Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the

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Transcriptome analysisof genes involved in lipid biosynthesis in the developing embryo of pecan (Carya illinoinensis) Ruimin Huang, Youjun Huang, Zhichao Sun, Jianqin Huang, and Zhengjia Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 01 May 2017 Downloaded from http://pubs.acs.org on May 2, 2017

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Journal of Agricultural and Food Chemistry

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Transcriptome analysis of genes involved in lipid biosynthesis in the developing embryo of

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pecan (Carya illinoinensis)

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Ruimin Huang†, Youjun Huang†, Zhichao Sun†, Jianqin Huang†, Zhengjia Wang†*

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†

6

Zhejiang Agriculture and Forestry University, Hangzhou 311300, China

State Key Laboratory of Subtropical Silviculture, School of Forestry and Biotechnology,

7 8

*

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Tel: 0086(0)571 63743856

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Fax: 0086(0)571 63732738

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E-mail: [email protected]

The corresponding author

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Authorship of the paper Designing the work: R.M.H., Z.J.W., J.Q.H.; running the

15

experiments: Z.C.S., Z.J.W., R.M.H.; data analysis and statistics: R.M.H., Y.J.H, Z.C.S.,

16

J.Q.H.; article writing and revising: R.M.H., Z.J.W., Y.J.H

17 18 19 20 21 22

1

ACS Paragon Plus Environment


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ABSTRACT

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Pecan (Carya illinoinensis) is an important woody tree species because of high content of

25

healthy

26

oil biosynthesis in developing pecan seeds remains largely unclear. Our analyses revealed that

27

mature pecan embryo accumulated more than 80% oil, in which 90% were unsaturated fatty

28

acids with abundant oleic acid. RNA-sequencing generated 84,643 unigenes in three cDNA

29

libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering

30

(DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes

31

for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, seven for oil bodies, and five

32

for transcription factors involved in oil synthesis. The genes associated with fatty acid

33

synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the

34

biosynthesis of oil began to increase, while crude fat contents increased from 16.61% to

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74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two

36

FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologs

37

were not detected. Consequently, we inferred that the linolenic acid in developing pecan

38

embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic

39

reticula. During pecan embryo development, different unigenes are expressed for plastidial

40

and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic

41

glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in

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five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well

43

consistent with their expression in transcriptomic analyses. Our data would be important for

44

the metabolic

oil

in

its

nut.

engineering

Thus

far,

of pecans to

the

pathways

increase

and

key

oil contents and

2


genes

related

modify fatty

to

acid

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compositions.

46 47

KEYWORDS

48

Embryogenesis, lipid biosynthesis, unsaturated fatty acids, fatty acid desaturase, glycolysis

49 50

INTRODUCTION

51

Nuts are delicious and nutritious foods which are rich in unsaturated fatty

52

acids, proteins, fiber, minerals, vitamins, and many other bioactive substances, including

53

phytosterols and phenolic antioxidants.1 Nuts contain a large amount of mono- and

54

poly-unsaturated fatty acids. It is noteworthy that linoleic acid and linolenic acid are essential

55

fatty acids for maintaining optimal health of human beings.2-4

56

Lipid biosynthesis of nuts depends on the correct spatial and temporal activity of many

57

gene products. These genes perform their functions in three continuous processes.5-7 The first

58

process involves de novo biosynthesis of fatty acids in plastids. This initial step is mainly

59

catalyzed by a complex so-called the fatty acid synthase. The second process results in the

60

synthesis of triacylglycerol (TAG) in the endoplasmic reticulum.8 During the final process,

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TAG combines with oleosin to form oil bodies which are released from the endoplasmic

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reticulum into the cytoplasm.

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In plastids, acetyl-CoA carboxylase (ACCase) catalyzes the formation of malonyl-CoA

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from acetyl-CoA. Subsequently, plastidial acetyl-CoA and malonyl-CoA are converted to

65

a long-chain acyl molecule via a series of reactions catalyzed by several enzymes,

66

with an acyl carrier protein (ACP) as a cofactor. Fatty acid synthase uses acetyl-CoA

3



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as the starting unit and malonyl- CoA as the extender unit. Each condensation is catalyzed

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by 3-ketoacyl-ACP synthase (KAS),9 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl-ACP

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dehydratase (HAD), and enoyl-ACP reductase (ENR). Stearoyl-ACP desaturase (SAD)

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converts saturated fatty acids containing ACP (18:0-ACP) to monounsaturated fatty acids

71

containing ACP (18:1-ACP). Fatty acyl-ACP thioesterase (FAT) then hydrolyzes the

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acyl groups to release free fatty acids.10

73

TAGs

are

assembled

in

the

endoplasmic

reticulum

74

using glycerol-3-phosphate and acyl- CoA as the primary substrates. Several acyl-transferases,

75

including glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase

76

(LPAAT) and diacylglycerol acyltransferase (DGAT), are involved in TAG biosynthesis.

77

In mature embryos, the resulting TAGs can be stored as oil bodies surrounded by a membrane

78

composed of a layer of phospholipids embedded with several proteins (i.e., oleosin, caleosin,

79

and steroleosin).11

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In addition, some transcription factors, such as WRINKLED1 (WRI1), FUSCA3 (FUS3),

81

ABSCISIC ACID3 (ABI3), and LEAFY COTYLEDON (LEC1 and LEC2) regulate embryo

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development.12 When these genes are overexpressed in Arabidopsis thaliana, the expression

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of numerous genes involved in fatty acid biosynthesis is increased suggesting a role in

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positive regulation of fatty acid biosynthesis. There is functional redundancy among these

85

transcription factors, which are regulated by a complex network involving hormones and

86

metabolic signaling pathways.13, 14 LEC2 and LECl can increase the production of FUS3 and

87

ABI3.

88

The search for genes controlling quantitative features of unsaturated fatty acids

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accumulation is of fundamental importance. The SAD and FAD genes, which play critical

90

roles in fatty acid desaturation, generally have multiple copies in plants.15,16 The copy number

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of gene is different in different plants. There are significant differences in sequence

92

characteristics, expression regulation and function between different copies of the same gene

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in the same plant. However, our knowledge for the formation mechanism of the unsaturated

94

fatty acid in the seed remains unclear.

95

Pecan [Carya illinoinensis (Wangenh.) K. Koch] belongs to the Juglandaceae family, and

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is originated from North America.17 Pecan embryos contain a large amount of oil (i.e., more

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than 70% of the dry weight). The edible pressed oil extracted from pecan nuts is abundant in

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protein, unsaturated fatty acids, vitamins, and other minerals. Pecan lipids are mainly

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composed of unsaturated fatty acids (more than 90%), with oleic (52.52–74.09%) and linoleic

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(17.69–37.52%) acids representing the main unsaturated fatty acids.18 Pecan oil is higher in

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monounsaturated fatty acids concentration than olive oil.19 Huang et al.20 reported that a

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combination of high SAD and low FAD2 contents facilitates the accumulation of oleic acid in

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hickory (Carya cathayensis Sarg.). However, the reasons of high oil and monounsaturated

104

fatty

105

we conducted sequencing

106

assembly experiments to identify the pathways and key genes related to oil accumulation

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during embryogenesis in pecan. Our data would provide new perspectives relevant to

108

future studies on the metabolic engineering of pecan to increase oil contents and modify fatty

109

acid compositions.

acid

contents

in

pecan

have

not

and

been

de

110

5


investigated. In novo

this

study,

transcriptome


111

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MATERIALS AND METHODS

112 113

Plant material

114 115

Fruits were collected from 12-year-old pecan [Carya illinoinensis (Wangenh.) K. Koch]

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cultivar ‘Pawnee’ trees in Xin’chang (29°N, 120°W), China. Samples were collected during

117

the seed development period from mid-August to mid-October, 2014. Nuts were harvested at

118

105 days after flowering (DAF) (i.e., early cotyledon stage) and then every 15 days until 165

119

DAF (i.e., maturity). Pecan fruits were removed from each side of the tree (i.e., south, east,

120

west, and north), for a total of 20 fruits per developmental stage per tree. After removing

121

the pericarp

122

lipid analyses and RNA extractions for transcriptome sequencing.

and

testa

(seed

coat), we

froze

the embryo in liquid

nitrogen

for

123 124

Lipid Analysis

125 126

Embryos harvested at 120, 135, 150, and 165 DAF were oven-dried at 85 °C to a

127

constant weight. Total lipids were extracted from freeze-dried powder at 50 °C for 8 h using

128

petroleum ether as a solvent. Fatty acid methyl esters were analyzed according to the

129

ISO 5509 method using the GC-2014C gas chromatograph (Shimadzu, Kyoto, Japan). Lipid

130

analyses were completed using a completely random experimental design, with three

131

biological replicates for each tissue and developmental stage.

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RNA Extraction and cDNA Library Construction

134 135

Total

RNA was extracted from embryos using the

136

Germantown,

137

to eliminate any contaminating genomic DNA. For RNA-sequencing (RNA-seq), we selected

138

the following three developmental stages based on embryo size and oil content: early

139

cotyledon stage (105 DAF), mid- cotyledon stage (120 DAF), and maturity (165

140

DAF). mRNA was purified from 1 µg total RNA, fragmented, and then used to prepare a

141

cDNA library with

142

CA, USA). Samples were then clustered and sequenced using the HiSeq 2500 Sequencing

143

System (Illumina). Deep-sequencing was completed with a 100-cycle paired-end run.

MD,

USA), and

then

RNeasy

treated with

the TruSeq RNA Sample

Prep

Kit

Mini

DNase

kit (Qiagen, I

(Illumina, San

(Qiagen)

Diego,

144 145

RNA-Seq Data Analysis

146 147

The quality of the RNA-seq reads were assessed with FastQC (version 0.10.1; Babraham

148

Bioinformatics, Cambridge, UK). Reads were assembled using the default parameters of the

149

Trinity program (r2013-02-25). Transcript abundance was calculated with RSEM as

150

fragments per kilobase of exon per million fragments mapped (FPKM).21 The Cuffdiff 2

151

algorithm22 was then used to analyze the differential gene and isoform expression levels (false

152

discovery rate ≤ 0.05). The over-representation of Gene Ontology and Kyoto Encyclopedia of

153

Genes and Genomes pathways were assessed using Fisher’s exact test (false discovery rate ≤

154

0.05).

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155 156

Differentially Expressed Genes

157 158

The expression levels of the unigenes from three samples, embryos collected at 105 (S1)

159

and 120 (S2) DAF and embryos at 105 (S1) and 165 (S5) DAF were compared. Genes were

160

considered differentially expressed if expression values exhibited a more than 2-fold change.

161 162

qPCR Analysis

163 164

cDNAs were synthesized using the PrimeScript RT reagent with gDNA Eraser (Takara,

165

Dalian, China) and RNAs from the five developmental stages. qPCRs were performed in

166

Mastercycler

167

Green qPCR SuperMix-UDG with ROX (Invitrogen, C11744-500, Carlsbad, CA, USA). The

168

reactions were carried out three times using independent samples. Primers for 18 key genes

169

related to lipid biosynthesis were used in the qPCR (Table S1), in which the 18S rRNA gene

170

of pecan was used as the reference gene. The gene expression levels were calculated as

171

−2−∆∆Ct.

ep

realplex

using

the

Platinum

172 173

RESULTS AND DISCUSSIONS

174 175

Morphological Characteristics and Oil Content of Developing Embryos

176

8


SYBR

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Pecan embryos developed between 105 to 165 DAF (Figure l). Additionally, the oil

178

content increased from less than 12% to more than 80% as the embryos developed. During

179

the early cotyledon stage (stage 1; 105 DAF), embryos were watery and transparent, with a

180

diameter less than 5 mm. In the mid-cotyledon stage (stage 2; 120 DAF), the embryo diameter

181

increased to about 10 mm. However, embryos remained watery and transparent, with

182

relatively low oil contents (i.e., 11.61%). During the late cotyledon stage (stage 3; 135 DAF),

183

the embryo diameter increased to approximately 15 mm. The embryos developed a flavescent

184

surface surrounding fleshy insides, and the oil content at this stage was 74.75%. In the full

185

cotyledon stage (stage 4; 150 DAF) and at maturity (stage 5; 165 DAF), the embryos grew

186

slightly, while the oil content was 81.89%. This suggests that the accumulation of oil in

187

embryos was one of the main features of stages 2 and 3.

188

We

investigated

the fatty

acid

compositions

of pecan oil

at

different

189

developmental stages (Figure 2). Palmitic acid and stearic acid, which are two of the main

190

components of saturated fatty acids, were maintained at relatively low levels (< 20%) in

191

stages 3–5. In contrast, the unsaturated fatty acid content was 57.01% in stage 2, and quickly

192

increased to 91.95% in stage 3. The oleic acid content increased from 40.01% in stage 2 to

193

81.44% in stage 3, and then decreased slightly in stages 4 and 5. The abundance of linoleic

194

acid decreased from 12.45% in stage 2 to 9.60% in stage 3, after which it increased in stages 4

195

and 5. The linolenic acid content remained at relatively low levels (average 0.96%) in stages

196

2–5. Our data revealed that mature pecan embryo accumulated more than 80% oil. More than

197

90% of the oil was composed of unsaturated fatty acids (i.e., 71.22% oleic acid, 19.71%

198

linoleic acid, and 0.78% linolenic acid), while less than 10% of the oil consisted of saturated

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fatty acids (i.e., 5.62% palmitic acid, 2.17% stearic acid, and a trace amount of arachic acid).

200

Our results indicated that for pecan embryos, oil started to accumulate in stage 1, with rapid

201

increases in oil contents occurring in stage 2. The oil content was relatively stable in stage 5.

202

Therefore, to examine the genes associated with lipid biosynthesis during pecan

203

embryo development, the following sequencing analyses were completed using samples

204

from three developmental stages.

205 206

Transcriptome Sequencing and de Novo Assembly of Unigenes

207 208

The de novo assembly of sequencing reads was completed using the Trinity program.

209

The non-redundant contigs were 200–17,640 nt long. The assembled reads resulted in the

210

detection of 109,712 transcripts (no shorter than 200 bp), and the unigene dataset included

211

84,643 sequences, with a mean length of about 1,824 bp (data not shown). We produced a

212

scatter

213

were tentatively annotated according to the known sequences of the closest matches. We

214

annotated 39,483 (46.65%) unigenes based on the information in public databases.

215

The relatively low annotation rate may have been a consequence of the limited genomic

216

information

217

hits revealed that 27.58% of the unigenes were highly homologous to previously deposited

218

sequences (< 1.0E-50). We also observed that 19.06% of the unigenes had an E-value

219

that ranged from 1.0E-5 to 1.0E-50 (Figure 3B). Additionally, the best match for the unigenes

220

were mostly from Vitis species (11.75%), poplar (Populus; 5.68%), castor bean (Ricinus;

plot

of

transcript

available

for

size

pecan.

distributions

The E-value

(Figure

3A). These

distribution

10


of

the

genes

top

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5.52%), and Glycine max (2.03%) (Figure 3C). These results were consistent with those from

222

earlier studies, and confirm that pecan trees are woody perennials.

223 224

Identification of Lipid-related Genes in the Developing Pecan Embryo

225 226

This study involved a detailed analysis of genes with key roles in lipid biosynthesis. The

227

oil biosynthesis model of the pecan embryo is presented in Figure 4. We identified 153

228

unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis,

229

34 for TAG biosynthesis, seven for oil bodies, and five for transcription factors involved in oil

230

synthesis (Table 1). Among the 153 unigenes, five from stage 1, 17 from stage 2, and nine

231

from stage 3 had FPKM values greater than 100. Additional investigations of highly

232

expressed genes may be necessary to elucidate their functions in pecan embryos. In the S2

233

versus S1 comparison, 101 unigenes showed significantly different expression, including 67

234

up-regulated unigenes and 34 down-regulated unigenes. In the S5 versus S1 comparison,

235

there were 37 up-regulated and 49 down-regulated unigenes .The genes associated with fatty

236

acid synthesis were most highly expressed in stage 2, while some genes related to TAG

237

assembly, such as LPAAT and DGAT1, were most abundantly expressed in stage 5.

238 239

Unigenes Related to Fatty Acid Biosynthesis

240 241

In plastids, the pyruvate dehydrogenase complex (PDHC), which consists of four

242

enzyme subunits (i.e., E1α, E1β, E2, and E3), provides the acetyl-CoA precursor required for

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243

de novo fatty acid synthesis.23 Our analysis revealed that the genes encoding the four subunits

244

were most abundantly expressed in stage 2 (Figure 5A). Highly active pyruvate

245

dehydrogenase stimulates oil accumulation in seeds. 24

246

Acetyl-CoA carboxylase is a rate-limiting enzyme for de novo fatty acid synthesis.

247

It catalyzes the first metabolic step of fatty acid biosynthesis in the plastid by adding one

248

carboxyl group toacetyl-CoA to form malonyl-CoA. ACCase in plastids consists of the

249

following four subunits: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP),

250

α-carboxyltransferase

251

the expression of the genes encoding these subunits exhibited a coordinated temporal pattern,

252

with the highest expression levels observed in stage 2. This may partially explain why oil

253

content increased from 11.61% (stage 2) to 74.75% (stage 3). The CTβ gene exhibited the

254

lowest expression level among the four subunit genes. Sasaki et al.25 reported that a lack of

255

CTβ subunit in plastids limits ACCase production and activity. The overexpression of the

256

gene encoding the CTβ subunit in plastids is expected to regulate the quantity of fatty acid

257

synthesized. Additional research is required to determine whether CTβ is important for fatty

258

acid biosynthesis in pecan.

(CTα),

and

β-carboxyltransferase

(CTβ).

Figure

5B

shows

259

Plastidial acetyl-CoA and malonyl-CoA are converted to long-chain acyl molecules via

260

a series of reactions catalyzed by several enzymes, with ACP as a cofactor. The temporal

261

expression patterns for the genes encoding the accumulation of oil in the embryo.26 We

262

observed that the ACP gene was most highly expressed in stage 2 (Figure 5D), implying that

263

fatty acid synthesis was fastest in this stage.

264

There are three types of KAS in plastids, namely KASI–III. KASI is highly active in

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265

catalyzing the production of acyl-ACP with chain lengths of C2-C14, but is less active in the

266

production of 16:0-ACP, and almost inactive for catalyzing the generation of 18:0-ACP.

267

KASII influences the conversion of 16:0-ACP to 18: 0-ACP, while KASIII helps acetyl-CoA

268

combine with malonyl-ACP to form 4:0-ACP.9 Our data indicated that KASI and KASII

269

expression levels exhibited a coordinated temporal pattern (Figure 5C), with the highest levels

270

in stage 2. KASIII was expressed at much lower levels than KASI and KASII, and was only

271

slightly up-regulated in stage 2.

272

Fatty acyl-ACP thioesterase (FAT) is the major determinant of chain length (i.e., ratio of

273

16 C to 18 C fatty acids) and abundance of saturated fatty acids.27 There are two distinct but

274

related thioesterases in higher plants: FATA is an acyl-specific thioesterase specific for

275

18:1 fatty acids,28 whereas saturated acyl-ACP is the primary substrate of FATB

276

thioesterases.29 In this study, three FATB paralogs and one FATA unigene were detected in the

277

transcriptomes of developing pecan embryos. The expression levels of FATA and FATB

278

exhibited opposing trends. The expression of FATA was highest in stage 2, while the FATB

279

expression level was down-regulated in the same stage (Figure 5E). Therefore, the FATA and

280

FATB expression levels influenced the synthesis of 16C and 18 C fatty acids in developing

281

pecan embryos. This may partially explain why oleic acid content increased from 40.01%

282

(stage 2) to 81.44% (stage 3).

283 284

Unigenes Related to Triacylglycerol and Oil Bodies

285 286

Diacylglycerol acyltransferase catalyzes the last acylation step in the conversion of

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287

diacylglycerol to TAG, which may be an important step in the accumulation of storage lipids

288

in plants.30 Three different DGAT gene family members have been detected in oilseed

289

plants, with DGAT1 and DGAT2 being expressed mostly in seeds.31 Another enzyme

290

catalyzing the acyl-CoA–independent synthesis of TAG via phospholipid:DAG transacylase

291

activity

292

(PDAT) gene family.32

293

complements DGAT activity. Our data revealed that DGAT1 was most abundantly expressed

294

in stage 5, while PDAT expression was steadily down-regulated during embryo development.

295

This may have resulted because of some functional overlap between DGAT and PDAT

296

(Figure 5F), and PDAT activity may be more important in the early stages.

has

been

identified In

in

developing

the

phospholipid:diacylglycerol acyltransferase

Arabidopsis

thaliana

seeds,

PDAT partially

In mature seeds, TAG can be stored as oil bodies. Oleosin is the most abundant structural

297 298

protein

in oil

bodies. It stabilizes

oil

bodies through

299

repulsion, preventing oil bodies from fusing.33, 34 Caleosin is involved in the synthesis and

300

metabolism of oil bodies. Steroleosin is involved in signal transductions regulated in plant.

301

35,36

302

3, 3, and 1 unigene, respectively (Table 1). The expression levels of oleosin, caleosin and

303

steroleosin in pecan embryos were all higher in stages 2 and 5 than in stage 1. Additionally,

304

all oleosin unigenes were up-regulated between stages 1 and 2, which was consistent with the

305

oil content changes.

charge

According to the unigene annotations, oleosin, caleosin and steroleosin were encoded by

306 307

increased spacing and

Unigenes Related to Fatty Acid Desaturation

308

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309

Mature pecan embryos consist of 71.22% oleic acid, 19.71% linoleic acid, and 0.78%

310

linolenic acid. Fatty acid desaturase is a key enzyme for controlling oleic acid contents

311

(Figure 6), while SAD is crucial for the de novo synthesis of unsaturated fatty acids in

312

plants.37 This influences the proportion of saturated and unsaturated fatty acids in pecan oil.

313

Our results indicated that SAD abundance was considerably higher in stage 2 than in the other

314

two stages (Figure 6). We detected four unigenes encoding SAD (Ug000260, Ug001498,

315

Ug000155, Ug050371). Two of these unigenes (i.e., Ug000260 and Ug000155) had FPKM

316

values greater than 500 in stage 2 (Table 1), which may be partially responsible for the

317

increase in oleic acid content from 40.01% (stage 2) to 81.44% (stage 3).

318

Many types of enzyme participate in fatty acid desaturation in plants. ∆12-Desaturase

319

(i.e., FAD2 and FAD6) desaturates oleic acid (18:1) to form linoleic acid (18:2), whereas

320

∆15-desaturase (i.e., FAD3, FAD7, and FAD8) desaturates linoleic acid (18:2) to form

321

α-linolenic acid (18:3) (Figure 6).38 In this study, we detected two, one, two, and two unigenes

322

encoding FAD2, FAD6, FAD7, and FAD8, respectively (Table 2). The two FAD2-encoding

323

unigenes (i.e., Ug000251 and Ug001772) were highly expressed, which may have important

324

consequences for linoleic acid content. These two unigenes may have been involved in the

325

increase in linoleic acid content from 9.60% (stage 3) to 19.71% (stage 5).

326

We did not detect any FAD3 homologs during the analysis of the transcriptome

327

sequencing data. We assumed that the linolenic acid in developing pecan embryos was

328

predominantly generated by FAD7 and FAD8 in plastids rather than by FAD3 in endoplasmic

329

reticula, which was similar to what has been observed for hickory20 and tree peony39 embryos.

330

Additionally, the linolenic acid content remained relatively low (i.e., average 0.96%) in stages

15



331

2–5, implying there was limited FAD7- and FAD8-catalyzed desaturation of linoleic acid

332

(18:2) to form α-linolenic acid (18:3).

333 334

Detection of Transcription Factors Involved in Lipid Synthesis

335 336

A set of transcription factors (i.e., LEC1, LEC2, ABI3, FUS3, and WRI1) with key roles

337

in seed oil synthesis and deposition was identified in previous studies (Figure 7). Table 3

338

shows LEC1 and LEC2 expression levels decreased during embryo development, with

339

undetectable levels in stage 5. In contrast, the expression of ABI3, which is essential for seed

340

maturation, was up-regulated. FUS3 expression was up- regulated in stage 2 and

341

down-regulated in stage 5 to levels considerably lower than those of stages 1 and 2. This gene

342

expression pattern was consistent with the rapid accumulation of oil in the early stages. FUS3

343

may be important for plastidial fatty synthesis during pecan embryo development. We did not

344

detect WRI1 transcripts in any stage, suggesting a lack of importance for this transcription

345

factor during pecan embryo development.

346 347

Cytosolic and Plastidial Glycolysis

348 349

Glycolysis provided precursors and energy for lipid synthesis. Some glycolysis- related

350

unigenes were expressed in the cytosol or plastids. Cytosolic glycolysis produced some

351

intermediate products that were transported to the plastid. For example, the phosphate

352

translocator transported phosphoenolpyruvate to the plastid. There are three irreversible

16


Page 16 of 47

Page 17 of 47


353

reactions in anaerobic glycolysis. These reactions are catalyzed by hexokinase (HXK),

354

phosphofructokinase (PFK), and pyruvate kinase (PKP).40 During pecan embryo development,

355

different unigenes were expressed in cytosolic and plastidial glycolysis (Figure 8). In the

356

cytosol, HXK and PFK were the most abundantly expressed genes in stages 1 and 3,

357

respectively. In contrast, in plastids, HXK, PFK, and PKP were the most highly expressed

358

genes in stage 2. Additionally, most of the genes associated with plastidial glycolysis were

359

highly expressed in stages 1 or 2, and down-regulated in stage 5. This expression pattern was

360

similar to that of genes related to fatty acid biosynthesis. This suggests that plastidial

361

glycolysis may be related to lipid biosynthesis. Furthermore, plastidial glycolysis provided

362

considerable amounts of precursors and energy for fatty acid biosynthesis, resulting in energy

363

being stored primarily in oil rather than in sugar in pecan embryos.

364 365

Quantitative Analysis of Lipid-related Genes

366 367

18 important genes associated with lipid biosynthesis were evaluated in five stages of

368

developing embryos using qPCR. Furthermore, the expression levels of 18 genes measured by

369

FPKM values were relatively high in transcriptomic datas.

370

Figure 9A-J shows the expression profiles of 10 genes associated with oil content. The

371

expression levels of Ug001051 (PDH-E1-α)(Figure 9A),Ug000604 (PDH-E1-β)(Figure 9B),

372

Ug000847(PDH-E3)(Figure

373

(ACC/BC)(Figure 9F) and Ug001705 (ACC/BCCP)(Figure 9G) were expressed maximally in

374

stage 2. The expression of Ug002028 (PDH-E2)(Figure 9C) was high in stage 2 and 4.The

9D),

Ug000023

(ACC/CTα)(Figure

17


9E),

Ug001595


375

high expression levels of PDHC and ACCase may partially explain why oil content increased

376

from 11.61% (stage 2) to 74.75% (stage 3). The expression of Ug009718 (DGAT1)(Figure 9H)

377

and Ug007373 (DGAT2)(Figure 9I) reached a maximum in stage 4; Ug006002

378

(PDAT)(Figure 9J) reached a minimum in stage 4.

379

Figure 9K-R shows the expression profiles of 8 genes associated with fatty acid

380

compositions. Ug000260 (SAD)(Figure 9K) and Ug000155 (SAD)(Figure 9M) were

381

transcribed at high levels in stage 2, 3 and 4. Ug001498 (SAD)(Figure 9L) reached the

382

maximum level of expression in stage 2. The high expression of SAD increased in oleic acid

383

content from 40.01% (stage 2) to 81.44% (stage 3). The expression of Ug000251

384

(FAD2)(Figure 9N) decreased before increasing to reach a maximum in stage 4. The

385

expression of Ug001772 (FAD2)(Figure 9O) was high in stage 1 and 2 and then decreased

386

sharply in stage 3.This expression pattern was similar to their digital expression in

387

transcriptomic datas. This may have resulted because of Ug001772 (FAD2) activity may be

388

more important in the early stages and Ug000251 (FAD2) be more important when the

389

embryos became mature. The expression of Ug002487 (FATA)(Figure 9P) was the highest in

390

stage 3 .The expression of Ug011130 (FATB)(Figure 9Q) and Ug002471 (FATB)(Figure 9R)

391

were relatively low in stage 3. The expression levels of FATA and FATB exhibited opposing

392

trends. The qPCR data were well consistent with their digital expression in transcriptomic

393

analyses. The finding further explain why oleic acid content increased from 40.01% (stage 2)

394

to 81.44% (stage 3).

395

In summary, mature pecan embryo accumulated more than 80% oil, with unsaturated

396

fatty acids being the main component (e.g., high oleic acid contents). The expression of lipid

18


Page 18 of 47

Page 19 of 47


397

biosynthesis genes influences oil content and lipid composition in pecan embryos. In stage 2,

398

the expression levels of the genes encoding ACCase, ACP, KASI, KASII, SAD, FATA, FAD2,

399

oleosin, and caleosin were up-regulated relative to the levels in stage 1. Additionally, oil

400

started to accumulate, while crude fat content increased from 16.61% (stage 2) to 74.45%

401

(stage 3). The increased KASII contribute to the conversion of stearic acid from palmitic acid

402

convert palmitic acid (C16:0) to stearic acid. Additionally, the increased production of SAD

403

and FATA and decreased production of FATB resulted in higher oleic acid contents. Increased

404

FAD2 abundance increased the proportion of polyunsaturated fatty acids in lipids. In stage 5,

405

the expression of many genes associated with lipid biosynthesis was considerably

406

down-regulated, which decreased the rate of lipid synthesis and the oil content remained

407

relatively stable. The qPCR data were well consistent with their digital expression in

408

transcriptomic analyses. Compare and contrast the previous data from developing ‘Sumner’

409

pecan embryos,41 the expression profiles of the genes encoding ACCase, ACP, HAD, KASI,

410

FATA, DGAT, PDAT and oleosin were well consistent. The results showed that the expression

411

of the genes involved in lipid biosynthesis in the developing embryo of pecan in different

412

varieties was similar. Our data provide potentially useful molecular information for future

413

studies on pecan embryo development, particularly regarding oil accumulation. Our findings

414

may be relevant for the metabolic engineering of pecan to increase oil contents and modify oil

415

compositions.

416 417

ACKNOWLEDGEMENT

418

19



Page 20 of 47

419

We thank Hikmet, Kumar, Zhengguo Lin, Lili Song, Liangshen Zhang and Hailan Liu

420

for critical reading and insightful comments. Yi Gan, Mingquan Ding, Tong Zhang and Jian

421

Li for giving helpful suggestions of the manucript; This work was supported by the National

422

High Technology Research and Development program of China (863 Program,

423

2013AA102605), and the Natural Science Foundation of China (31570666) .

424 425

ABBREVIATIONS USED

426 427

DAF, days after flowering; qPCR, quantitative PCR; TAG, triacylglycerol; ACCase,

428

acetyl-CoA carboxylase; ACP, acyl carrier protein ; KAS, 3-ketoacyl-ACP synthase; KAR,

429

3-ketoacyl-ACP

430

enoyl-ACPreductase; SAD, stearoyl-ACP desaturase; FAT, fatty acyl-ACP thioesterase; GPAT,

431

glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidate acyltransferase; DGAT,

432

diacylglycerol acyltransferase; WRI1, WRINKLED1; FUS3, FUSCA3; ABI3, ABSCISIC

433

ACID3; LEC, LEAFY COTYLEDON; PDHC, pyruvate dehydrogenase complex; FPKM,

434

kilobase per million mapped reads; BC, biotin carboxylase; BCCP, biotin carboxyl carrier

435

protein;

436

phospholipid:diacylglycerol acyltransferase; DAG, 1,2-Diacylglycerol; TAG, triacylglycerol;

437

ALDO, non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase;

438

BASS1, sodium bile acid symporter family; CINV, neutral invertase; ENO, enolase; FBA,

439

fructose-bisphosphate

440

glyceraldehyde-3-phosphatedehydrogenase

reductase;

CTα,

HAD,

3-hydroxyacyl-ACP

α-carboxyltransferase;

aldolase;

CTβ,

FBP,

dehydratase;

β-carboxyltransferase;

fructose-1,6-bisphosphatase C-2;

GLT,

20


glucose

I;

transporter;

ENR,

PDAT,

GAPC, GPT,

Page 21 of 47


441

glucose6-Phosphate; HXK, hexokinase; MDH, malate dehydrogenase; ME, malate

442

dehydrogenase; PDHC, pyruvate dehydrogenase complex; PEPC, phosphoenolpyruvate

443

carboxylase; PFK, 6-phosphofructokinase 1; PFP, pyrophosphate--fructose-6-phosphate

444

1-phosphotransferase beta subunit, putative; PGI, phospho-glucose (Glc) isomerase; PGK,

445

phosphoglyceratekinase;

446

pyruvatekinase; PKP, pyruvate kinase beta subunit; PPT, phosphoenolpyruvate/phosphate

447

translocator; SUS, sucrose synthase; TIM, triosephosphate isomerase; TPT, triose-phosphate ⁄

448

phosphate translocator.

PGM,

phosphoglucomutase;

PGMI,

phosphoglycerate;

PK,

449 450 451

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protein (ACP) thioesterase gene from Brassica juncea. Biochem. Soc. T. 2000, 28 (6),

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Acyl-Acyl Carrier Proteins. Arch. Biochem. Biophys. 1995, 316 (1), 612-618.

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the rate of triacylglycerol synthesis. Biochim. Biophys. Acta 1988, 958 (1), 125-129.

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Oleosin protein is synthesised by ER-bound ribosomes. Plant Physiol. Bioch. 1999, 37

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Murphy, D. J.; Rogers, J. C.; Mundy, D. J. Caleosins: Ca2+-binding proteins

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Fofana, B.; Cloutier, S.; Duguid, S.; Ching, J.; Rampitsch, C. Gene expression of

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stearoyl-ACP desaturase and delta12 fatty acid desaturase 2 is modulated during seed

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development of flax (Linum usitatissimum). Lipids 2006, 41 (7), 705-712.

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Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the

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composition of developing tree peony (Paeonia section Moutan DC.) seeds and

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transcriptome analysis during seed development. Bmc Genomics 2015, 16 (1), 208.

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(41) Mattison, C. P.; Rai, R.; Settlage, R.; Hinchliffe, D. J.; Madison, C.; Bland, J. M.; Brashear,

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S.; Graham, C. J.; Tarver, M. R.; Florane, C. RNA-Seq Analysis of Developing Pecan

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(Carya illinoinensis) Embryos Reveals Parallel Expression Patterns Among Allergen and

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Lipid Metabolism Genes. J. Agric. Food Chem. 2017, 65(7), 1443−1455.

577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594

27



Page 28 of 47

595

Table 1. Identification of Lipid-related Genes in the Developing Pecan Embryo. The numbers

596

under the S1, S2, and S5columns represent the FPKM values of each unigene identified in the

597

pecan embryo transcriptomes. Red and green colors represent the differential expression of

598

corresponding unigenes being up-regulated/more transcripts and down-regulated/fewer

599

transcripts in the embryos, respectively. Unigene was differentially expressed if FPKM values

600

between two samples were ≥2-fold difference. The expressed unigenes with RPKM values >

601

100 are highlighted with bold font.

602 KEGG enzyme

S2/

S5/

S1

S1

0.44

2.40

0.95

2.31

0.65

6.97

1.97

0.32

2.33

0.65

7.24

2.00

147.75

197.46

54.79

1.34

0.37

Ug000604

81.61

355.39

18.08

4.35

0.22

Ug002028

45.39

92.60

3.26

2.04

0.07

Ug000847

118.83

183.82

23.28

1.55

0.20

Ug000023

28.53

2353.39

216.60

82.48

7.59

Ug077832

0.00

2.63

0.00

Ug016281

2.79

1.26

14.56

0.45

5.21

Ug042935

0.54

1.16

0.12

2.13

0.21

Ug069808

0.00

0.79

0.62

acetyl-CoA carboxylase,

Ug001595

62.26

72.92

41.19

1.17

0.66

biotin

Ug045595

0.56

0.54

0.13

0.95

0.22

carboxylase subunit

Ug053995

0.40

1.93

0.00

4.78

0.00

[EC:6.4.1.2 6.3.4.14]

Ug055115

1.72

0.00

0.00

0.00

0.00

annotation[international

S1

S2

S5

Ug075759

0.00

0.00

1.91

pyruvate dehydrogenase E1

Ug050654

0.46

1.10

component

Ug066149

0.33

alpha subunit [EC:1.2.4.1]

Ug054227 Ug001051

enzyme name]

PDH-E1-α

Unigene ID

pyruvate dehydrogenase E1

PDH-E1-β

component beta subunit [EC:1.2.4.1] pyruvate dehydrogenase E2 component

PDH-E2

(dihydrolipoamide acetyltransferase) [EC:2.3.1.12] dihydrolipoamide

PDH-E3

dehydrogenase [EC:1.8.1.4] acetyl-CoA carboxylase

ACC/CTα

carboxyl transferase subunit alpha [EC:6.4.1.2]

ACC/BC

28


Page 29 of 47


ACC/BCC P

Ug058656

0.47

0.87

0.43

1.83

0.90

acetyl-CoA carboxylase

Ug001705

63.72

246.04

65.30

3.86

1.02

biotin

Ug008462

20.33

37.59

0.00

1.85

0.00

carboxyl carrier protein

Ug011252

32.17

13.24

3.03

0.41

0.09


Ug052224

2.42

2.84

0.00

1.17

0.00

biotin carboxyl

Ug038420

0.49

1.54

0.15

3.14

0.30

Ug023294

0.43

1.32

1.00

3.08

2.34

Ug033193

0.19

0.99

0.58

5.22

3.06

Ug035560

0.39

1.25

0.69

3.23

1.77

Ug039572

0.25

1.75

0.82

6.93

3.25

Ug000228

288.55

947.21

44.75

3.28

0.16

Ug035628

0.52

3.67

0.81

7.06

1.55

Ug050249

0.00

2.51

0.21

Ug030974

4.09

3.18

0.00

0.78

0.00

Ug030695

0.44

1.47

0.95

3.34

2.16

[acyl-carrier-protein]

Ug030940

1.07

0.00

0.00

0.00

0.00

S-malonyltransferase

Ug037583

0.64

1.27

0.44

1.99

0.69

[EC:2.3.1.39]

Ug042958

0.56

0.39

0.44

0.71

0.79

Ug045242

1.88

1.42

0.00

0.76

0.00

Ug047972

0.41

0.75

0.19

1.83

0.46

Ug058642

0.67

1.53

1.23

2.30

1.84

Ug071448

0.00

0.00

2.00


ACC/CTβ

carboxyl transferase subunit beta [EC:6.4.1.2]

ACP

MAT

KAS III

acyl carrier protein 4

3-oxoacyl-[acyl-carrier-protei n] synthase III [EC:2.3.1.180]

Ug067439

0.38

0.45

0.76

1.19

2.00

Ug048491

3.43

0.00

0.00

0.00

0.00

Ug053896

0.34

0.83

0.69

2.41

2.01

Ug060062

0.43

1.04

0.43

2.40

0.99

Ug068554

0.00

5.07

0.00

Ug004284

56.44

72.95

9.00

1.29

0.16

Ug078489

0.00

5.16

0.00

Ug002729

19.16

8.49

10.00

0.44

0.52

Ug028341

0.00

1.47

0.27

Ug035670

0.54

1.55

0.57

2.89

1.06

Ug036401

0.37

1.79

0.39

4.79

1.04

Ug036983

0.62

1.62

0.43

2.60

0.69

Ug037576

0.37

1.50

0.95

4.07

2.56

Ug038262

0.82

0.61

0.44

0.74

0.54

Ug041144

0.29

1.28

0.61

4.42

2.11

Ug042586

0.54

1.35

0.34

2.52

0.64

Ug046326

0.38

1.53

0.00

4.08

0.00

Ug046514

0.32

1.19

0.11

3.72

0.34

3-hydroxyacyl-[acyl-carrier-p

HAD

rotein] dehydratase [EC:4.2.1.59]

3-oxoacyl-[acyl-carrier

KAR

protein] reductase [EC:1.1.1.100]

29



ENR

KASI

KASII

FATB

LACS

0.38

1.41

0.20

3.67

0.53

Ug047524

0.00

1.04

0.00

Ug047935

0.36

1.28

Ug049918

0.19

1.23

0.20

3.53

0.55

0.35

6.59

1.89

Ug051886

0.46

1.29

0.12

2.79

0.25

Ug054452

0.26

0.65

0.25

2.49

0.98

Ug054977

0.23

1.16

0.30

4.99

1.27

Ug056508

0.28

0.69

0.55

2.45

1.95

Ug059761

0.00

0.88

0.00

Ug059838

0.00

1.36

0.00

Ug060530

0.50

0.93

0.00

1.87

0.00

Ug062377

0.20

0.50

0.42

2.47

2.08

Ug066125

1.14

1.26

0.00

1.10

0.00

Ug067256

0.60

1.31

0.00

2.20

0.00

Ug070413

0.24

1.16

0.47

4.84

1.99

Ug074184

0.00

0.71

0.54

Ug001277

23.35

54.16

11.94

2.32

0.51

0.42

1.97

0.00

4.68

0.00

Ug049287

7.60

6.03

2.35

0.79

0.31

Ug000823

31.45

109.85

66.21

3.49

2.11

Ug024636

6.06

3.97

0.41

0.65

0.07

3-oxoacyl-[acyl-carrier-protei

Ug044879

1.44

0.00

5.61

0.00

3.91

n] synthase II [EC:2.3.1.179]

Ug029931

0.24

1.84

0.94

7.59

3.90

Ug035121

0.54

1.47

0.51

2.73

0.95

Ug059632

0.42

1.07

0.00

2.56

0.00

reductase I

3-oxoacyl-[acyl-carrier-protei

Ug002782

22.94

104.63

60.77

4.56

2.65

n] synthase II [EC:2.3.1.179]

Ug062855

1.57

3.34

2.83

2.12

1.80

Ug000260

59.33

539.78

84.85

9.10

1.43

Ug001498

36.20

27.65

34.26

0.76

0.95

Ug000155

154.78

995.27

1.62

6.43

0.01

Ug050371

2.41

0.00

0.00

0.00

0.00

Ug002487

12.03

41.52

17.18

3.45

1.43

Ug011130

0.57

1.67

78.12

2.96

Ug022490

0.00

1.75

11.60

Ug002471

59.60

41.61

31.63

0.70

0.53

Ug014691

6.00

0.73

36.18

0.12

6.03

Ug000753

12.72

121.87

30.19

9.58

2.37

Ug009542

27.72

2.90

15.71

0.10

0.57

Ug014992

13.32

8.15

4.13

0.61

0.31

Ug001785

29.26

35.22

190.33

1.20

6.50

desaturase [EC:1.14.19.2 1.14.19.11 1.14.19.26]

FATA

Ug046542

Ug047749

enoyl-[acyl-carrier protein]

acyl-[acyl-carrier-protein]

SAD

Page 30 of 47

fatty acyl-ACP thioesterase A [EC:3.1.2.14]

fatty acyl-ACP thioesterase B [EC:3.1.2.14 3.1.2.21]

long-chain acyl-CoA synthetase [EC:6.2.1.3]

30


138.0 8

Page 31 of 47


ABCAT

GPAT

Ug007130

22.64

12.14

2.58

0.54

0.11

Ug017638

4.58

2.00

2.24

0.44

0.49

Ug012494

17.72

1.00

23.73

0.06

1.34

Ug004037

36.51

20.50

40.26

0.56

1.10

Ug005307

18.41

10.52

44.85

0.57

2.44

Ug006482

53.26

0.54

0.00

0.01

0.00

Ug020647

10.02

0.00

1.76

0.00

0.18

glycerol-3-phosphate

Ug034565

5.19

1.26

2.35

0.24

0.45

acyltransferase [EC:2.3.1.15]

Ug004369

36.63

17.23

4.11

0.47

0.11

Ug004921

29.64

32.48

45.48

1.10

1.53

Ug037032

1.92

0.40

1.02

0.21

0.53

lysocardiolipin and

Ug020363

2.09

1.24

13.25

0.59

6.35

lysophospholipid

Ug009714

18.59

9.17

10.13

0.49

0.54

Ug019864

7.05

1.39

6.68

0.20

0.95

Ug022890

5.94

0.52

2.05

0.09

0.35

ABC transporter D family member 1

acyltransferase [EC:2.3.1.2.3.1.51] lysophospholipid acyltransferase [EC:2.3.1.51 2.3.1.23 2.3.1.-] Ug023476

4.16

0.00

0.16

0.00

0.04

Ug039723

0.99

4.60

0.00

4.64

0.00

Ug046512

0.00

3.18

0.66

Ug006688

10.61

6.05

11.60

0.57

1.09

Ug019087

8.14

8.15

5.65

1.00

0.69

Ug037330

0.07

1.63

0.81

23.08

11.46

Ug048565

0.45

0.75

0.44

1.65

0.97

Ug048607

0.67

1.13

0.44

1.68

0.65

Ug055336

0.32

0.39

0.28

1.21

0.88

Ug058796

0.00

0.00

1.42

Ug007147

11.54

2.15

86.92

0.19

7.53

Ug016509

6.21

0.00

21.56

0.00

3.47

Ug010046

23.24

14.13

16.75

0.61

0.72

ethanolaminephosphotransfer

Ug019984

10.84

2.77

4.22

0.26

0.39

ase [EC:2.7.8.1]

Ug016963

10.04

13.26

16.26

1.32

1.62

acyl-lipid omega-6 desaturase

Ug000251

19.56

431.20

171.27

22.04

8.76

Ug001772

159.58

30.43

34.69

0.19

0.22

Ug019864

7.05

1.39

6.68

0.20

0.95

LPAAT 1-acyl-sn-glycerol-3-phospha te acyltransferase [EC:2.3.1.51]

phospholipase A2 / LPA acyltransferase [EC:3.1.1.3 3.1.1.13 3.1.1.4 2.3.1.51]

PAP

CPT

FAD2

phosphatidate phosphatase LPIN [EC:3.1.3.4]

(Delta-12 desaturase) [EC:1.14.19.6 1.14.19.22] lysophospholipid

LPCAT

acyltransferase [EC:2.3.1.51 2.3.1.23 2.3.1.-]

31



diacylglycerol

DGAT1

Page 32 of 47

Ug018331

3.93

0.00

15.66

0.00

3.98

Ug009718

10.11

20.32

37.98

2.01

3.76

Ug007373

18.99

20.93

18.79

1.10

0.99

Ug006002

38.83

6.87

12.01

0.18

0.31

O-acyltransferase 1 [EC:2.3.1.20 2.3.1.75 2.3.1.76] 2-acylglycerol

DGAT2

O-acyltransferase 2 [EC:2.3.1.22] phospholipid:diacylglycerol

PDAT

oleosin

caleosin

STERO

acyltransferase

Ug015317

4.49

2.04

2.99

0.45

0.67

[EC:2.3.1.158]

Ug007926

9.91

14.81

6.71

1.50

0.68

Ug000025

83.12

3255.39

1881.54

39.17

22.64

Ug000051

24.87

1362.20

1120.17

54.76

45.03

Ug075103

7.52

385.75

272.12

51.32

36.20

oleosin 1

peroxygenase [EC:1.11.2.3]

hydroxysteroid dehydrogenase 5

Ug000079

49.01

1480.43

787.89

30.20

16.07

Ug014047

14.90

2.43

15.83

0.16

1.06

Ug009689

14.77

20.15

28.14

1.36

1.91

Ug000191

4.55

454.98

1424.20

99.95

603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

32


312.8 7

Page 33 of 47


628

Table 2. Identification of unigenes related to fatty acid desaturation.

629 KEGG enzyme

annotation[international enzyme name] acyl-[acyl-carrier-protein]

SAD

desaturase [EC:1.14.19.2 1.14.19.11 1.14.19.26]

Unigene

S2/

S5/

S1

S1

84.85

9.10

1.43

34.26

0.76

0.95

S1

S2

S5

Ug000260

59.33

539.78

Ug001498

36.20

27.65

ID

Ug000155

154.78

995.27

1.62

6.43

0.01

Ug050371

2.41

0.00

0.00

0.00

0.00

Ug011761

6.89

16.95

4.69

2.46

0.68

Ug005069

20.14

92.26

11.30

4.58

0.56

Ug035990

2.00

6.07

0.00

3.03

0.00

Ug011603

23.84

5.38

5.34

0.23

0.22

Ug028194

2.14

2.05

3.78

0.96

1.77

acyl-lipid omega-6 desaturase

FAD6

(Delta-12 desaturase) [EC:1.14.19.23 1.14.19.45] acyl-lipid omega-3 desaturase

FAD7

[EC:1.14.19.25 1.14.19.35 1.14.19.36] acyl-lipid omega-3 desaturase

FAD8

[EC:1.14.19.25 1.14.19.35 1.14.19.36]

630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 33



654 655

Page 34 of 47

Table 3. Identification of transcription factors involved in lipid synthesis. KEGG enzyme

annotation[international enzyme name]

LEC1

LEC2

ABI3

FUS3

nuclear transcription factor Y subunit B-6 B3 domain-containing transcription factor LEC2

Unigene

S2/

S5/

S1

S1

0.00

0.24

0.00

0.00

0.22

0.00

S1

S2

S5

Ug013330

19.41

4.60

Ug025787

4.50

1.01

ID

B3 domain-containing

Ug000911

29.02

67.96

102.41

2.34

3.53

transcription factor ABI3

Ug003413

32.94

53.30

74.60

1.62

2.26

Ug015187

3.42

26.26

0.45

7.68

0.13

B3 domain-containing transcription factor FUS3

656 657 658 659 660 661 662 663 664 665 666 667 668 669 670

34


Page 35 of 47


671

Figure captions

672

Figure 1. Morphological characteristics and oil accumulation in the process of pecan embryo

673

development. (A) Morphological characteristics in stage 1-5. (B) Oil accumulation in stage

674

2-5. S1-S5 indicate 5 temporal developmental stages.

675 676

Figure 2. Changes in the fatty acid composition in the developing embryo of pecan.

677 678

Figure 3. Characteristics of Illumina reads and homology search of assembled contigs. (A)

679

Size distribution of pecan Illumina reads. (B) E-value distribution of best BLASTX hits for

680

each unigenes. (C) Species distribution of top BLAST hits of pecan sequences with other

681

plant species.

682 683

Figure 4. Transcriptional specialization of lipid-related genes in the developing pecan embryo.

684

The three squares in each horizontal row correspond to three developmental stages. Full

685

names of the genes are listed in Table 1. Twenty-eight unigenes encoding KAR are not shown.

686

G-3-P,

687

1,2-Diacylglycerol; TAG, Triacylglycerol

Glycerol-3-P;

LPA,

1-Acylglycerol-3P;

PA,

1,2-Diacylglycerol-3P;

DAG,

688 689

Figure 5. Nonpartitioned expression pattern (reads per kilobase per million mapped reads,

690

FPKM) of several fatty acid and TAG biosynthesis genes in developing embryo of pecan.

691 692

Figure 6. The biosynthetic pathway of unsaturated fatty acid in pecan embryo development.

35



Page 36 of 47

693

The value at the fore indicates the ratio of total FPKM in S2 to that in S1 and the value at the

694

back indicates the ratio of total FPKM in S5 to that in S1.

695 696

Figure 7. The regulation model for seed oil accumulation by transcriptional factors. The value

697

at the fore indicates the ratio of total FPKM in S2 to that in S1 and the value at the back

698

indicates the ratio of total FPKM in S5 to that in S1.

699 700

Figure 8. The differential transcription of glycolysis genes. The two-way arrow indicates a

701

reversible reaction and the one-way arrow indicates an irreversible reaction. The solid circle

702

in yellow indicates a translocator. The value at the fore indicates the ratio of total FPKM in S2

703

to that in S1 and the value at the back indicates the ratio of total FPKM in S5 to that in S1.

704 705

Figure 9. Expression profiles of 18 unigenes associated with fatty acid compositions. (A)

706

Ug001051 (PDH-E1-α); (B) Ug000604 (PDH-E1-β); (C) Ug002028

707

Ug000847(PDH-E3); (E) Ug000023 (ACC/CTα); (F) Ug001595 (ACC/BC); (G) Ug001705

708

(ACC/BCCP); (H) Ug009718 (DGAT1); (I) Ug007373 (DGAT2); (J) Ug006002 (PDAT); (K)

709

Ug000260 (SAD); (L) Ug001498 (SAD); (M) Ug000155 (SAD); (N) Ug000251 (FAD2); (O)

710

Ug001772 (FAD2); (P) Ug002487 (FATA); (Q) Ug011130 (FATB)); (R) Ug002471 (FATB).

711

QPCR was used to quantify the mRNA levels using the total RNAs from five embryo

712

developmental stages.

713

36


(PDH-E2);

(D)

Page 37 of 47


714 715

Figure 1

716 717

37



718 719

Figure 2

720 721

38


Page 38 of 47

Page 39 of 47


722 723

Figure 3

724 725

39



726 727

Figure 4

728 729

40


Page 40 of 47

Page 41 of 47


730 731

Figure 5

732 733

41



734 735

Figure 6

736 737

42


Page 42 of 47

Page 43 of 47


738 739

Figure 7

740 741

43



742 743

Figure 8

744

44


Page 44 of 47

Page 45 of 47


745 746

Figure 9

747

45



748

TOC Graphic

The three squares in each horizontal row correspond to three developmental stages.

749 750

Brief Summary

751 752

153 unigenes associated with lipid biosynthesis were identified, including 107 unigenes for

753

fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, seven for oil bodies, and five for

754

transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis

755

were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil

756

began to increase, while crude fat contents increased from 16.61% to 74.45% (165 DAF). We

757

identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible

758

for unsaturated fatty acid biosynthesis. However, FAD3 homologs were not detected. 46


Page 46 of 47

Page 47 of 47


759

Consequently, we assumed that the linolenic acid in developing pecan embryos is generated

760

by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula.

47


Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the

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