Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen

Journal of Integrative Plant Biology 2011, 53 (10): 835–844

Research Article

Gene Expression Profile Changes in Germinating Rice ∗

Dongli He1 , Chao Han1,2 and Pingfang Yang1 1 Key

Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China 2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China ∗ Corresponding author Tel(Fax): +86 27 8751 0956; E-mail: [email protected] Available online on 12 September 2011 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/j.1744-7909.2011.01074.x

Abstract Water absorption is a prerequisite for seed germination. During imbibition, water influx causes the resumption of many physiological and metabolic processes in growing seed. In order to obtain more complete knowledge about the mechanism of seed germination, two-dimensional gel electrophoresis was applied to investigate the protein profile changes of rice seed during the first 48 h of imbibition. Thirtynine differentially expressed proteins were identified, including 19 down-regulated and 20 up-regulated proteins. Storage proteins and some seed development- and desiccation-associated proteins were down regulated. The changed patterns of these proteins indicated extensive mobilization of seed reserves. By contrast, catabolism-associated proteins were up regulated upon imbibition. Semi-quantitative real time polymerase chain reaction analysis showed that most of the genes encoding the down- or upregulated proteins were also down or up regulated at mRNA level. The expression of these genes was largely consistent at mRNA and protein levels. In providing additional information concerning gene regulation in early plant life, this study will facilitate understanding of the molecular mechanisms of seed germination. Keywords:

rice seed; two-dimensional gel electrophoresis; matrix assisted laser desorption; ionization-time of flight; semi-quantitative real time polymerase chain reaction; germination; proteomics; imbibition. He D, Han C, Yang P (2011) Gene expression profile changes in germinating rice. J. Integr. Plant Biol. 53(10), 835–844.

Introduction Rice seed is the staple food for more than half of the world’s population, so its formation and subsequent germination have attracted extensive research. Seed germination is one of the most complex physiological processes in both rice and general plant growth and development. Large amounts of physiological and biochemical studies have been performed in order to elucidate the mechanism of seed germination (North 2010; Penfield 2009), but there remains a long road ahead before we obtain full understanding of its intricacies. Under optimal germination conditions, the rice seed weights increased rapidly during the first 20 h imbibition (phase I), and then experienced a stable stage until 50 h (phase II), after that, the seed weights continued to increase (Yang et al.

2007). The water uptake restores the metabolic activity of the seed from the physiological quiescent status and leads to extensive physiological and biochemical changes (Bewley 1997), including carbohydrate metabolism, signal transduction, gene expression and regulation of redox homeostasis. The embryo and endosperm play different roles in rice seed germination. The embryo contains most of the seed’s genetic information and development abilities. Upon imbibition, the embryo produces the phytohormone gibberellic acid, which is perceived by the aleurone layer initiating a signaling cascade that leads to synthesis and release of a variety of hydrolytic enzymes into the starchy endosperm for the degradation of storage compounds (Bethke et al. 1997). Degradation of the reserves will supply energy and carbon sources to the developing embryo for seedling establishment. In addition to  C

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gibberellic acid (GA), ABA is another phytohormone that plays a central role in the regulation of seed germination. ABA is accumulated at the later stage of seed maturation (Leung et al. 1998) and affects seed development, maturation and dormancy in many plant species (An and Lin 2011; Fkelstein 2008). Following the genome completion of rice subspecies indica and japonica (Yu et al. 2002; Goff et al. 2002), several extensive studies have examined the effects of gene expression and gene function on transcript level. However, transcription seems not to be required for seed germination (Rajjou et al. 2004; He et al. 2011). Therefore, studies at protein level may be more valuable in clarifying gene’s function, because protein is the final product of gene and directly executes the physiological function. Indeed, with protein isolation and sequencing methods improving rapidly over the last two decades (May et al. 2011), an increasing number of proteomic studies have been performed to analyze seed germination (Gallardo et al. 2002; Fu et al. 2011; Ostergaard et al. 2004; Sheoran et al. 2005). Recently, Sun and his coworkers applied a proteomic approach to examine the role of GA and ABA in the modulation of protein expression levels during rice (Oryza sativa cv. Dongjin) seed germination, revealing that proteins in the embryo rather than the endosperm are sensitive to exogenous phytohormones (Kim et al. 2008). They then utilized two-dimensional gel electrophoresis (2-DE) in conjunction with mass spectrometry to unravel the changes in embryo proteins of germinating rice seeds (Kim et al. 2009). Yang and his coworkers employed a 2-DE proteomics approach to study whole germinating rice (O. sativa cv. 9311) seeds. In this study, 148 differentially expressed proteins were identified during the 72 h germination process, which revealed that the degradation of storage proteins occurred mainly in germination phase II (Yang et al. 2007). Recently, we utilized 1-DE in conjunction with LC-MS/MS to analyze the 24 h imbibition seed and displayed a protein profile including 673 proteins. Through the profiling, we constructed a model of the metabolic and regulatory pathways for germinating rice seeds at phase II (He et al. 2011). Since phase II is significant for seed germination, it is important to explore the changes in gene expression in seeds during this period. In this study, we applied comparative proteomic techniques to further study changes in the protein profile of rice seeds within 48 hours of imbibition. Thirty-nine differentially expressed proteins were identified. Using semi-quantitative real time-PCR (qRT-PCR), we showed the expression of the genes encoding 10 of these differentially displayed proteins were basically consistent at mRNA and protein level. These results may help us to understand the style of gene expression during seed germination and the manner in which expression style affects germination.

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Results and Discussion Protein profile of rice seed and its changes in the imbibition process Given suitable environmental conditions (such as water, atmosphere and temperature), seeds will germinate. In our study, the peeled off rice seeds began to germinate after one day’s imbibition. Most of the rice seeds germinated after 48 h imbibition (Figure 1). See protein at different stages of imbibition (0 h, 12 h, 24 h and 48 h) was extracted and subjected to 2DE analysis in parallel. Because most plant proteins are acidic, in order to magnify the discrepancy, isoelectric focusing was always performed with a range of pH 4–7, however, we found that even so, fewer proteins exited out of this span. So the first dimensional electrophoresis was run in the range of pH 3–10. After CBB R-250 staining, each gel showed more than 1 000 protein spots with the mass weight range of 14–120 kDa (Figure 2A). All experiments were carried out in triplicate. Comparative analysis of the 2-DE maps was performed using ImageMaster 2D Platinum software. The proteome profiles were much alike in general (Figure 2), but 49 proteins were detected as having changed more than 2-fold in abundance during the imbibition process. Among these proteins, 25 were down-regulated (Figure 2A) and 24 were up-regulated including the induced proteins (Figure 2D). The changed protein levels show the dynamic variation of rice seed germination and may play important roles in this complicated physiological process.

Identification of the differentially expressed protein spots All the 49 changed proteins were analyzed through MALDITOF MS and database searching. According to the criteria described previously (Yang et al. 2007), 39 proteins including 19

Figure 1. Germination of rice seeds. Rice (Oryza sativa L. japonica. cv. Nipponbare) seeds were dehulled and imbibed in distilled water (changing the water every 24 h) at 26 oC

for 0 h, 12 h, 24 h and 48 h.

Gene Expression Changes in Rice 837

Figure 2. 2-DE maps of the rice during imbibition. The range of pH 3–10 was applied for the first dimension and 12% gel was used for the second dimension. Arrows indicate the differentially displayed proteins (variation >2 fold). D means down-regulated, U means up-regulated.

down-regulated and 20 up-regulated types were identified (Table 1 and 2). The success rate of identification was 79.6%. More accurate annotation of the rice genomic sequence will increase the identification rate. Among the down-regulated proteins, there are storage proteins and seed development- and desiccation-associated proteins. The former group included glutelin (spots D7, D8 and D9), globulin (spots D6 and D16) and seed allergen (spot D23), degradation of the storage proteins also result in the accumulation of some fragments of globulin (spots U16, U22 and U23). The storage proteins are important nutrient contents in cereal seed. These proteins were synthesized in the process of seed development, and reserved in mature rice seeds. While in the germination process, they were degraded as the sources of sustenance and energy for seedling growth. The latter group included early embryogenesis abundant protein (spots D12 and D13), late embryogenesis abundant protein (LEA; spots D10, D24 and D25) and ABA-induced protein (spots D11 and D17).

At the late stage of seed maturation, abscisic acid-induced protein (spots D11 and D17), LEA and some stress related proteins (spots D3 and D21) play important roles in keeping the seed alive while undergoing desiccation. These proteins contribute a great deal to seed development and maturation. When it comes to the germination process, they are not as important as in maturation. Previous studies have also shown that these proteins are rapidly degraded upon imbibition (Jiang and Kermode 1994; Han et al. 1996). The change patterns of these proteins showed an extensive mobilization of the reserves. We have examined the content of free amino acids of germination rice seeds within 72 h imbibition (Figure 3), showing that the content increased slightly during the first 24 h, then decreased and kept constant at about 0.6%. This implied that during seed germination, rapid release of free amino acids from endogenous proteins was important for new function, proteins synthesis and other physiological requirements.

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Table 1. Identification of the down-regulated proteins through MALDI-TOF MS No.

Exp. MW(kD)/pI

Rice protein name

D3

110.0/5.66

orthophosphate dikinase

21

103.59/5.98

BAA22420

D4

97.0/4.42

hypothetical protein

22

48.32/4.85

EEE51235

D5

91.3/5.72

putative seed maturation protein

26

92.27/5.37

BAD25449

D6

57.6/6.92

globulin-like protein

22

52.38/6.78

AAM33459

D7

45.0/6.42

glutelin type I precursor

25

56.78/9.09

CAA29149

D8

45.0/7.02

glutelin type I precursor

25

56.78/9.09

CAA29149

D9

45.0/8.12

glutelin type I precursor

25

56.78/9.09

CAA29149

D10

43.2/4.05

putative Late embryogenesis abundant protein D-34

33

27.91/4.19

BAD61622

D11

37.2/4.92

Putative abscisic acid-induced protein

49

27.06/4.95

NP_001049030

D12

42.0/6.01

putative early embryogenesis protein

19

58.35/8.72

BAD08864

D13

36.4/5.86

putative early embryogenesis protein

23

58.35/8.72

BAD08864

D16

29.5/5.62

putative globulin (with alternative splicing)

13

63.85/8.35

AAS07324

D17

25.4/4.91

Putative abscisic acid-induced protein

34

27.06/4.95

NP_001049030

D20

20.3/6.52

auxin-binding protein precursor

39

22.88/6.04

AAX81926

D21

17.0/5.91

putative cold regulated protein

50

17.59/5.43

AAT47029

D22

16.0/6.01

OSJNBa0018J19.13

38

17.29/5.44

CAE04446

D23

15.8/6.53

seed allergen RA17 storage protein

36

17.44/6.92

NP_001059184

D24

15.2/4.86

putative late embryogenesis abundant protein LEA14-A

64

16.11/5.00

NP_001042461

D25

15.0/5.35

Emp1:

37

10.16/5.57

CAA44836

Uptake of water by the dry mature seed is the prerequisite for germination. With the influx of water, the quiescent seeds enter into a metabolically active stage. Some of the first changes upon imbibition are the resumption of respiratory activity, the reestablishment mitochondria function from the germination progress (Howell et al. 2006), and the increased release of carbon dioxide over a short period of time (He et al. 2011). In accordance with this, the alpha-amylase (spot U10) rapidly increased its abundance after 24 hours’ imbibition. Some other proteins involved in the glycolysis pathway, such as pyruvate orthophosphate dikinase (spot U1), fructokinase (spot U15) and phosphoglycerate kinase (spot U12) were also detected as upregulated proteins. The tricarboxylic acid (TCA) cycle plays key role in maintaining energy supply for seed germination. However, we did not detect the changes in the enzymes involved in TCA cycle, implying that the TCA cycle-associated enzymes existed in the mature dry seed in inactive form, where upon imbibition, they were activated, thereafter catalyzing the degradation of carbohydrates (Salon et al. 1988). Although proteins of the TCA cycle (succinyl-CoA ligase and cytoplasmic malate dehydrogenase) have also been identified as stably accumulated materials during the germination process (Sun et al. 2009), the expression variation seems not apparent (less than two fold). At the early stage of germination, the structures of seeds restrict gas diffusion, which results in an internal deficiency of oxygen. This oxygen deficiency may result in more pyruvate production than can be utilized for activities of the TCA cycle and the electron transport chain. The redundant pyruvate may

Cov. (%)

Theo. MW(kD)/pI

Accession No.

be degraded through other ways. Pyruvate decarboxylase (PDC, U3), which was up-regulated in our studies, catalyzes the irreversible conversion of pyruvate to acetaldehyde and CO 2 . Acetaldehyde can be converted into acetate and then enter into other pathways (Gass et al. 2005). We confirmed the aerobic respiration efficiency of the early stage of germination was very low and increased sharply during 48 h to 72 h (He et al. 2011), which implied the seeds’ restrictions were gradually removed along with imbibition and making it possible to absorb more oxygen into the cells. It also indicated anaerobic respiration might exist as the major pathway to provide enough energy for various metabolisms at the early stages of germination. Germination not only consumes reserves, but also bestows the plant with defenses and rebuilds the plant’s morphology. Allergenic proteins (U24 and D23) were detected in our results, proteins which belong to the alpha-amylase inhibitor (AAIs) and seed storage (SS) protein subfamilyies which are mainly present in the seeds of a variety of plants. AAIs play an important role in the natural defenses of plants against insects and pathogens and impede the digestion of plant starch and proteins by inhibiting digestive alpha-amylases and proteinases (Marchler-Bauer et al. 2011). The distinct expression patterns indicated they executed at different stage. It is known that allergenicity of rice is partly dependent on globulin and albumin fraction proteins (Shibasaki et al. 1979). We also found some globulin fragments (U16, U22 and U23) were up-regulated in seed imbibition, such as the allergens in rice seeds, this degradation product might not only supply substance but also

Gene Expression Changes in Rice 839

Table 2. Identification of the up-regulated proteins through MALDI-TOF MS No.

Exp. MW(kD)/pI

Rice protein name

Cov. (%)

Theo. MW(kD)/pI

Accession No.

U1

91.6/5.56

cytosolic pyruvate orthophosphate dikinase

35

97.25/5.42

CAA06247

U2

77.5/6.27

OSJNBb0059K02.15

31

64.15/6.83

CAE04505

U3

75.8/6.02

pyruvate decarboxylase

26

66.05/5.86

BAC20138

U5

64.0/5.96

putative UDP-glucose dehydrogenase

24

53.44/5.79

AAK16194

U6

63.4/6.21

putative UDP-glucose dehydrogenase

27

53.44/5.79

AAK16194

U9

57.2/6.01

dTDP-glucose 4,6-dehydratase

22

44.82/5.85

BAB85329

U10

54.6/4.92

alpha-amylase

28

47.90/5.06

CAA34516

U11

47.2/5.95

S-adenosyl-L-methionine synthetase

33

42.77/5.68

AAC05590

U12

46.8/6.31

putative cytosolic phosphoglycerate kinase 1

29

42.31/6.19

BAD45421

U13

46.6/6.90

Putative phosphoserine aminotransferase

26

45.30/8.53

AAM51827

U14

46.4/5.86

Similar to Globulin 1 (Fragment)

23

57.92/8.72

NP_001060907

U15

35.8/4.98

putative fructokinase

26

35.39/5.02

NP_001060837

U16

31.4/6.69

Fragment of globulin-like protein

14

U17

31.4/6.81

33-kDa secretory protein

31

30.49/8.20

AAC36744

U18

28.4/4.73

14–3-3-like protein

33

28.70/4.78

NP_001061856

U19

25.4/5.01

OSJNBb0118P14.11

36

33.67/4.91

CAD40673

U20

25.0/6.71

Unnamed protein

27

20.27/6.60

U22

17.8/4.43

Fragment of putative globulin protein

13

U23

16.2/4.58

Fragment of putative globulin protein

14

U24

14.8/6.82

allergen RAG2 precursor

37

17.73/8.58

U24

14.8/6.82

allergen RAG2 precursor

37

17.73/8.58

NP_001059185

D23

15.8/6.53

seed allergen RA17 storage protein

36

17.44/6.92

NP_001059184

improve defense for seedling growth. Within the germination process, the cell signal transduction pathways (U18), cell wall biosynthesis (Johansson et al. 2002; U6), nucleotide sugars metabolism (U9), amino acid biosynthesis (U13) related proteins accumulated quickly and the metabolic and regulatory network began to recover. Modifications exist at different gene expression levels. In our results, it is interesting to discover that some protein spots were identified to have the same accession number, but they didn’t share an identical molecular weight and/or isoelectric point, such as spots D7, D8, D9 and U5 and U6, indicating that modifications (eg. phosphorylation) might have occurred in some proteins, so the isoelectric points were changed. D12, D13 and U22, U23 may undergo alternative mRNA splicing at post transcript level. It is also possible they were processed or degraded from the same protein. This phenomenon was also observed in other proteome experiments (Yang et al. 2007; Kim et al. 2008).

AAM33459

AAA72362 AAS07324 AAS07324 NP_001059185

regulated by GA or ABA signal. Promoters of these 10 genes were obtained from http://rapdb.dna.affrc.go.jp/ and GA/ABA responsive cis-elements within those DNA sequences were identified with PLACE and PLANTCARE softwares. All promoters were defined as 2 kb sequences before the translation start code ‘ATG’ of genes, except the pOs12g0529400(453 bp),

Cis-elements analysis of the promoters of selected genes Many GA- and ABA-responsive functional proteins are involved in the regulation of seed germination. From the genes encoding the differentially displayed proteins, we selected 10 candidate genes (Figure 4A). to explore whether they were

Figure 3. Free amino acid analysis of rice seeds during imbibition by Hitachi model L-8800 amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan) using ninhydrin methods. Values were means with the SD (n = 3); ∗ P < 0.05.

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Figure 4. Quantitative changes of the expression of 10 selected genes at protein (A) and RNA (B) levels during rice seed imbibition. The protein intensity was detected by ImageMasterTM 2D Platinum software. mRNA levels were quantified by semi-qRT-PCR using a CFX96 real time system and the 2−C T Method. The y-axis is the relative quantities of gene expression at protein (A) or mRNA (B) level. The Values were means with the SD (n = 3); P < 0.01.

pOs08g0136700(1556 bp) and pOs08g0430500 (1608 bp) for the small gaps between the gene and the adjoining upstream gene. Quantity, location and core sequence of each cis-element were determined and summarized (Table 3). Analysis indicated most the promoters for other genes had core sequences of ABRE and GARE cis-elements. Some promoters had more than two corresponding sites, such as the Os06g0341300 promoter (encoding putative late embryogenesis abundant protein, D34) containing 10 ABRE sites, which implied that those genes are more likely regulated by ABA during seed development and maturation (Gomez-Porras et al. ´ 2007). The promoter of pOs12g0529400 (encoding an auxinbinding protein precursor) had no cis-element corresponding to the known hormone.

Auxin-binding protein (ABP) is involved in the auxin transport within the cell and can trigger early modification of ion fluxes across the plasma membrane in response to auxin (David et al. 2007). ABP1 expression had been confirmed to be regulated by IAA and ABA associating with the cambium periodicity in Eucommia ulmoides Oliv. (Hou et al. 2006), indicating that there might be other special sites on the promoter for the recognition of corresponding trans-regulation factors. As an important regulating factor, 14–3-3 protein (U18, Os08g0430500) is involved in many signaling pathways (Schoonheim et al. 2009). Promoter analysis indicated the pOs08g0430500 had 7 ABRE and 8 GARE sites. It is possible that the promoters might be coordinately regulated by these two hormones. The analysis also demonstrated some promoters also had

Gene Expression Changes in Rice 841

Table 3. ABA and GA responsive cis-elements within the 10 selected promoters (2 kb upstream from ATG of the representative genes or genome sequence gaps) identified through Place (http://www.dna.affrc.go.jp/PLACE/signalscan.html) and plantcare (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/).

Protein ID

ABA

Rice gene number (Oryza sativa cv.)

D5

Os02g0530600

D10

Os06g0341300

Core sequence

GA

Number (site, strand)

ACGTG

4(−317,+; −1197, +; −1753,+;−1860,-)

MACGYGB

2(−1566,-; −1817,+)

ACGTG

9(−898,-;857.-;−810,-;−246,+;−217,-;

Core sequence

Number (site, strand)

CCTTTT

3(−448,+; −802,+; −806,+)

CCTTTT

1(−1739,-)

TCTGTTG

2(−1110,+; −1139,+)

CCTTTT

2(−868,+;−1426,-)

201, +;- 194,-; −116,+; −62,+;) D11

D12

Os03g0159600

Os08g0127900

D20

Os12g0529400

D21

Os05g0218100

U10

Os02g0765600

U11

Os01g0323600

MACGYGB

1(−183,+;)

ACGTG

4(−1269,+;−1678,+; −1761,-;−1917,+)

CCGCCGCGCT

2(−937,+; −1120,+)

MACGYGB

3(−1007,-; −1816,-; −1872,-)

ACGTG

4(−1976,-;−962,-;−930,-;−199,-)

MACGYGB

1(−1938,+)

ACGTG

3(−321,-; −354,-; −1947,+)

CCTTTT

2(−1636,+;−633,-)

MACGYGB

1(−1984,-;)

TTTTTTCC

1(−416,+)

ACGTG

2(−313,-; −1139,-)

CCTTTT

2(−8,+;−1788,-)

MACGYGB

1(−1519,-;)

TTTTTTCC

1(−576,-)

ACGTG

1(−155,+)

AAACAGA

1(−1287,-; −1371,+)

CCTTTT

2(−566,-;−1275,+)

TTTTTTCC

1(−1011,-)

AAACAGA

1(−1159,-)

TCTGTTG

1(−1790,+)

U17

Os08g0136700

ACGTG

1(−1404,+)

CCTTTT

1(−1354,-)

U18

Os08g0430500

ACGTG

6(−473,+; −533,+; −676,-; −1614,-;

CCTTTT

5(−416,+; −638,+;

−1735,-; −1872,+) MACGYGB

1(−629,+)

cis-elements responding to other hormones’ (such as auxin, JA and SA; data not shown) signals, and suggested those genes might be co-regulated by two or more hormones and involved in the cross-talk of signal transduction.

Semi qRT-PCR analysis of expression profile of the selected genes To further show the expression style of the genes encoding the differentially displayed proteins, semi qRT-PCR were applied to examine the expression of 10 selected genes at mRNA level using specific primers (Table 4). with rice ubiquitin as the internal control. Six of the ten selected candidates are seed maturation related proteins and were down-regulated upon the imbibition, while the other 4 were up-regulated. Total RNA were extracted from rice seed embryos (after 0 h, 12 h, 24 h and 48 h imbibition) and then reverse transcripted for semi-qRT-PCR analysis. Results are presented in Figure 4B.

CCTTTT TTTTTTCC

−685,+; −669,-; −1415,-) 1(−424,+)

AAACAGA

1(−1158,+)

TCTGTTG

1(−614,-)

Of the 10 genes, the expression profiles of 9 genes at mRNA levels were fully consistent with protein levels. Some down-regulated proteins (D5, D12, D20 and D21) also had high mRNA levels in dry seeds, but decreased quickly during germination. Those mRNA might be superabundant during seed development and maturation or stocked for emergency. The mRNA levels of induced proteins (U10, U17 and U18) were significantly increased during germination. Otherwise, posttranscriptional regulation resulted in discrepant expression between protein and mRNA (such as Os01g0323600 encoding S-adenosylmethionine synthetase). S-adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-adenosylmethionine (AdoMet) from methionine and ATP. AdoMet is a main methyl group donor and plays a central role in regulatory effects on DNA transcription and chromosome structure. It is also involved in the biosynthetic pathway of many secondary metabolites (Layer et al. 2004). The mRNA levels of S-adenosyl-L- methionine synthetase were

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Table 4. Specific primers used for semi-quantitative RT-PCR analysis. Rice gene number (Oryza sativa cv.)

Forward primer (5’→3’)

Reverse primer (5’→3’)

Amplicon size (bp)

Os02g0530600

AGGTTGGCGATGTGGTTTAC

TGCGTCTGAGCAAACTATGG

Os06g0341300

CGACGCTGAGTGAACCAGT

GCAACAGGTGACATCACACG

230 bp 140 bp

Os03g0159600

AGCGGAGATGCGCAACAAGC

CGTCGCGACACGAGCTGTTT

154 bp

Os08g0127900

GTGACCGACGAGGAGATGAT

AGCTTCTGGCTGTTGAGGAC

170 bp

Os12g0529400

TGGAATGAAGGAGGTGGAAG

ACCTGGTGCGGATCATTTAC

215 bp

Os05g0218100

GGCCTCCTTCGGGTTAGTAG

CGCAAGCTGAGGAAGCCGAAGG

187 bp

Os02g0765600

CAGGGCTACGCATACATCCT

GATCACCTTGCCATCGATCT

166 bp

Os01g0323600

AAGATCCCCGACAAGGAGAT

CATACTTGAGCGGCTTGACA

183 bp

Os08g0136700

CAGTGGGTAGGAGTGGGCTA

CTGCCTCGAGTGGGAAGTAG

233 bp

Os08g0430500

CCATTGAACAGAAGGAGGAGGGTCG GCAGCAGTAGATGAGGGCACAAGG

151 bp

Os01g0328400a(UBQ5)

ACCACTTCGACCGCCACTACT

69 bp

high in dry rice seed, and showed a transient down-regulation after imbibition for 12 h, and were then further induced during germination, indicating that rapid synthesis of this synthetase is absolutely necessary for seed germination and seedlings. In summary, seed germination is a complicated and multiphase physiological process in which comprehensive biochemical and gene expressional changes occur. In addition to the storage proteins, many ABA-responsive proteins were also degraded during germination. To the contrary, many up-regulated proteins were GA-responsive. Moreover, the expression of these imbibition responsive genes was consistent at mRNA and protein level, which implied the specific regulatory style of gene expression during seed germination.

Materials and Methods Rice seeds and imbibition Rice (Oryza sativa L. japonica. cv. Nipponbare) seeds were dehulled and washed with distilled water three times, then imbibed in distilled water (changing the water every 24 h) at 26 o C on plates; statistics were performed on the seeds germination rate and seeds were collected at intervals of 0 h, 12 h, 24 h and 48 h imbibition respectively. The seeds were stored at −80 ◦ C until used for proteins extraction.

Protein extraction Proteins of seeds at different time points of imbibition were extracted as follows separately. 0.25 g seeds were ground in a precooled mortar with 2 mL cold homogenization buffer containing 20 mM Tris/HCl (pH 7.5), 250 mM sucrose, 10 mM ethylene glycol-bis(b-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA), 1 mM PMSF, 1 mM DTT, and 1% Triton X-100 (Yang et al. 2007). After homogenization, the homogenate was shifted into a microtube and centrifuged at 15 000 g for 15 min at 4 o C. The pellet was discarded, and the supernatant was mixed

ACGCCTAAGCCTGCTGGTT

with 1/4 volume 50% cold trichloroacetic acid and kept in an ice bath for 30 min. It was then centrifuged at 15 000 g for 15 min at 4 o C and the supernatant discarded. The pellets were washed with acetone 3 times, centrifuged and vacuumdried. The drying powder was solubilized in sample buffer containing 8 M urea, 4% Triton X-100, 2% ampholine pH 3.5–10 (Amersham Biosciences, Uppsala, Sweden) and 20 mM DTT.

Two-dimensional gel electrophoresis (2-DE) Two-dimensional electrophoresis was carried out as previously described (Yang et al. 2007). For the first dimension, 4% gel containing 8 M urea, 5% carrier ampholyte (one part pH 3.5–10, one part pH 5–8) and 2% NP-40 in 3 mm diameter glass tube were used. Isoelectric focusing (IEF) was performed at 200 V, 400 V, 800 V for 30 min, 15 h and 1 h respectively. After the first dimensional run, gels were incubated in equilibration buffer (0.05 M Tris-HCl pH 6.8, 2.5% SDS, 10% (v/v) glycerol and 5% 2-mercaptoethanol) for 15 min twice. The second SDS-PAGE was run in 15% resolving gels. After electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB) R-250.

Image scanning and analysis The stained gels were scanned at 300 dots per inch (dpi) resolution using a UMAX Power Look 2100XL scanner (Maxium Tech Inc., Taipei, China). The transparency mode was used to obtain a greyscale image. The image analysis was performed with ImageMasterTM 2D Platinum software (Amersham Bioscience). The optimized parameters were as followed: silency 2.5, partial threshold 4 and minimum area 50.

MALDI-TOF MS analysis and database searching Protein spots showing apparent variation (>twofold) during the period of imbibition were excised from the CBB stained gels. The proteins were then digested according to the method described before (Yang et al. 2007). After digestion, the

Gene Expression Changes in Rice 843

peptides were collected, and the gels were washed with 0.1% TFA in 50% acetonitrile for three times to collect the remaining peptides. The peptides were desalted by ZipTipC 18TM pipette tips (Millipore, Bedford, MA, USA) and cocrystallized with one volume of saturated α-cyano-4-hydroxycinnamic acid in 50% v/v acetonitrile containing 1% TFA. Tryptic peptide masses were measured with a MALDI-TOF mass spectrometer (Shimadzu Biotech, Kyoto, Japan). The acquired peptide mass fingerprinting (PMF) data were searched in the NCBI database using the Mascot software available at (http://www.matrixscience.com). The searching parameters were set as previously described (Yang et al. 2007): peptide masses were assumed to be monoisotopic, 0.5 Da was used as mass accuracy, a maximum of one missing cleavage site and modification of cysteines to carboxyamidomethyl cysteine (Cys_CAM) by iodoacetamide were considered. Oryza sativa was chosen for the taxonomic category. Only the best matches with sequence coverage no less than 12% were selected.

Extraction and analysis of free amino acids of germination seeds One hundred of de-hulled germination rice seeds from each stage were selected for analysis. The seeds were ground in a mortar added 80% ethanol (10 mL), after incubation for 30 min at 80 ◦ C and then were centrifuged at 12 000 g for 10 min at 10 ◦ C. The residue was re-extracted three times with 80% ethanol. The combined supernatants were diluted to 40 mL with 80% ethanol. An aliquot of each extract (2 mL) was concentrated in a rotary evaporator at 30 ◦ C and the residue was then dissolved in 0.2 M HCl (1.0 mL). This extract was used for free amino acids analysis. The analysis was carried out on a Hitachi model L-8800 amino acid analyzer (Hitachi Co. Ltd.,Tokyo, Japan) using ninhydrin methods as previously described (Matsuyama et al. 2009). Three independent analyses were carried out for each sample.

RNA isolation and semi-quantitative real time PCR analysis Total RNA was extracted from rice seed embryos (after imbibition of 0 h, 12 h, 24 h and 48 h) using TRIpure reagent (Bioteke China) as described by the manufacturer. Firststrand cDNA was synthesized with oligo (dT) primers using a Rever Tra Ace-a-First Strand cDNA synthesis kit (Toyobo, http://www.toyobo.co.jp/e/) according to the manufacturer’s instructions. Transcript levels of each selected gene were measured through semi qRT-PCR. The PCR solution system was in a 20 µl final volume containing 10 µl 2× iQTM SYBR Green Supermix (Bio-Rad), 200 nmol of specific forward and reverse primers, and 10 ng cDNA as template. PCR was performed on the C1000TM thermal cycler combined with a CFX96TM detection module (Bio-Rad, CA, USA). The amplifi-

cation program was as follows: 95 ◦ C for 10 min, and 45 cycles of 95 ◦ C for 10 s, 64 ◦ C for 10 s and 72 ◦ C for 30 s. The specificity of amplicons was verified by melting curve analysis (65 ◦ C to 95 ◦ C) after 40 cycles. The data were normalized to the amplication of the rice ubiquitin gene (Jain et al. 2006). For each sample, the mean value from triplicate experiments was adapted to calculate the transcript abundance. Gene specific primer sequences used for RT-PCR were listed in Table 1.

Identification of promoter region and cis-elements analysis of relevant promoter Two kilobase sequences before the translation start code ‘ATG’ of gene or genome sequence gaps of two adjoining gene in the same direction were defined as promoter regions in this report. Promoter regions were obtained from http://rapdb.dna.affrc.go.jp/ and cis-elements within those DNA sequences were identified through PLACE http://www.dna.affrc.go.jp/PLACE/signalscan.html/ and PLANTCARE http://bioinformatics.psb.ugent.be/webtools/plan tcare/html/ softwares. Numbers, location and core sequence of aimed cis-elements were summarized.

Acknowledgements We are grateful to Dr. Bing Yi in Huazhong Agriculture University for his help in qRT-PCR analysis and Ms. Li Wang for the MALDI-TOF MS analysis. This work was supported by the 100 Talents Program of the Chinese Academy of Sciences.

Received 7 Jun. 2011

Accepted 1 Sept. 2011

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(Co-Editor: Jianmin Wan)