Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses

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Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses Xiangrong Youa, b, c , Lingxia Wangb , Wenyu Liangb, d , Yonghong Gaib , Xiaoyan Wangb , Wei Chena, b,⁎ a

Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Corps, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China b College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China c Institute of Agro-food Science and Technology, Guangxi Academy of Agricultural Sciences, Nanning 530007, PR China d School of Life Sciences, Ningxia University, Yinchuan 750021, PR China

AR TIC LE I N FO

ABS TR ACT

Article history:

Two-dimensional gel electrophoresis (2-DE) was used to analyze the proteins related to

Received 6 August 2011

floral reversion in Dimocarpus longan Lour. Proteins were extracted from buds undergoing

Accepted 24 October 2011

the normal process of flowering and from those undergoing floral reversion in three

Available online 7 November 2011

developing stages in D. longan. Differentially expressed proteins were identified from the

Keywords:

ionization-time of flying-mass spectroscopy and protein database search. A total of 39

D. longan

proteins, including 18 up-regulated and 21 down-regulated proteins, were classified into

Flowering reversion

different categories, such as energy and substance metabolism, protein translation, sec-

2-DE

ondary metabolism, phytohormone, cytoskeleton structure, regulation, and stress tolerance.

Differential proteins

Among these, the largest functional class was associated with primary metabolism. Down-

Ultrastructure

regulated proteins were involved in photosynthesis, transcription, and translation, whereas

gels after 2-DE analysis, which were confirmed using matrix-assisted laser desorption/

up-regulated proteins were involved in respiration. Decreased flavonoid synthesis and upregulated GA20ox might be involved in the floral reversion process. Up-regulated 14-3-3 proteins played a role in the regulation of floral reversion in D. longan by responding to abiotic stress. Observations via transmission electron microscopy revealed the ultrastructure changes in shedding buds undergoing floral reversion. Overall, the results provided insights into the molecular basis for the floral reversion mechanism in D. longan. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Longan (Dimocarpus longan Lour.) is an important subtropical fruit tree that originated in South China. Biennial bearing is the most serious problem that affects longan fruit products. A number of factors affect D. longan fruit yield, with floral reversion as one of the most challenging problems that influence it. Both internal and external factors affect floral

reversion, such as temperature, photoperiod, light quality, light intensity, and herbicides [1,2]. The introduction of an exogenous gene even induces floral reversion [3]. Sisi Chen et al. [4] indicated that high temperature and moisture in winter are major factors that cause D. longan flowering reversion. During this process, normal flowers stop developing halfway through, and they form floral spikes with leaves. Some even revert to vegetative branches.

⁎ Corresponding author at: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China. Tel.: +86 591 83718915; fax: +86 591 83847208. E-mail address: [email protected] (W. Chen). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.10.023

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The reversion of flowering occurs when the production of vegetative structures is resumed in a meristem after floral development has been initiated. Leaf production can possibly resume at any time during flower initiation as long as the shoot apical meristem has not yet become completely determined for flower formation [5,6]. To date, flowering and reversion are explained in terms of a “balance model” with some similarities to enabler and promoter interactions. Studies on Arabidopsis, Zea mays, Glycine max, and Lycopersicon esculentum showed that a disruption in internal balance such as in hormone levels and gene and protein expression levels induces reversion [7–10]. The floral reversion mechanism in D. longan is complicated, including a series of physiological and biochemical processes. However all these processes are based on the function of different proteins. The biological meaning of a protein can be inferred from its differential presence in the buds of floral reversion and normal flowering. In the present study, anatomic and proteomic approaches were used to investigate ultrastructural changes and the differential protein expression in different stages of normal flowering and reversion in D. longan in an attempt to describe the flower development and reversion process. The results can give insights into the flowering and reversion processes of D. longan on a molecular basis.

2.

Materials and methods

2.1.

Plant materials

Bud samples were collected from 25-year-old normally developing “longyou” longan trees. Normal flowering buds from

corresponding development stages (Fig. 1A) and floral reversion buds from three development stages (Fig. 1B) were collected, immediately kept in liquid nitrogen, and stored at −80 °C for further analysis.

2.2. Sample microscopy

preparation

for

transmission

electron

Flower buds were fixed in a mixture of 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for several days at 4 °C. After washing with rinsing buffer (0.1 M phosphate buffer, pH 7.2), the samples were post-fixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1.5 h at 4 °C. They were dehydrated in a graded alcohol and acetone series, and embedded in epoxy resin 618. Ultrathin sections (80 nm) were cut with a microtome using a diamond knife. The sections on grids were stained with aqueous uranyl acetate followed by a lead electron staining solution for 5 min. The sections were observed using a JEM-1010 electron microscope and photographed.

2.3.

Preparation of total protein extraction

Proteins of longan buds were extracted according to Saravanan and Rose [11]. Briefly, 1 g of buds was ground in 4 ml of precooled homogenization buffer containing 100 mM Tris– HCl (pH 8.0), 50 mM L-ascorbic acid, 100 mM KCl, 50 mM disodium tetraborate decahydrate, 1%Triton X-100, 2% βmercaptoethanol, and 1 mM PMSF. After homogenization, mixed with 4 ml tris-buffered phenol (pH8.0), the homogenate was centrifuged at 15,000 g for 10 min at 4 °C. The phenol

Fig. 1 – Different development stages of longan normal flowering and floral reversion buds. A1–3: Normal flowering bud development stages (A1: shows vegetative lateral branch growing under flower buds at the first stage; A2–A3: show vegetative lateral branch abscised while flower buds developing at the second and third stages). B1–3: floral reversion bud development stages (show vegetative lateral branch growing under flower buds at all these three stages and flower development has been blocked).

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phase was collected and mixed with 5 volumes of 0.1 mM ammonium acetate/methanol at −20 °C for 2 h. Then it was centrifuged at 15,000 g for 20 min at 4 °C. The pellet was washed once with cold methanol and then twice with acetone containing β-mercaptoethanol. After centrifugation, the pellet was air-dried at −20 °C. The dried power was solubilized in a sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte3-10, and 40 mM DTT, and the protein content was measured according to the procedure of Bradford (Bradford 1976). The protein solution could be used for 2-DE or stored in −80 °C until used.

2.4.

1101

Two-dimensional gel electrophoresis

IEF was performed using an IPGphor IEF System (AmershamPharmacia Biotech). Approximate 1000 mg proteins were mixed with rehydration buffer (8 M urea, 4% CHAPS, 65 mM DTT, and 0.5% Ipg buffer pH 4–7, final volume of 0.8 ml), and the samples were loaded on IPG strips (24 cm, pH 4–7) respectively, and rehydrated for 12 h (20 °C, 30 V), then performed at 20 °C in a stepwise manner: 200 V (1 h), 500 V (1 h), 1000 V (1 h), gradient 8000 V (0.5 h) and finally 8000 V until the total V hours reached at least 42,000 Vh. After IEF, the strips were equilibrated in equilibration

Fig. 2 – TEM analysis of longan flower buds. (A, B) Normal flower buds; (C–F) flower buds undergoing reversion. A. Small vacuole stage, ×20,000; B. large vacuole stage, ×10,000; C. observation of cell wall dissolution and paramural bodies, × 20,000; D. fine structure of paramural bodies, ×50,000; E. observation of clear and more ER, × 25,000; F. cell structure deformation and membrane invagination, ×25,000. N nucleus; NC nucleolus; Mt mitochondria; W cell wall; V vacuole; M membrane; P paramural bodies; ER endoplasmic reticulum.

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buffer I (50 mM Tris–HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 1% DTT) for 15 min, and then in equilibration buffer II (50 mM Tris–HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 2.5% iodoacetamide) for 15 min with gentle shaking. After equilibration, strips were applied to 11% (w/v) SDS-PAGE gels and sealed with agarose sealing solution (0.5% w/v agarose in SDS buffer plus a few grains of Bromphenol Blue). Protein samples were separated by SDS gel electrophoresis with SDS buffer (25 mM Tris, 192 mM Gly, and 0.1% w/v SDS) in Ettan DALT-six System (Amersham Biosciences) at 18 °C. Electrophoresis was carried out first at 10 mA per gel for 1 h and 30 mA per gel with a maximum of 50 V for approximately 10 h. After electrophoresis, the gels were stained with CBB-R250 with gentle shaking for 2 h, then transferred to 1% (v/v) acetic acid destain with gentle shaking for about 1 d till the gel background was clear.

2.5.

Image acquisition and data analysis

CBB-stained gels were analyzed with PDQuest 2-D Analysis Software (version 7.0; Bio-Rad). Specifically, spot detection, spot measurement, background subtraction and spot matching were performed. Following automatic spot detection, gel images were carefully edited. Prior to performing spot matching between gel images, one gel image was selected as the reference gel. After automatic matching, the unmatched spots of the member gels were added to the reference gel. The amount of a protein spot was expressed as the volume of that spot which was defined as the sum of the intensities of all the pixels that make up the spot. In order to correct the variability due to CBB-staining and to reflect the quantitative variations in intensity of protein spots, the spot volumes were normalized as a percentage of the total volume in all of the spots present in the gel. The resulting data from image analysis were transferred to Quantity One software for querying protein spots showing quantitative or qualitative variations. Statistical analysis of the data was performed using Microsoft Excel 2003. SD was calculated from three spots from different gels and used as error bars.

2.6.

In-gel tryptic digestion

The protein spots were manually excised from the CBB-stained gels and then transferred to V-bottom 96-well microplates loaded with 100 μl of 50% ACN/25 mM ammonium bicarbonate solution per well. After being destained for 1 h, gel plugs were dehydrated with 100 μl of 100% ACN for 20 min and then thoroughly dried in a SpeedVac concentrator (Thermo Savant, USA) for 30 min. The dried gel particles were rehydrated at 4 °C for 45 min with 2 μl/ well trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate, and then incubated at 37 °C for 12 h. After trypsin digestion, the peptide mixtures were extracted with 8 μl extraction solution (50% ACN/0.5% TFA) per well at 37 °C for 1 h. Finally, the extracts were dried under the protection of N2.

2.7.

Protein MALDI-TOF/TOF analysis

The peptides were eluted with 0.8 μl matrix solution (α-cyano-4hydroxy-cinnamic acid (CHCA, Sigma, St. Louis, MO, USA) in 0.1% TFA, 50% ACN) before they were spotted on the target plate. Samples were allowed to air-dry and analyzed by 4700 MALDI-TOF/ TOF Proteomics Analyzer (Applied Biosystems, Foster City, CA,

USA). The UV laser was operated at a 200 Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 20 kV. Myoglobin digested by trypsin was used to calibrate the mass instrument with internal calibration mode. All acquired spectra of samples were processed using 4700 Explore™ software (Applied Biosystems) in a default mode. Parent mass peaks with mass range of 700–3200 Da and minimum S/N 20 were picked out for tandem TOF/TOF analysis. Combined MS and MS/MS spectra were submitted to MASCOT (V2.1, Matrix Science, London, U.K.) by GPS Explorer software (V3.6, Applied Biosystems) and searched with the following parameters: NCBInr database (release date: 2006.03.18), taxonomy restrictions to viridiplantae, trypsin digest with one missing cleavage, none fixed modifications, MS tolerance of 0.2 Da, MS/MS tolerance of 0.6 Da, and possible oxidation of methionine. Known contaminant ions (keratin) were excluded. Total of 4,736,044 sequences and 1,634,373,987 residues in the database were actually searched. MASCOT protein scores (based on combined MS and MS/MS spectra) of greater than 66 were considered statistically significant (more than 2-fold and p<0.05). The individual MS/ MS spectrum with statistically significant (confidence interval>95%) best ion score (based on MS/MS spectra) was accepted. To eliminate the redundancy of proteins that appeared in the database under different names and accession numbers, the singleprotein member was first chosen according to corresponding theoretical and experimental MW/pI, then the highest protein score (top rank) was singled out from the multi-protein family.

2.8.

Western blot analysis

The proteins of normal flowering and floral reversion buds were prepared and suspended in a total volume of 1 ml of protein sample mixture (62.5 ml Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.001% bromphenol blue). The mixtures were centrifuged for 5 min at 14,000 g. The proteins (50 μg per lane) were separated with 12.5% SDS-PAGE and subsequently electroblotted onto a nitrocellulose membrane using a blot-transfer buffer (50 mM Tris-base, 40 mM Gly, 20% v/v methanol, and 1% v/v SDS). Duplicate blots were blocked for 2 h in TTBS (50 mM Tris–HCl, pH 8.2, 150 mM NaCl, and 0.1% v/v Tween 20) containing 5% w/v nonfat dry milk. The membrane was then incubated for 2 h with rabbit polyclonal antibody raised against 14-3-3 protein and Gibberellin 20-oxidase (GA20ox) (Santa Cruz, USA). The antibody was diluted in TTBs to a ratio of 1:500. The membrane was washed (3× 15 min) with TTBs and then incubated with a secondary goat anti-rabbit IgG conjugated with horseradish peroxidase (diluted in TTBs to a 1:5000 proportion). After the blots were washed with TTBs, immunoblot signals were detected with DAB (Boster, Wuhan China). The immunodetection image was scanned, and the intensities were quantified using Quantity software (Bio-Rad, USA).

3.

Results

3.1.

Transmission electron microscopy observations

Transmission electron microscopy analysis was performed to observe changes in cell ultrastructure corresponding to D. longan flowering reversion. The cell structure of normal flower

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buds is illustrated in Fig. 2A–B, whereas the profiles of reversion buds are shown in Fig. 2C–F. In normal flower buds, the cell walls and membrane systems (i.e., the cell membrane and the nuclear membrane) were intact. The cells contained a number of different organelles, i.e., round or oval mitochondria and small (Fig. 2A) or large vacuoles (Fig. 2B) interspaced around the nucleus. By contrast, the cell structure in floral reversion buds showed clear changes in the cell wall. In some sites, the wall had dissolved, and in the most serious

Mr

IEF pH 7

97.4 kD→

4

1103

situations, a large number of paramural bodies gave the cell wall a roughened appearance, suggesting the possibility of collapsing. The internal cell structure was also largely disorganized. Fig. 2C and D shows the structure of paramural bodies. Fig. 2E shows the presence of apparent short strands of the endoplasmic reticulum (ER), indicating an increase in the amount of ER. Cell deformation and membrane invagination, which potentially resulted from the disruption of the cell wall, are shown in Fig. 2F.

IEF pH 7

A1

B1

A2

B2

A3

B3

4

66.2 kD→ 39.2 kD→ 26.6 kD→ 21.5 kD → 14.4 kD → 97.4 kD→ 66.2 kD→ 39.2 kD→ 26.6 kD→ 21.5 kD→ 14.4 kD→ 97.4 kD→ 66.2 kD→ 39.2 kD→ 26.6 kD→ 21.5 kD→ 14.4 kD→ Fig. 3 – 2-DE gel pattern of longan bud proteins of different development stages of normal flowering and flowering reversion. A1: Normal flowering bud development stage I; A2: normal flowering bud development stage II; A3: normal flowering bud development stage III; B1: floral reversion bud development stage I; B2: floral reversion bud development stage II; B3: floral reversion bud development stage II.

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3.2. Two-dimensional gel electrophoresis (2-DE) protein profiles of floral reversion and normal flowering buds in different development stages Whole proteins from the three stages of the developing buds undergoing floral reversion and normal flowering were resolved and detected using 2-DE followed by colloidal Coomassie blue staining. Initial analyses were performed using immobilized pH gradient (IPG) strips ranging from pH 3 to 10 (data not shown). The region at pH 4 to 7 was observed on the proteome map to be a highly dense area; therefore, additional analyses with IPG strips at pH 4 to 7 were performed to improve spot resolution (Fig. 3). After 2-DE, gel images were taken and analyzed using PDQuest™ 2-D Analysis software (Bio-Rad, Hercules, CA,

USA). About 800 protein spots were detected on each gel, with the match rate of whole proteins on each stage of floral reversion and normal flowering remaining at 76% to 78%. The protein variance on the 2-D maps of the three stages of development of the floral reversion buds was concentrated on pI 5–6.5 and molecular weight 30–66.2 kDa; a similar variance was not shown on the protein maps of normal flowering buds (Fig. 4). The bud proteins on each development stage of floral reversion and normal flowering were compared. In all three development stages, the spots had differential regulation of floral reversion compared with the three normal flowering stages selected, and 58 differential spots (24 up-regulated and 34 down-regulated proteins) were found. Each differentially expressed protein spot changed in abundance more than two-fold (Fig. 5).

Fig. 4 – PDQuest analysis result of longan bud protein 2-DE gel pattern of normal flowering (A) and flowering reversion (B).

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Fig. 5 – Partial magnified pictures of differential proteins on 2-D gels of normal flowering (A) and floral reversion (B) longan flower buds.

3.3. Identification expressed protein

and

classification

of

differentially

Each of the different regulated protein spots was excised and identified using matrix-assisted laser desorption/ionizationtime of flying-mass spectroscopy. A total of 39 proteins comprising 18 up-regulated and 21 down-regulated protein spots

were identified (Table 1), with an identification rate of 65.5%. Protein spots 28 and 29 and protein spots 36 and 37 (most likely due to post-translation modifications or genetic isoforms) were identified as the same kind of protein, respectively, because one unique protein was often represented by more than one spot on the 2-DE gel. Taking this into account, 37 proteins were identified and classified into one of the seven functional categories

Spot Protein no. a change b

Protein name c

Down

Elongation factor EF-2 [Arabidopsis thaliana]

2

Down

4

gi| 6056373

Matched peptide sequences (m/z) e

Matched Theoretical Experimental Score/ Fold peptides f MW (kDa)/ MW (kDa)/ threshold i change j pI g pI h

STLTDSLVAAAGIIAQEVAGDVR (2257.29) STGISLYYEMTDESLK (1836.93) LWGENFFDPATR (1452.74) VENLYEGPLDDQYANAIR (2080.07) FSVSPVVR (890.58) ILAEEFGWDK (1207.65) DSVVAGFQWASK (1294.68) RVIYASQITAKPR (1502.74)

8

94.18/5.89

91.55/6.35

154/66

3.01

RNA binding/aconitate gi| hydratase/hydro-lyase/ 15233349 iron ion binding/lyase [Arabidopsis thaliana]

LPYSIR (748.47) ILLESAIR (914.61) QVEIPFKPAR (1184.72) INPLVPVDLVIDHSVQVDVAR (2298.36) SDDTVSMIEAYLR (1515.76) GTFANIR (778.45) FDTEVELAYFDHGGILQYVIR (2485.31)

7

98.09/5.98

89.6/6.33

91/66

2.08

Up

Putative aconitase [Prunus avium]

gi| 34851120

YYSLPALNDPR (1308.75) INPLVPVDLVIDHSVQVDVAR (2298.41) DGVTATDLVLTVTQMLR (1833.09)

3

98.82/6.01

93.5/6.23

72/66

2.07

7

Down

ATP binding/protein binding [Arabidopsis thaliana]

gi| 30685661

DGNTLLKEMQIQNPTAIMIAR (2389.46) EMQIQNPTAIMIAR (1615.93) KPEEAIDLFMVEIMHMR (2089.17) LVEGLVLDHGSR (1294.80) SEINAGFFYSNAEQR (1732.91) GIDPPSLDLLAR (1266.78) NPNSCTILIK (1102.56) DGLRSVK (774.37) QLINSGPVIASQLLLVDEVIR (2277.50)

9

58.89/5.88

59.67/6.29

272/66

3.06

8

Down

Ribulose 1,5bisphosphate carboxylase [Lagerstroemia speciosa]

gi| 21303372

DTDILAAFR (1021.59) TFQGPPHGIQVER (1465.81) GGLDFTKDDENVNSQPFMR (2170.05) DDENVNSQPFMR (1451.68) ELGVPIVMHDYLTGGFTQNTSLAHYCR (3022.53) AMHAVIDR (912.52) NHGMHFR (898.47) DITLGFVDLLR (1261.77)

8

46.38/6.99

54.56/6.61

267/66

2.30

Spots %volme variations (p < 0.05) k

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1

Accession no. d

1106

Table 1 – Identification of differential proteins in longan flower buds of floral reversion and normal flowering with MALDI-TOF/TOF and database searching.

Down

DNA-binding protein GBP16 [Oryza sativa]

gi| 2511541

AADVLAAANTAAEVALR (1626.95) VDDAEFEENEVYAIDIVTSTGEGKPK (2855.42) FIFSEISQK (1098.62) FPIMPFTAR (1079.62)

4

43.13/6.48

52.37/6.61

128/66

2.70

10

Down

Chloroplast translational elongation factor Tu [Pelargonium graveolens]

gi| 12830555

ILDEALAGDNVGLLLR (1682.00)

1

51.28/6.12

47.99/6.6

110/66

2.50

11

Up

Malate dehydrogenase [Lupinus albus]

gi| 27462764

VLVTGAAGQIGYALVPMIAR (2000.29) NISCLTR (806.36)

2

35,524/6.39

37.4/6.65

76/66

2.46

12

Down

Chalcone synthase [Dendrobium hybrid cultivar]

gi| 53794420

AEGPATVLAIGTSTPPNALYQADYPDYYFR (3261.74)

1

43.20/6.32

46.53/6.55

68/66

16.60

15

Up

Putative plastidic glutamine synthetase [Spiraea nipponica]

gi| 26892040

IIAEYIWIGGTGVDVR (1762.03) EHISAYGEGNER (1361.69) HETASINTFSWGVANR (1789.93) GYLEDR (752.43)

4

47.33/6.77

170/66

2.50

40/6.38

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9

1107

(continued on on next next page) (continued

Spot Protein no. a change b

Protein name c

Accession no. d

Matched peptide sequences (m/z) e

Matched Theoretical Experimental Score/ Fold peptides f MW (kDa)/ MW (kDa)/ threshold i change j pI g pI h

Down

Disulfide-isomerase precursor-like protein [Solanum tuberosum]

gi| 77745442

EIGHDR (726.37) YGVQGYPTIQWFPK (1683.95) AGEDYDGGR (939.45) NILATFA (749.45)

4

39.47/5.62

36.8/6.29

72/66

2.37

18

Down

Abscisic stress ripening-like protein [Prunus persica]

gi| 16588758

DPEHAHR (861.45) HKIEEEIAAAAAVGSGGFAFHEHHEK (2772.53) KHHHLF (818.48)

3

20.75/5.68

24.05/6.45

401/66

3.04

19

Down

Cytosolic phosphoglycerate kinase [Pisum sativum]

gi| 9230771

LVAQIPEGGVLLLENVR (1820.18) NDPEFAK (820.36) LASLADLYVNDAFGTAHR (1934.11) GVSLLLPTDVVIADK (1539.93)

4

42.26/5.73

45.07/6.08

88/66

7.12

20

Down

Flavanone 3hydroxylase [Daucus carota]

gi| 5924375

GGFIVSSHLQGEAVQDWR (1986.02) LMGLGCKLLEVLSEAMGLDK (2152.11) HTDPGTITLLLQDQVGGLQATR (2334.31)

3

41.08/5.98

38.6/6.07

171/66

11.55

21

Up

Gibberellin 20-oxidase 1 [Gossypium hirsutum]

gi| 74273629

DEDERPK (888.47) LVSEMTR (835.51) GGFIVSSHLQGEAVQDWR (1986.12) SRDYSR (783.40) WPDKPEGWVEVTK (1570.90) HTDPGTITLLLQDQVGGLQATR (2334.44) DLELAR (716.46)

7

41.38/5.35

39.23/5.89

248/66

8.79

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16

Spots %volme variations (p < 0.05) k

1108

Table 1 (continued)

40.84/5.43

39.23/5.77

259/66

9.46

Reversibly glycosylated gi| 18077708 polypeptide [Gossypium hirsutum]

4 VPEGFDYELYNR (1501.81) GYPFSLR (839.52) ELIGPAMYFGLMGDGQPIGR (2122.14) LADAMVTWIEAWDELNPSGDISAKIPNGASK (3315.85)

40.82/6.01

40.69/6.19

89/66

3.65

Down

Leucoanthocyanidin dioxgenase [Vitis labrusca × Vitis vinifera]

gi| 22266677

ILSVLSLGLGLEEGR (1555.97) EVGGMEELLLQK (1345.69) IILKPLPETVSETEPPLFPPR (2373.42)

3

40.17/5.63

46.53/5.86

215/66

2.01

25

Down

Vacuolar H+-ATPase A1 gi| subunit isoform; V27883932 ATPase A1 subunit isoform [Lycopersicon esculentum]

11

68.53/5.10

65.87/5.54

149/66

4.51

26

Up

gi| Cytosolic 9230771 phosphoglycerate kinase [Pisum sativum]

ESEYGYLRK (1144.62) VGHDNLIGEIIR (1335.79) GVSVPALDK (885.50) VALPPDAMGK (1014.54) EASIYTGITIAEYFR (1733.94) WAEALR (745.44) LAEMPADSGYPAYLAAR (1795.97) KHFPSVNWLISYSK (1705.83) FCPFYK (804.35) NIIHFYNLANQAVER (1802.01) LGDLFYR(883.52) YSLKPLVPR (1072.74) LVAQIPEGGVLLLENVR (1820.16) LASLADLYVNDAFGTAHR (1934.09)

3

42.26/5.73

45.07/6.15

74/66

6.63

Up

Flavanone 3 betahydroxylase [Malus × domestica]

23

Up

24

gi| 62632851

MAPATTTLTSIAHEK (1571.82) GGFIVSSHLQGEAVQDWR (1986.08) KYSDELMGLACK (1357.74) HTDPGTITLLLQDQVGGLQATR (2334.37)

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4

22

1109

(continued on on next next page) page) (continued

Spot Protein no. a change b

Protein name c

Accession no. d

Matched peptide sequences (m/z) e

Matched Theoretical Experimental Score/ Fold peptides f MW (kDa)/ MW (kDa)/ threshold i change j pI g pI h

Up

EIF4A-2; ATPdependent helicase [Arabidopsis thaliana]

gi| 15221761

GIYAYGFEKPSAIQQR (1828.09) ILQAGVHVVVGTPGR (1502.90) VFDMLR (780.47) MFVLDEADEMLSR (1555.84)

4

46.73/5.45

53.83/5.74

94/66

7.93

28

Down

Peroxidase [Nicotiana tabacum]

gi| 14031049

DSVFLSGGPDYDLPLGR (1807.97) YYVDLMNR (1089.55)

2

39.04/5.99

47.26/5.65

101/66

*2.17

29

Down

Peroxidase [Nicotiana tabacum]

gi| 14031049

DSVFLSGGPDYDLPLGR (1807.97) YYVDLMNR (1089.55)

2

39.04/5.99

44.71/5.65

101/66

*2.57

30

Up

ATP synthase subunit beta, mitochondrial precursor

gi|114421

AVQYATSAAAPASQPSTPPKSGSEPSGK (2672.48) VLNTGSPITVPVGR (1409.90) EAPAFVEQATEQQILVTGIK (2172.24) VVDLLAPYQR (1173.73) TVLIMELINNVAK (1457.89) AHGGFSVFAGVGER (1390.77) VGLTGLTVAEHFR (1399.85) DAEGQDVLLFIDNIFR (1865.03) FTQANSEVSALLGR (1492.86) QISELGIYPAVDPLDSTSR (2061.14) MLSPHILGEDHYNTARGVQK (2266.25)

11

59.82/5.95

57.84/5.5

276/66

5.76

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 9 9 –11 1 8

27

Spots %volme variations (p < 0.05) k

1110

Table 1 (continued)

RuBisCO large subunit- gi| binding protein subunit 2506277 beta, chloroplast precursor

VVAAGANPVLITR (1280.85) SAENSLYVVEGMQFDR (1844.94) DLINILEDAIR (1284.79) APGFGER (733.43) SQYLDDIAILTGGTVIR (1835.09)

5

62.95/5.85

62.59/5.31

277/66

*3.24

32

Up

Alpha tubulin gi| [Physcomitrella patens] 25396550

AVFLDLEPTVIDEVR (1716.00) QLFHPEQLISGK (1396.82) SLDIERPTYTNLNR (1691.94) LVSQVISSLTASLR (1473.93) FDGALNVDVTEFQTNLVPYPR (2395.32) AFVHWYVGEGMEEGEFSEAR (2330.13)

6

49.54/4.96

57.12/5.31

473/66

2.43

33

Up

Glucose-6-phosphate isomerase [Lycopersicon esculentum]

gi| 51340062

15

67.62/5.49

61.86/5.34

235/66

2.02

34

Down

Chaperonine 60 K alpha chain — rape (fragment)

gi|99801

10

57.63/4.99

62.59/5.09

282/66

*2.94

35

Up

Tubulin beta-3 chain (Beta-3 tubulin)

gi| 74053562

AFKDMVDLEK (1211.62) GAIANPDEGR (999.52) MVGHYWLR (1061.57) SGGTPETR (804.34) QGVAITQENSLLDNTAR (1830.00) IEGWLAR (844.49) FPMFDWVGGR (1227.60) TSEMSAVGLLPAALQGIDIR (2042.18) DSLLLFSR (950.55) EFDLDGNR (965.47) GSTDQHAYIQQLR (1516.80) ESITVTVQEVTPR (1458.83) SVGALVALYER (1177.70) AVGIYASLVNINAYHQPGVEAGK (2371.34) QPVEPLTLDEIAER (1609.77) FSVRANVK (920.62) VVNDGVTIAR (1043.65) AIELPDAMENAGAALIR (1754.99) TNDSAGDGTTTASVLAR (1636.86) VGPDGVLSIESSSSFETTVEVEEGMEIDR (3098.60) GYISPQFVTNPEK (1479.81) LLVEFENAR (1090.65) APLLIIAEDVTGEALATLVVNK (2250.40) APGFGER (733.42) VGAATETELEDR (1290.71) AVLMDLEPGTMDSVR (1633.87) SGPYGQIFRPDNFVFGQSGAGNNWAK (2814.46) GHYTEGAELIDSVLDVVR (1973.10) IREEYPDR (1077.56) FPGQLNSDLR (1146.64) LAVNLIPFPR (1139.75) LHFFMVGFAPLTSR (1622.94) YLTASAMFR (1059.58) MASTFIGNSTSIQEMFR (1920.01) VSEQFTAMFR (1215.63)

10

50.09/4.73

57.12/5.03

265/66

3.21

(continued on on next next page) (continued

1111

Down

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 9 9 –1 11 8

31

Spot Protein no. a change b

Protein name c

Accession no. d

Matched peptide sequences (m/z) e

Matched Theoretical Experimental Score/ Fold peptides f MW (kDa)/ MW (kDa)/ threshold i change j pI g pI h

Up

Transaldolase [Lycopersicon esculentum]

gi| 10441272

YEAVIDAYLDGLEASGLSDLSR (2357.28) VTSVASFFVSR (1199.71) IGTPEALDLR (1084.66)

3

47.57/5.65

51.64/5.12

188/66

5.83

37

Up

Transaldolase [Lycopersicon esculentum]

gi| 10441272

YEAVIDAYLDGLEASGLSDLSR (2357.28) VTSVASFFVSR (1199.71) IGTPEALDLR (1084.66)

3

47.57/5.65

51.64/5.05

182/66

6.21

38

Down

Putative 40S Ribosomal protein [Oryza sativa]

gi| 15217294

FAQYTGAHAIAGR (1362.79) HTPGTFTNQLQTSFSEPR (2048.07) LLILTDPR (940.65) WDVMVDLFFYR (1490.79)

4

33.12/4.86

46.16/5.43

130/66

2.61

39

Down

Salt tolerance protein [Sesuvium portulacastrum]

gi| 56112332

AHGSETVK (828.36) DFGSALWDMIR (1310.73)

2

38.43/5.10

88/66

7.73

35/4.84

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 9 9 –11 1 8

36

Spots %volme variations (p < 0.05) k

1112

Table 1 (continued)

Up

Similar to late embryogenesis abundant proteins [Arabidopsis thaliana]

41

Up

43

48

gi| 21592483

DFGSALWDMIR (1310.71)

1

35.89/4.71

38.3/4.76

75/66

3.71

14-3-3 protein [Solanum gi| lycopersicum] 22217852

DSTLIMQLLR (1189.76) SAQDIALAELAPTHPIR (1803.11) LGLALNFSVFYYEILNSPDR (2331.34)

3

29.24/4.79

28.4/4.52

83/66

3.12

Down

Bis(5′-nucleosyl)tetraphosphatase [Arabidopsis thaliana]

gi| 15220667

WYLVRLR (1005.62) LRNDEDEK (1018.57) WAKPEEVVEQAVDYK (1790.98)

3

19.82/4.76

26.3/5.62

73/66

2.15

Down

Eukaryotic translation initiation factor 5A-2 (eIF-5A-2) (eIF-4D)

gi|124226

TYPQQAGTIR (1134.63) VVEVSTSKTGK (1134.63) TDYQLIDISEDGFVSLLTENGNTK (2672.34) DDLRLPTDDNLLTQIK (1870.05) LPTDDNLLTQIKDGFAEGK (2075.06)

5

17.35/5.60

18.94/5.93

94/66

13.33

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 9 9 –1 11 8

40

(continued on on next next page) (continued

1113

1114

Table 1 (continued) Spot Protein no. a change b

Protein name c

Accession no. d gi|123378

Up

DNA-binding protein MNB1B (HMG1-like protein)

59

Up

EIF4A1; ATP-dependent gi| helicase [Arabidopsis 79313227 thaliana]

a

Matched Theoretical Experimental Score/ Fold peptides f MW (kDa)/ MW (kDa)/ threshold i change j pI g pI h

RAPSAFFVFMEEFR (1733.93) APSAFFVFMEEFR (1577.82)

2

17.14/5.90

22.08/5.73

89/66

5.54

GIYAYGFEKPSAIQQR (1827.98) ILQAGVHVVVGTPGR (1502.76) MFVLDEADEMLSR (1571.77) IQVGVFSATMPPEALEITR (2075.06) FMSKPVR (864.47) KVDWLTDK (1004.58) VLITTDLLAR (1114.70) GVAINFVTR (976.58) DDERMLCLR (1166.62) MLCLRTWPICCEGR (1680.92) TWPICCEGRK (1192.48)

11

46.92/5.98

52.74/5.59

71/66

2.06

Spots %volme variations (p < 0.05) k

Spot number corresponds to the 2-DE gel in Figs. 3 and 4. Up- or down-regulation of flower bud proteins expressed on floral reversion compared to normal flowering. c Names and species of the proteins obtained via the MASCOT software from the NCBI database. d Accession number from the NCBInr database. e The sequences of all the identified peptides with the corresponding m/z ratio in brackets. f The total number of identified peptide. g Experimental molecular mass (Mr) and isoelectric point (pI) estimated in comparison to a 2D gel with marker proteins. h Theoretical molecular mass (Mr) and isoelectric point (pI) of the homologous protein calculated with a tool available at NCBInr database. i MOWSE score probability (protein score) for the entire protein and for ions complemented by the percentage of the confidence index (C.I.). j Protein change (more than 2-fold and p < 0.05) of up-regulation or down-regulation of floral reversion compared to normal flowering at average volume of three development times. The values which are significantly changed at one corresponding development stage are marked by asterisks (*). k X-axis denotes three development times (stages I, II and III), and Y-axis denotes the relative levels of protein expression (normalized vol.% of spots), values are expressed as the mean of three replications ± standard deviation. Black column shows normal flowering and white column shows floral reversion. b

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 9 9 –11 1 8

53

Matched peptide sequences (m/z) e

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 9 9 –1 11 8

established by Bevan et al. (Fig. 6) [12]. The largest functional class was associated with two categories: metabolism (28%) and protein translation (28%). Secondary metabolism was the third largest category (13%), followed by proteins related to cytoskeleton structure (8%). Those related to regulation (5%) and stress tolerance (5%) were under minor categories among the identified proteins. Other categories included unclassified proteins (8%) and unknown proteins (5%). Among the identified proteins, those related to substance and energy metabolism, such as cytosolic phosphoglycerate kinase, RuBisCO, and RuBisCO large subunit-binding protein subunit beta, were down-regulated on the floral reversion D. longan buds. By contrast, aconitase, malate dehydrogenase, glucose-6-phosphate isomerase, transaldolase, ATP-binding protein, and ATP synthase were up-regulated on floral reversion buds. The proteins related to transcription and translation included chloroplast translational elongation factor Tu, elongation factor-2 EF-2, eukaryotic translation initiation factor 5A-2, disulfide-isomerase precursor-like protein, RNAbinding protein, DNA-binding protein GBP16, ATP-dependent helicase, 40S ribosomal protein, chaperonine 60 K alpha chain, bis(5′-nucleosyl)-tetraphosphatase, and plastidic glutamine synthetase. Those related to secondary metabolism, such as chalcone synthase, flavonoid 3-hydroxylase, and anthyocyanidin synthase, were down-regulated on floral reversion buds. Also, the proteins of cytoskeleton were all down-regulated except for α-tubulin. The proteins related to regulation, such as 14-3-3 protein and DNA-binding protein MNB1B, and those related to stress tolerance, such as late embryogenesis-abundant protein, were all up-regulated on floral reversion D. longan buds. Other unclassified proteins like peroxidase were down-regulated, whereas gibberellin 20-oxidase was up-regulated.

3.4.

Western blot of 14-3-3 protein and GA20ox

According to the 2-D results, the open reading frame of the two up-regulated proteins 14-3-3 and GA20ox was cloned and expressed in E. coli. The products were purified and collected, and an antibody was obtained by immunity. D. longan 14-3-3 protein and GA20ox expressed in E. coli were identified using Western blot, and both foreign proteins had immunobinding blot on homologue MW. Semi-quantitative Western blot analysis showed that the expression volume of GA20ox in floral reversion buds was about twice as that in normal flowering buds (Fig. 7) according to the 2-D results. By Other function 8%

Unknown 5%

Secondary metabolism 13%

Cytoskeleton 8%

Substence and energy metabolism 28%

Regulation 5% Transcription and translation 28%

Stress tolerance 5%

Fig. 6 – Functional categorization of differential expressioned proteins of normal flowering and floral reversion on longan.

1115

Fig. 7 – Western blotting profile of 14-3-3 protein and GA-20oxidase in longan normal flowering and floral reversion buds. A1–A3: normal flowering; B1–B3: floral reversion.

contrast, the semi-quantitative Western blot analysis of 143-3 protein only had immunobinding blot on floral reversion buds; the results showed the same trends as the 2-D results.

4.

Discussion

Longan flowering reversion can cause flower bud abscission after vegetative lateral branch bolting [4]. In the present study, reversion buds were characterized by ultrastructural changes that included cell wall dissolution, cell membrane invagination, and ER multiplication, among others. These features are similar to those occurring in the abscission processes of plant organs [13–15]. The changes in ER were significant, as this is the primary organelle of the endomembrane system and is responsible for the synthesis and maturation of proteins used for secretion in the plasma membrane. It is also responsible for the transport of proteins to various organelles in the endocytic and exocytic pathways [16]. However, the increase in ER observed in reversion buds in the present study might indicate the synthesis of protein for the translocation of cells from a deteriorating abscission region to the plant body. The phenomenon of floral reversion in D. longan involves a series of physiological and biochemical processes. However, the metabolic variations in flower buds might be the direct factor that influences it. In the present study, proteins related to photosynthesis were down-regulated, whereas those related to respiration were up-regulated in D. longan floral reversion buds, indicating a decrease in photosynthesis and an increase in respiration. The decrease in photosynthesis directly reduced carbon assimilation, whereas the increase in respiration resulted in the consumption of more photosynthetic products and thus a reduction in the carbon to nitrogen ratio (C/N). C/N is a very important theory for plant flowering control [17–19]. An adequate C/N is a prerequisite for the proper formation of floral organisms [20]. Research on D. longan [21] showed that ringing treat increased the content of total sugar and amylum, leading to a high C/N which accelerated flowering. The results suggested that down-regulated proteins related to photosynthesis and up-regulated proteins related to respiration on floral reversion buds might be unfavorable to flower transition. The down-regulation of proteins related to transcription and translation indicated the improper and inadequate protein expression in floral transition organisms. Down-regulated elongation factors might display various defects in vegetative and reproductive development, such as early bolting of an increased number of leaves and inflorescences and abnormal flower and

1116

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 9 9 –11 1 8

leaf structures [22]. The translation initiation factor 5A-2 might play an important role in floral transition during reproductive development. An eIF-5A deletion mutant causes defective sporogenesis and abnormal development in floral organs, leading to the abortion of both female and male germline cells on Arabidopsis [23]. This result indicated that the down-regulation of the protein translation control factor might be one of the main reasons for D. longan floral reversion. The proteins chalcone synthase (CHS), flavonoid 3hydroxylase (F3H) and flavanone 3 beta-hydroxylase (F′3H) are involved in the pathway of flavonoids. Mainly participating in flavonoid synthesis, they were down-regulated in floral reversion buds. Flavonoids not only protect plants from the harmful effects of UV irradiation but also play a crucial role in their sexual reproduction process [24]. Antisense CHS gene under the control of the cauliflower mosaic virus 35S promoter was expressed in petunia. The transgenic plants were sterile males due to an arrest in male gametophyte development, indicating that flavonoids played an essential role in male gametophyte development [24]. Ma and Zu [25] studied the dynamic changes in endogenous hormones and flavonoids in Catharanthus roseus as the plants transitioned from the vegetative to reproductive stages. The contents of flavonoids in the leaves were evidently decreased during the first flower bud differentiation stage, implying that parts of the flavonoids in the leaves were transplanted and consumed during the first flower bud differentiation stage. This finding also indicated that flavonoids played an important role in regulating and controlling the change process from vegetative growth to reproductive growth for C. roseus. Li et al. [26] investigated the changes in quality and quantity of Flos Lonicerace during flowering stages. The results showed that the contents of total flavonoids in the first flowering time were higher than those in the second flowering time, implying that the accumulation of flavonoids was important for early floral transition. We presumed that the down-regulation of flavonoid synthesis proteins was unfavorable for floral transition and differentiation. The regulation factor 14-3-3 protein was up-regulated on floral-resistant D. longan buds. The biological roles of 14-3-3 complexes were demonstrated in signal transduction, subcellular targeting, and cell cycle control. They can also act as an allosteric cofactor modulating the catalytic activity of their binding partners. In plants, 14-3-3 binding proteins include a variety of key metabolic enzymes, such as nitrate reductase, sucrose-phosphate synthase, and plasma membrane H+-ATPase [27,28]. Therefore, interactions with 14-3-3 proteins are subject to environmental control through signaling pathways which have an effect on 14-3-3 binding sites. There are multiple levels at which 14-3-3 proteins may play roles in stress responses in higher plants because they regulate the activities of many proteins involved in signal transduction [29].14-3-3 proteins exhibited diverse patterns of gene expression in response to salt stress and potassium and iron deficiencies through regulation of H+-ATPase activity in the plasma membranes of S. lycopersicum [30], Z. mays [31], and barley (Hordeum disticum) [32]. The reduced expression of an Arabidopsis thaliana 14-3-3 protein, GF14 lambda, led to increased sensitivity to low temperatures, indicating that the 14-3-3 protein was required for cold tolerance in plants [33].

Overexpression of the GF14 lambda gene in cotton (Gossypium hirsutum) demonstrated a “stay-green” phenotype and improved water stress tolerance. These transgenic plants wilted less and maintained a higher photosynthesis than segregated non-transgenic control plants under water-deficit conditions. [34] In the current work, the flowering reversion in D. longan can also be considered as a result of abiotic stress (high temperature in winter), and the up-regulation of 14-3-3 protein may be a response to environmental factors. GA20ox is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. The rice (Oryza sativa L.) GA20ox gene OsGA20ox2 (SD1) is well known as the “Green Revolution gene,” and the loss of function mutation in this locus causes semi-dwarfism. Analysis of a mutant rice cultivar showed that the endogenous GA level increased due to the up-regulation of the OsGA20ox1 gene. The final stature of the mutant reflected internode overgrowth and was approximately twice that of its wild-type parent [35]. Transgenic plants of Nicotiana tabacum overexpressing GA20ox cDNA (CcGA20ox1) from citrus fruits were up to twice as tall as the control plants [36]. In some short day plants, such as malus, lichi, and longan, GAs are rather considered as a flowering inhibitor than promoter. “Redchief” and “Red Fuji” apple trees (Malus domestica Mill) were treated with exogenous GAs during the flower bud differentiation phase. The results showed that the contents of GAs and IAA in spur buds remarkably increased, whereas the node number of flower buds decreased. The inhibitor effect of GA treatment was only detected in the critical period of floral differentiation, whereas it was not found on leaf buds with identical treatment [37]. Similar results were found on loquat (Eriobotrya japonica Lind1.) [38] and orange trees [39]. Research on changes in litchi endogenous hormones in the process of flower sex determination showed that lower concentrations of GA were beneficial for sexual organ development, whereas higher concentrations inhibited corresponding sexual organ development [40]. Other studies also showed that the activity of endogenous GA1/3 was inhibited on the ringing treatment of D. longan trees. The treatment promoted flower differentiation, indicating that the lower level of endogenous GA was beneficial in increasing the flowering rate of D. longan [41]. In the present study, the up-regulation of GA20ox in D. longan floral reversion buds might be one of the main factors that induced floral reversion. The higher expression level of GA20ox led to increased endogenous GA, inhibition of floral transition, and promotion of vegetative growth, resulting in branch elongation and inflorescence. The detection of up-regulated cytoskeleton proteins on floral reversion buds further confirmed this result. The flower reversion identically led to elongated inflorescence and sparse fruits in D. longan trees [42]. Taken together, the data demonstrated that the up or down-regulated expression of proteins in flower buds from a number of functional categories was modulated as a result of D. longan floral reversion. Among a series of physiological regulated processes, the imbalance in photosynthesis and respiration, the decrease in flavonoid synthesis, the upregulation of GA-20-oxidase, and the down-regulation of proteins related to transcription and translation might have directly resulted in floral reversion in D. longan. The upregulation of the 14-3-3 protein can be suggested as a

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 9 9 –1 11 8

response to abiotic stress. The results on ultrastructural changes in reversion buds also explained the shedding of some reversion buds in D. longan. The proteomics-based approach to this biological problem revealed a number of targets for better understanding floral reversion in D. longan.

Acknowledgments This work was supported by the National Natural Science Grant of China (Award no. 30571293); The Ph.D. Programs Foundation of Ministry of Education of China (Award no. 200803890009) and Natural Science Foundation of Fujian Province, China (2007J0045). We also would like to thank Dr. Ray R. Ming of the Department of Plant Biology, University of Illinois at UrbanaChampaign (USA) for the critical reviewing of the manuscript.

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