A flax fibre proteome: identification of proteins enriched in bast fibres

1471-2229-8-52 1471-2229 Research article A flax fibre proteome: identification of proteins enriched in bast fibres Hotte SC Naomi nhotte@ualberta.ca Deyholos K Michael deyholos@ualberta.ca

Department of Biological Sciences, Edmonton, T6G 2E9, Canada

BMC Plant Biology 1471-2229 2008 8 1 52 http://www.biomedcentral.com/1471-2229/8/52 18447950 10.1186/1471-2229-8-52 07 11 2007 30 4 2008 30 4 2008 2008 Hotte and Deyholos; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Bast fibres from the phloem tissues of flax are scientifically interesting and economically useful due in part to a dynamic system of secondary cell wall deposition. To better understand the molecular mechanisms underlying the process of cell wall development in flax, we extracted proteins from individually dissected phloem fibres (i.e. individual cells) at an early stage of secondary cell wall development, and compared these extracts to protein extracts from surrounding, non-fibre cells of the cortex, using fluorescent (DiGE) labels and 2D-gel electrophoresis, with identities assigned to some proteins by mass spectrometry.

Results

The abundance of many proteins in fibres was notably different from the surrounding non-fibre cells of the cortex, with approximately 13% of the 1,850 detectable spots being significantly (> 1.5 fold, p ≤ 0.05) enriched in fibres. Following mass spectrometry, we assigned identity to 114 spots, of which 51 were significantly enriched in fibres. We observed that a K⁺channel subunit, annexins, porins, secretory pathway components, β-amylase, β-galactosidase and pectin and galactan biosynthetic enzymes were among the most highly enriched proteins detected in developing flax fibres, with many of these proteins showing electrophoretic patterns consistent with post-translational modifications.

Conclusion

The fibre-enriched proteins we identified are consistent with the dynamic process of secondary wall deposition previously suggested by histological and biochemical analyses, and particularly the importance of galactans and the secretory pathway in this process. The apparent abundance of β-amylase suggests that starch may be an unappreciated source of materials for cell wall biogenesis in flax bast fibres. Furthermore, our observations confirm previous reports that correlate accumulation proteins such as annexins, and specific heat shock proteins with secondary cell wall deposition.

Background

Flax (Linum usitatissimum L.) has attracted human attention since the beginning of agriculture 12. This is due in part to the unusual properties of the bast (i.e. phloem) fibres, which because of their great length and high tensile strength have found use in textiles and many other products 3. Fibre length is achieved almost entirely through intrusive growth, which is a process limited to very few cell types in plants 45. The elongation stage is succeeded by a dynamic process of secondary wall deposition, in which a matrix of galactose-rich polymer in the nascent wall is gradually and centripetally replaced by highly crystalline cellulose 6. Because secondary wall deposition increases the tensile strength of cells, fibres which have undergone even the very first stages of cell wall thickening can be distinguished mechanically by their resistance to breakage at the "snap-point" of the stem 7. The snap-point thus defines an important developmental transition from cell elongation to cell wall thickening.

Previously, we and others have produced libraries of cDNAs from fibre-bearing peels of flax and hemp stems 89. In addition to containing bast fibres at various stages of development, these peels also contained many other cell types, including those associated with cambium and transport phloem. Analysis of these libraries by cDNA microarray hybridization and other techniques identified distinct patterns of expression of transcripts of polysaccharide-related enzymes in stem peels during fibre elongation and cell wall deposition. However, due to inherent technical and biological limitations, it is known that in many circumstances, abundance of transcripts and proteins for a given gene may not be highly correlated 1011. This well-established limitation on the biological relevance of transcriptome analysis led us to complement our previous work with a survey of the proteins present in developing flax fibres during the onset of secondary wall deposition. This is similar to a proteomics approaches used to study secondary cell wall development of other cell types in other species 1213141516. For this study of the proteome, we also increased the specificity of our analysis by extracting proteins from phloem fibres that had been individually dissected from the snap point of growing stems, and comparing their abundance to proteins in the surrounding, non-fibre cells of the cortex from the same stems. The objective of this study is therefore to identify those proteins that contribute to the interesting pattern of cell wall deposition in flax fibres.

Results and discussion

Separation of fibre and non-fibre proteins

To increase our understanding of the proteins that contribute to the unique properties of flax bast fibres, we extracted proteins from ultimate fibres (i.e. individual cells) dissected from the snap-point region of vegetative stems (21–24 days post germination) (Figure 1). The snap-point is the stem region in which secondary wall deposition begins 7. We also collected the surrounding non-fibre cells (consisting predominantly of parenchyma, sieve elements, and companion cells) from the cortex of the snap-point. Throughout the remainder of this report, will refer to the ultimate bast fibres we collected from the snap-point as simply "fibres", and the surrounding, non-fibre cells of the cortex as the "non-fibre fraction". By labelling proteins from fibres and the non-fibre fraction with contrasting fluorescent dyes, and separating the mixture of the two samples simultaneously using 2D gel electrophoresis (DiGE), we were able to identify proteins that were more abundant in fibres as compared to the non-fibre fraction (Figure 2).

Figure 1

A typical flax plant at the time of fibre extraction

A typical flax plant at the time of fibre extraction. (A) The 3 cm region of the stem from which fibres were dissected is indicated by the bracket. (B) Detail of a transverse section of fresh stem tissues at the time of harvest. This hand section was obtained from just below the snap-point to demonstrate the arrangement of tissues within the stem, i.e. transverse sectioning was not used when obtaining tissues for protein analysis. A bracket indicates the region of the cortex from which the fibre and non-fibre fractions would be obtained. The position of representative fibres within the cortex is shown by arrowheads. The scale bar is 100 μm. (C) Stem tissues during dissection. Fibres from which surrounding, non-fibres cells been partially removed are indicated by arrowheads. A fully dissected fibre, comprising a single cell is indicated by the arrow. This fibre is representative of the cells from which proteins were extracted. The scale bar is 100 μm.

Figure 2

Representative analytical DiGE gel

Representative analytical DiGE gel. Proteins extracted from fibre and surrounding non-fibre tissues were fluorescently labeled with red and green dyes, respectively, and were mixed then separated simultaneously using 2D gel electrophoresis. Labels correspond to protein spot numbers used in Table 1 and in the text. The pH range of the first dimension separation is from 3 (left) to 10 (right).

In each of four replicate gels, we detected an average of 1850 distinct protein spots from fibres, and 1695 spots from the non-fibre fraction. In total, 558 protein spots differed in fluorescent signal intensity by at least 1.5 fold (p ≤ 0.05) between the samples, with 246 spots (13% of total detected) enriched in fibres and 312 spots (18% of total) enriched in the non-fibre fraction (Figure 3). The distinctive protein profiles of fibres and the non-fibre fraction were also evident from visual inspection of the DiGE gel image (Figure 2). Phloem fibres therefore appear to express a complement of proteins that is distinct from surrounding cell types in the stem.

Figure 3

Frequency distribution of mean intensity ratios for all spots

Frequency distribution of mean intensity ratios for all spots. A mean ratio near 1 meant the spot was found in equal abundance in both tissues; spots represented to the right of this point on the axis had higher signal intensity in fibre tissues, while spots represented to the left were more intense in non-fibre tissues. The grey and black regions of each bar show the portion of spots for which p > 0.05 and p ≤ 0.05, respectively, in a t-test of the significance of differences in intensity between fibre and non-fibre tissues.

Protein identification by LC/MS

We picked 190 protein spots that were enriched in fibre samples for identification by mass spectrometry. Spots were selected based on criteria of large spot volume, high fold-enrichment of signals, and well-focused spot morphology. For comparison, we also selected an additional 50 spots that were enriched in non-fibre fractions or that were similarly abundant in both types of protein samples. Although the patterns of fold-enrichment that we report were reproducible within the statistical parameters indicated (Table 1), individual ratios should not be extrapolated quantitatively to whole proteins, in part because some proteins may be represented by more than one spot.

Table 1

Protein identities based on peptide matches to Genbank protein databases

fold enrich.^b

spot ID#

func. cat.^a

protein identity

Genbank ID

fibre

non-fibre

p-value^c

Mowse score

pep. count^d

C&E

aconitate hydratase

4586021

1.5

0.14

C&E

aconitate hydratase

4586021

1.5

0.08

C&E

β-amylase

1771782

8.8

< 0.01

C&E

ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

168312

1.5

0.25

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

168312

2.0^e

0.08

180

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

1834444

6.1

< 0.01

129

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

2687483

5.4

< 0.01

130

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

6983900

2.9

< 0.01

232

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

1817560

3.3

< 0.01

250

C&E

enolase

9581744

1.1

0.65

265

C&E

enolase

8919731

1.1

0.93

158

C&E

enolase

9581744

3.4

0.02

206

C&E

ribulose-1,5-bisphosphate carboxylase, large subunit

4098530

2.8

0.04

103

C&E

fumarate hydratase

108708038

2.5

0.01

C&E

fumarate hydratase

15226618

1.6

0.33

100

C&E

6-phosphogluconate dehydrogenase

2529229

1.5

0.19

100

C&E

citrate synthase

11066954

3.7

< 0.01

123

C&E

phosphoglycerate kinase

1161600

1.2

0.56

257

C&E

phosphoglycerate kinase

92872324

1.7

0.06

426

C&E

ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

66735801

C&E

rubisco activase

13430332

6.1

< 0.01

C&E

rubisco activase

170129

5.2

< 0.01

C&E

phosphoglycerate kinase

3328122

2.9

0.02

250

C&E

fructose-bisphosphate aldolase

15227981

1.1

0.82

155

C&E

fructose-bisphosphate aldolase

20204

2.4

0.03

102

C&E

fructose-bisphosphate aldolase

15227981

1.1

0.6

116

C&E

fructose-bisphosphate aldolase

20204

1.3

0.04

177

C&E

rubisco activase

4261547

2.2

0.03

C&E

succinate-CoA ligase

15225353

2.3

0.02

253

C&E

glyceraldehyde-3-phosphate dehydrogenase

120666

2.6

0.01

C&E

glyceraldehyde-3-phosphate dehydrogenase

3023813

1.1

0.49

C&E

glyceraldehyde-3-phosphate dehydrogenase

74419004

3.8

< 0.01

215

C&E

malate dehydrogenase

18297

1.6

0.17

241

C&E

malate dehydrogenase

18297

1.4

0.26

138

C&E

malate dehydrogenase

10334493

3.3

< 0.01

296

C&E

fructokinase

31652274

6.7

< 0.01

142

C&E

fructokinase

31652274

2.2

< 0.01

154

C&E

kinase/ribokinase, potential fructokinase

15224669

0.01

208

ATP

AAA-ATPase

86212372

1.6

0.24

322

ATP

ATPase, transitional endoplasmic reticulum

7378614

1.2^e

0.65

101

ATP

vacuolar proton-ATPase

50251203

2.6

0.02

585

ATP

ATP binding

15221770

0.87

100

ATP

F1 ATPase

12986

1.6

0.05

143

ATP

ATP synthase β subunit

21684923

2.0^e

0.08

192

ATP

ATP synthase β subunit

19685

0.99

675

ATP

ATP synthase β subunit

56784991

1.8

0.06

307

ATP

F1-ATPase gammma subunit

303626

0.66

105

ATP

vacuolar V-H⁺ATPase subunit E

5733660

1.8

0.01

106

ATP

vacuolar V-H⁺ATPase subunit E

5733660

1.1

0.82

100

CWP

β-galactosidase

115437888

8.4

< 0.01

CWP

β-galactosidase

3641863

8.9

< 0.01

CWP

β-galactosidase

3641863

5.4

< 0.01

105

CWP

β-galactosidase

3641863

8.8

< 0.01

CWP

β-galactosidase

34913072

9.3

< 0.01

CWP

MUCILAGE-MODIFIED 4

42562732

4.1

< 0.01

CWP

rhamnose biosynthetic enzyme

108707484

6.6

< 0.01

100

CWP

phosphoglucomutase

12585309

1.8

0.15

170

CWP

phosphoglucomutase

6272281

3.7

0.02

122

CWP

UDP-glucose pyrophosphorylase

6136112

1.4

0.37

CWP

UDP-glucose pyrophosphorylase

82659609

3.5

0.01

166

CWP

UDP-glucose pyrophosphorylase

9280626

1.6^e

0.1

129

CWP

β-galactosidase

3641863

1.2

0.51

CWP

NAD-dependent epimerase/dehydratase (UXS6)

15226950

6.1

< 0.01

109

CWP

UDP-glucose 4-epimerase

12643850

1.1

0.84

101

CWP

GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase

12324315

2.3

< 0.01

155

104

CWP

dTDP-D-glucose 4,6-dehydratase-like

50253123

< 0.01

Met synthase

77556633

< 0.01

222

Met synthase

8439545

2.2

< 0.01

105

S-adenosyl-L-homocysteine hydrolase

1710838

1.6^e

0.1

174

serine hydroxymethyltransferase

11762130

2.2

0.02

129

Met adenosyltransferase

37051117

2.1

0.02

MemT

GDP dissociation inhibitor

8439465

0.13

212

MemT

GDP dissociation inhibitor

8439465

1.9

0.08

158

MemT

K⁺channel β-subunit

15219795

8.6

0.01

132

102

MemT

34 kDa outer mitochondrial membrane porin-like protein

83283993

1.7

103

MemT

36kDa porin I

515358

3.9

< 0.01

104

C&S

myosin heavy chain

108710464

2.5

0.05

C&S

myosin heavy chain

T00727

3.6

0.01

C&S

dynamin central region

92891191

3.1

0.09

C&S

dynamin-like

21593776

0.77

143

C&S

β-tubulin

295851

1.8

0.06

161

C&S

tubulin/FtsZ family, GTPase domain

62734655

1.7

0.06

367

C&S

actin

32186910

3.1

0.01

281

C&S

actin

15242516

1.5

0.21

459

P&AA

elongation factor EF-2

6056373

2.5

0.02

P&AA

ClpC protease

4105131

1.2^e

P&AA

ClpC protease

18423214

1.7

0.06

286

P&AA

HSP 90

1708314

1.7

0.01

312

P&AA

HSP 70-3

38325815

1.9

0.08

404

P&AA

HSP 70

62733235

1.7

0.11

612

P&AA

HSP 70

22636

0.04

100

P&AA

chaperonin CPN60-1

108706134

2.7

0.04

139

P&AA

chaperonin CPN60-1

108706134

1.5

0.04

327

P&AA

HSP 60

16221

2.1

0.02

140

P&AA

eukaryotic elongation factor 1A

24371059

2.3

0.02

227

P&AA

26S protease regulatory subunit

1709798

2.1

< 0.01

P&AA

translation initiation factor eIF-4A

475221

1.6

0.09

262

P&AA

26S proteasome subunit P45

92870338

1.9

0.11

P&AA

aminomethyltransferase

3334196

3.7

< 0.01

P&AA

elongation factor-1 alpha

396134

1.2

0.5

P&AA

glutamine synthetase

121341

1.7^e

0.26

119

P&AA

P0 ribosomal protein

1143507

2.5

< 0.01

155

P&AA

glutamate-ammonia ligase

99698

1.2^e

0.5

114

P&AA

eukaryotic translation initiation factor 5A

8778393

0.04

misc

nucleolar protein NOP5

108708132

1.4

0.37

misc

ferric leghemoglobin reductase

5823556

1.6

0.2

124

misc

calreticulin

3288109