies reporting that the expression of PEPC is upregulated in the 22

477

early induction period (Ikaran et al., 2015; Fan et al., 2014a). The molecular

478

mechanism in the microalgae is still unclear at present.

479

3.4 Correlations between gene expression and lipid content

480

The Spearman correlation analysis (using SPSS 19.0) was carried out to

481

determine the relevance between key gene expression level and lipid accumulation

482

under FA treatment conditions. The relation between gene transcription and lipid

483

content was concluded, and an insight into the potential targets was given in this study,

484

thereby providing an overall perspective on the potential mechanism of the lipid

485

accumulation response to FA induction. The relationship between gene expression

486

levels and the lipid biosynthesis was analyzed. Several gens, viz. pepc, DGAT1, KAS

487

III and rbcL showed insignificant (p >0.05) correlation with lipid accumulation.

488

Several genes that may play a primary role in lipid accumulation could be selected as

489

promising candidates for further genetic engineering. The analysis results also shows

490

that several genes (ME, accD, and GPAT) were significantly (p <0.05) correlated with

491

lipid accumulation. These genes encode the corresponding isoenzymes of

492

NADP-dependent ME, ACCase, and GPAT in the de novo TAG biosynthesis

493

pathway, and these isoenzymes likely influence lipid accumulation and thus could be

494

selected as modification candidates.

495

3.5. Biochemical activities of some key enzymes involved in Monoraphidium sp.

496

FXY-10 lipid biosynthesis

497 498

The ACC, ME, and PEPC activities in the FA-treated cells were determined to investigate the relevance between key enzymatic activity and lipid accumulation. 23

499

The correlation between ACCase activity and fatty acid synthesis of

500

Monoraphidium sp. FXY-10 under FA-treated conditions was determined. Figure 4A

501

shows the time course measurement of ACCase activity. As shown in Fig. 4A,

502

significant (p < 0.01) improvement in ACCase activity was observed on the whole

503

culture process for FA-treated cells relative to the control sample. ACCase is the first

504

committed enzyme for fatty acid synthesis leading to the carboxylation of acetyl-CoA

505

to form malonyl-CoA, and this step is indeed the rate-limiting step for fatty acid

506

biosynthesis (Fan et al., 2014a; Courchesne et al., 2009). Numerous studies have

507

shown that this caused an increase of the intracellular ACCase activity as a result of

508

enhanced fatty acid synthesis rate (Fan et al., 2014a; Courchesne et al., 2009; Ma et

509

al., 2016). Ma et al. (2016) reported that the highest oil content was obtained under

510

high light and nitrogen-deficient conditions, and ACCase activity increased by 3-fold,

511

which was much more than under low light and nitrogen-deficient conditions or high

512

light and nitrogen-adequate conditions (Ma et al., 2016). Considering the comparative

513

analysis of previous studies and our research results, we hypothesized that ACCase is

514

a key enzyme in the fatty acid synthesis of Monoraphidium sp. FXY-10.

515

ME has been postulated as the rate-limiting step for fatty acid biosynthesis, and

516

this enzyme catalyzes the conversion of malate into pyruvate and simultaneously

517

reduces NADP+ into NADPH (Ma et al., 2016). The importance of ME is dependent

518

on its capability to promote NADPH production and thus provides a unique source of

519

reducing power and cofactors for fatty acid synthesis (Xue et al., 2015; Courchesne et

520

al., 2009). The levels of ME were examined in cells cultured for 10 days under 24

521

FA-treated conditions, and these levels were increased remarkably in the first 6 days

522

(Fig. 4B). The ME enzymatic activities of Monoraphidium sp. FXY-10 on days 2, 4,

523

and 6 were increased by 2.1-, 4-, and 2.7-fold in the FA-treated culture than in the

524

control culture. The increased ME activity in the early induction phase provides more

525

NADPH for the oil accumulation in the later induced stage. Lipid content is enhanced

526

approximately by 2-fold, and ME is increased by a 3.5-fold (Xue et al., 2015). An

527

increase in ME activity is associated with a faster lipid accumulation (Courchesne et

528

al., 2009). The results of this study showed the same phenomenon, that is, the lipid

529

accumulation of Monoraphidium sp. FXY-10 under FA-treated conditions was

530

associated with the ME activity.

531

The PEPC activity was determined to understand the role of the PEPCase in fatty

532

acid synthesis in Monoraphidium sp. FXY-10 under FA treatment (Fig. 4D). The

533

enzyme activity level during FA treatment decreased by 1.4-fold to 1.8-fold in the

534

whole cultivation process. This finding indicated the negative role of increased PEPC

535

activity in fatty acid synthesis. Furthermore, Deng et al. (2014) reported that a

536

157%–184% increase in PEPCase activity leads to a 37% decrease in the TAG

537

content.

538

In the current study, the expression level of accD, PEPC, and ME was increased

539

in FA treatments. Compared with that in the control, in FA-induced conditions, the

540

ACCase and ME activities were significantly increased, whereas the PEPCase activity

541

was significantly reduced. However, in FA-treated conditions, the biochemical

542

activities of some lipid synthesis enzymes were inconsistent with the expression of 25

543

related genes. Chang et al. (2013) reported that the expression levels of starch

544

metabolism-related genes were inconsistent with activities of starch

545

metabolism-related enzymes, which is in line with the results of the present study.

546

The expression of enzyme genes is not directly related to enzyme activity.

547

Up-regulation of gene expression only indicates high number of synthetic peptides.

548

However, from the peptide chain to the enzyme has been restrained by some factors,

549

thereby directly affecting enzyme levels. Thus, gene expression and enzyme activity

550

presented different trends in FA-induced conditions

551 552

4. Conclusions

553

This study showed that FA addition altered cell physiology and metabolism.

554

Furthermore, the expression of seven key lipid biosynthesis-related genes increased to

555

varying degrees. Under FA addition, ACCase and ME activities were considerably

556

up-regulated, whereas the PEPCase activity was significantly down-regulated. This

557

finding was correlated well with the enhanced lipid accumulation. Our results help

558

enhance our understanding of the underlying molecular changes in response to FA

559

and provide insights into the regulatory mechanism of lipid metabolism in

560

Monoraphidium sp. FXY-10. The mechanism by which FA regulates lipid

561

biosynthesis is yet to be fully understood and needs further investigation.

562 563 564 565

Acknowledgements This work was funded by the National Natural Science Foundation of China (21266013 and 21666012), the Natural Science Foundation of Yunnan Province, 26

566

China (2010CD028).

567 568

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30

Figure captions

723 724 725

Fig. 1. Time course of biomass (a), protein (b), Chl a (c), and carbohydrate content (d),

726

reactive oxygen species (ROS) (e) of Monoraphidium sp. FXY-10 under fulvic acid

727

induced conditions. Vertical bars represent the means ± SD (n= 3). Single asterisks

728

denote statistically significant (p < 0.05) whilst double asterisks denote statistically

729

significant (p < 0.01).

730 731

Fig. 2. The lipid content of Monoraphidium sp. FXY-10 cultivated for 10 days under

732

fulvic acid induced conditions. Vertical bars represent the means ± SD (n= 3). Single

733

asterisks denote statistically significant (p < 0.05) whilst double asterisks denote

734

statistically significant (p < 0.01).

735 736

Fig.3. Expression of lipid biosynthesis-related genes in Monoraphidium sp. FXY-10

737

under fulvic acid induced conditions. (A) ribulose 1, 5-bisphosphate

738

carboxylase/oxygenase(RuBisCO), rbcL; (B) Malic enzyme, ME; (C) acetyl-CoA

739

carboxylase beta subunit, accD; (D) β-ketoacyl-ACP synthase, KAS III; (E)

740

glycerol-3-phosphate acyltransferase, GPAT; (F) Diacylglyrerol acyltransferase,

741

DGAT; (G) Phosphoenolpyruvate carboxylase, PEPC. Vertical bars represent the

742

means ± SD (n= 3).

743 744

Fig. 4. Time course of ACC (a), ME (b), and PEPC (c) enzyme activity of

745

Monoraphidium sp. FXY-10 under fulvic acid induced conditions. Vertical bars

746

represent the means ± SD (n= 3). Single asterisks denote statistically significant (p <

747

0.05) whilst double asterisks denote statistically significant (p < 0.01).

31

(a)

Control FA

Biomass (DWL-1)

2.2

2.1

2.0

1.9

1.8

(b)

Control FA

40

Protein (%, dry biomass weight)

2.3

**

38

36

**

34

** 32

**

30

28

0

2

4

6

8

10

2

4

6

8

10

Induction time (day)

Induction time (day) 54

(c)

Contorl FA

24 22

50

20

(d) **

Carbohydrate (% DW)

48

18

Chl a (µ g/mL)

Control FA

52

16 14 12 10

46 44

**

42 40 38 36

8

**

34 6 32

**

4 2

4

6

8

2

10

1200

**

1000

6

DCF-fluorescence intensity (1×10 cell)

(e)

Control FA

1100

900

**

800 700 600

**

500 400 300 200 2

4

6

8

10

Induction time (day)

748

4

6

Induction time (day)

Induction time (day)

Che et al. Fig. 1

749

32

8

10

58

Control FA

56

**

54

Lipid content (%)

52 50 48

**

46

**

44 42 40 38 36 2

4

6

Induction time (day)

750

Che et al. Fig. 2

751

33

8

10

A

3.0

B

rbcL

1.4

me

1.2

Relative expression level

Relative expression level

2.5

2.0

1.5

1.0

0.5

1.0 0.8 0.6 0.4 0.2

0.0

0.0 2

4

6

8

10

2

4

Cultivation time (day) C

2.0

accD

1.8

6

8

10

Cultivation time (day) D

KAS III

2.0

Relative expression level

Relative expression level

1.6 1.4 1.2 1.0 0.8 0.6 0.4

1.5

1.0

0.5

0.0

0.2 0.0 2

4

6

8

2

10

Cultivation time (day) E

8

10

DGAT1

1.1 1.0

Relative expression level

1.4

Relative expression level

6

Cultivation time (day) F

1.2

GPAT

1.6

4

1.2 1.0 0.8 0.6 0.4

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

2

4

6

8

10

2

2.0

pepc

1.8

Relative expression level

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 2

4

6

8

10

Cultivation time (day)

752

4

6

8

Cultivation time (day)

Cultivation time (day) G

Che et al. 34 Fig. 3

10

B

Control FA

220 200 180

**

160

**

140

**

**

120 100 2

4

6

8

ME enzyme activity (mmol/min/g wet weight)

ACC enzyme activity(µ mol/h/g fresh weight)

A

4.5 4.0

** 3.5 3.0 2.5 2.0 1.5

0.0

10

2

PEPC enzyme activity (nmol/min/g wet weight)

C

200 180

**

**

140

**

120

** 100

**

80 60 2

4

6

8

10

Induction time (day)

753 754

4

6

Induction time (day)

220

160

*

0.5

Control FA

240

**

1.0

Induction time (day) 260

Control FA

Che et al. Fig. 4

35

8

10

755

Table 1

756

Genes involved in fatty acid and triacylglycerol biosynthetic pathway in Monoraphidium sp.

757

FXY-10 and their respective primers used in qRT-PCR expression analysis Gene 18s RNA

Gene abb 18s RNA

primer GGGAGTATGGTCGCAAGG

Annealing

Length (bp) of

temp [°C]

production

57

242

57

110

57

292

57

209

57

326

57

291

57

125

57

213

GACTATTTAGCAGGCTGAGGT Malic enzyme

ME

TCGGCGTGAGCACTATCGGTG CGGACTGGTTGGTGGGGTTG

Phosphoenolpyruvate

PEPC

carboxylase Diacylglycerol

CGCAGCACCTCCGCCTTTGT DGAT1

acyltransferase Ribulose1,5-bisphosphate

rbcL

GTACCTGCCCAACCTCACCG GCACCAGCATGGACACCACC

GPAT

acyltransferase β-ketoacyl-ACP synthase

CAAGCCGCTCGCCCAGAT TCCACAGCCGCCAGAACT

carboxylase/oxygenase glycerol-3-phosphate

CCATCCCCTGGGTGTTTGCC

GTGGTGTTCCGCTACGC GAACGCCGAGTAGGAGG

KAS

III

TGCCAGACACCATCACAAACT TGACGCCAGCGATTACAGC

acetyl-CoA carboxylase beta subunit

accD

GGGCGTGATGGAGTTTG AGGTTGGCCTCGTTCTG GGCTCCTTCTTGGCAATG

758 759

36

760

ROS

Chlorophyll a Inoculating

Gene expression

Fresh algae Enzymatic activity

Heterotrophic cells Adding

Fulvic acid

Lyophilized algal powder Lipid Photoautotrophic induction culture by fulvic acid

Protein Carbohydrate

761

37

762

1.

FXY-10 accumulated a considerable amount of lipids under FA induction.

763

2.

Lipid and protein contents increased rapidly and carbohydrate content sharply declined.

764 765

3.

ME, accD and GPAT were significantly correlated with lipid accumulation.

766

4.

ACCase, ME, PEPCase activities were related to lipid accumulation.

767

5.

FA-induced strategy could be developed to produce microalgal lipids efficiently.

768 769

38