Seeking Transformation Markers: An Analysis of Differential Tissue Proteomes on the Rice Germplasm Generated from Transformation of Echinochloa crusgalli Genomic DNA Caifeng Zhao,†,‡ Bingran Zhao,§ Yan Ren,†,‡ Wei Tong,†,‡ Jingqiang Wang,†,‡ Kang Zhao,†,‡ Shaokun Shu,†,‡ Ningzhi Xu,†,‡ and Siqi Liu*,†,‡,| Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China, Beijing Proteomics Institute, Beijing 101300, China, National Hybrid Rice R&D Center, Changsha 410125, China, and The Department of Medicine, University of Louisville, Louisville, Kentucky 40202 Received September 25, 2006
The transformation of distally related genomic DNAs into plant was proposed as a novel technique to breed new cultivars. For example, a restorer rice line, RB207, was successfully developed and stabilized through the transformation of genomic DNAs of Echinochloa crusgalli (E. crusgalli) into a rice line, R207. Although the phenotypes of this variant line are apparently different from its receptor, the molecular bases are not elucidated yet. Herein, we have systematically studied the differential proteomes from the tissues of E. crusgalli, R207, and RB207 in an attempt to find an explanation regarding the phenotypic changes of RB207. The 2-DE method was employed to separate the leaf and embryo proteins of these plants followed by protein identification with mass spectrometry. In the leaf, 953 ( 15, 1084 ( 11, and 1091 ( 11 silver-stained spots were detected, whereas in the embryo, 986 ( 3, 884 ( 10, and 892 ( 14 spots were found from E. crusgalli, R207, and RB207, respectively. In comparison to the 2-DE images of the two rice lines, which showed many similarities, the ones of the E. crusgalli and rice were found to be so different that they were incomparable. There were some differentially expressed 2-DE spots between the two rice cultivars, 72 in leaf and 53 in embryo, respectively. The results of protein identification suggested that, regardless of leaves or embryos, none of the E. crusgalli genes were encoded in the new rice cultivar, RB207. The fact that 60% of the differentially expressed spots between R207 and RB207, however, were verified as the proteins involved in metabolism and photosynthesis makes a rather convincing argument that the DNA fragments transferred from E. crusgalli to rice are responsible for exerting the unknown influence to the expression of rice genes. Keywords: Echinochloa crusgalli • Rice • Leaf • Embryo • Genomic DNA transformation • Two-dimensional gel electrophoresis • Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry
Introduction Rice is the most important food crop for the people living in tropical and subtropical regions of the world. With the world population continuously increasing, rice yields must also grow to meet nutritional requirements. The enhancement of rice production is the essential strategy to ensure the rice supply. In the last 2 decades, breeding techniques used in the generation of hybrid rice have been significantly improved.1 Although the conventional hybrid breeding with inter-subspecific heterosis has been demonstrated as a feasible means to generate the varieties with high yields, it still faces its own limitations. * To whom correspondence should be addressed: Siqi Liu, Ph.D., Beijing Genomics Institute, Chinese Academy of Sciences, Beijing Airport Industrial Zone B-6, Beijing 101300, China. E-mail,
[email protected]; phone, 8610-8048-5325; fax, 8610-8048-5324. † Beijing Genomics Institute, Chinese Academy of Sciences. ‡ Beijing Proteomics Institute. § National Hybrid Rice R&D Center. | University of Louisville.
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For instance, the gene transfers within different rice cultivars are difficultly envisaged for the specific traits, such as resisting pests, improving grain quality, or protecting infections. Moreover, the traditional breeding methods are highly demanding in time and manpower. Current breeding techniques based upon molecular and genomic bases have become a main trend for developing new rice cultivars.2 The C3 plants, which fix atmospheric CO2 to the 3-carbon compound, 3-phosphoglycerate (3-PGA), through Calvin cycle by Rubisco, are the most abundant crop plants. The overall photorespiratory cycle in the C3 plant, however, is far from efficient for CO2 fixation.3 Since O2 competes with CO2 at the active site of Rubisco, almost half of the CO2 in the photorespiration cannot be fixed in the C3 plants. In contrast to the C3 plants, the C4 plants can fix atmospheric CO2 to the 4-carbon compound, oxaloacetate, through Hatch-Slack cycle. In the C4 plants, phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes the primary CO2 fixation, has a much higher CO2 affinity than Rubisco. Evidence suggests that the C4 plants 10.1021/pr0605015 CCC: $37.00
2007 American Chemical Society
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Seeking Transformation Markers Table 1. Means of Yield Traits of Variants and Their Receptors (n ) 20) material
plant height (cm)
length of leaf
width of leaf
leaf area
spikelets/ panicle
seeds/ panicle
1000 grain weight (g)
head rice (%)
length/width ratio of seed
E. crusgalli R207 RB207
156 107 127
60.06 ( 2.63 39.74 ( 2.96 57.38 ( 5.96
1.83 ( 0.07 1.88 ( 0.13 2.28 ( 0.16
85.94 ( 3.82 57.15 ( 6.94 101.49 ( 15.79
/ 205 ( 7.35 286 ( 12.8
/ 158 ( 6.09 226 ( 5.94
/ 22.3 ( 0.65 31.7 ( 0.51
/ 59.5 ( 0.93 69.1 ( 1.04
/ 2.9 ( 0.08 3.5 ( 0.07
exhibit many desirable agronomic traits, high rate of photosynthesis, fast growth, and high efficiency in water and mineral use.4 Thus, a great effort has been driven by a fascinating strategy, to transfer the C4 traits to the C3 plants.5 Rice is a typical C3 plant, whereas, Echinochloa crusgalli (E. crusgalli) is a C4 plant, which is a weed concomitantly grown with rice in fields. Importantly, E. crusgalli in a rice field generally exhibits fast growth with higher stems, and wider and darker green leaves. Hence, Zhao et al. proposed that, if some traits of E. crusgalli were transferred into rice, the new rice cultivar would have advantages of high grain yields with large panicles.6 Many approaches of gene transformation have been developed, which allow crop plants to accept several genes of interest using gene transformation. In 1987, Pena et al. first reported a new method to tranform exogenous DNA into cereal plants rather than using tissue culture techniques.7 After simply injecting aliquots of aqueous solution of pLGVneo1103 vector, which carried aminoglycoside phosphotransferase II gene (APT II), into floral tillers of the diploid rye, the seeds set on the injected tillers were screened for kanamycin resistance, and APT II was successfully transformed in 29% of the seeds. The authors postulated that the DNA was transported by the plant vascular system to the germ cells. This method, later called pollen-tube pathway of transformation, has been employed in many plants for gene transformation, such as wheat, soybean, and watermelon.8-10 On the basis of Pena’s method, Zhao et al. further modified this technique, called spike-stalk injection.11 According to the modified protocol, the exogenous DNA was injected into the uppermost internode of a rice stem when the recipient rice had undergone meiosis. By the use of this new technique, several useful germplasms of hybrid rice have been achieved. A typical sample was a stable rice strain, RB207, in which the genomic DNAs of E. crusgalli were introduced into R207, a restorer strain of the 3-line hybrid rice. This new rice cultivar exhibited significant phenotypic differences from its parents. In addition, Xing et al. adopted the analysis of amplified fragment length polymorphism (AFLP) to R207 and RB207 and found occurrence of 15.6% mutation rates between the two rice lines.12 The two original investigations suggested
that some traits in E. crusgalli (C4 plant) were likely transferred to rice (C3 plant). Although the transformed plants generated by pollen-tube pathway exhibit significant phenotypic alterations, notably, the genetic mechanisms for this type of gene transformation have not been fully elucidated. Regardless of which kind of phenotypic changes are observed in the transformed products, the phenomenon caused by simple injection of exogenous DNAs is not easily explained by the current theory of genetics or molecular biology. To elucidate the proper theoretic model for this transformation, a fundamental question of how to accurately determine the molecular makers shared by the variants and the receptors needs to be addressed. During the past several years, the field of proteomics has evolved considerably.13 As there are phenotypic differences between R207 and RB207, these trait changes are reasoned to correspond with the different expressions of proteins. We hypothesize, therefore, to attribute the phenotypic changes of the transformed rice line to the differential proteomes between R207 and RB207, qualitatively or quantitatively. In this communication, we have chosen two plant tissues, leaf and embryo, from three species, E. crusgalli, R207, and RB207, and measured their proteomes using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The proteomic data suggested that the E. crusgalli genes were not directly expressed in the transgenic rice tissues, at least at the relative abundant protein level; moreover, some differentially expressed proteins between R207 and RB207 were found, implying that the treatment of spike-stalk injection might somehow disturb the gene expression in rice.
Materials and Methods Chemicals. All chemicals employed for electrophoresis were purchased from Amersham Biosciences (Uppsala, Sweden). IPG strips were from Bio-Rad (Hercules, CA). All chemicals of analytical grade were from Sigma (St. Louis, MO). All HPLC grade solvents were from J. T. Baker (Phillipsburg, NJ). Modified trypsin (sequence grade) was obtained from Promega (Madison, WI).
Figure 1. Separation of the leaf proteins by 2-DE approach. (A) The image of 2-DE from E. crusgalli leaf proteins; (B) the image of 2-DE from R207 leaf proteins; (C) the image of 2-DE from RB207 leaf proteins. Journal of Proteome Research • Vol. 6, No. 4, 2007 1355
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Figure 2. Separation of the embryo proteins by 2-DE approach. (A) The image of 2-DE from E. crusgalli embryo proteins; (B) the image of 2-DE from R207 embryo proteins; (C) the image of 2-DE from RB207 embryo proteins.
Figure 3. The image comparison corresponding to several 2-DE spots with the significant changes of spot volume from the leaves. (A) Close-up comparison of the different 2-DE spots among E. crusgalli, R207, and RB207 leaves. According to the 2-DE images analysis of leaf, only two spots, nos. 914 and 927 unique in RB207 might come from E. crusgalli, as shown in the local images. (B) Spots nos. 3 and 8 of R207 leaf were increased 6.5- and 5.1-fold, respectively, as compared to RB207, and both spots were identified as glycine dehydrogenase. Spot no. 10 of R207 leaf was increased 5.7-fold, but with no protein matched upon the PMF signals. C) Spot no. 641 of R207 leaf was decreased 4.2-fold as compared to RB207, and it was identified as ATPase R subunit.
The Plant Materials. Three plant materials were examined in the proteomic analysis. R207 is a restorer line of the 3-line hybrid rice, which was used as a receptor for transfer of E. crusgalli genomic DNA. RB207 is a new rice germplasm, which was generated by the spike-stalk-injection method by introducing E. crusgalli genomic DNA into R207 and developed as a stable rice strain after seven generations.6 The E. crusgalli plant was collected from the farm at the National Hybrid Rice R&D Center, Changsha, China. The seeds of all materials were sown on May 23, 2003, in Changsha, Hunan Province, China (28°30′N, 113°74′E, altitude 40 m). Fifty seedlings of each material were transplanted on June 15, 2003, with the density of transplanted plants at 23.3 cm × 26.6 cm. The field was flooded to a depth of 5 cm and highly irrigated throughout the plant growth. Before planting, the soil was fertilized with 10 and 30 g/m2 phosphorus and potassium, respectively. Nitrogen from urea (7 g/m2) was supplied 10, 50, and 80 days after planting. Their traits were monitored and recorded during the growing period. For all of these plants, the leaves were collected at tillering phase, the seeds were harvested at the final matured stage, and the embryos were obtained by carefully removing the endosperms with scalpel. 1356
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Figure 4. The image comparison corresponding to several 2-DE spots with the significant changes of spot volume from the embryos. (A) The spot volume of spot no. 740 of R207 embryo was intensified 3.5-fold as compared to RB207, and it was identified as translocase. (B) Spot no. 187 of R207 embryo had its spot volume reduced 5.4-fold as compared to RB207, and it was identified as glucose-1-phosphate adenylyltransferase by MALDI-TOF MS.
Protein Extraction from Leaves and Embryos. The leaves and embryos of the three plants were ground to fine powder
Seeking Transformation Markers
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Figure 5. Identification of BRI1-KD interacting protein 114 by MALDI-TOF MS and LC-MS/MS. (A) MALDI-TOF MS spectrum of the trypsinized product from the 2-DE spot no. 962 of R207 leaf, 8 peptide fragments matched to BRI1-KD interacting protein 114 (gi|42733490); (B) LC-MS/MS spectrum, the parent ion 472.642+ was selected for MS/MS analysis, and the tandem mass spectrum was collected corresponding to the amino acid sequence, GLVGEIISR, by analyzing b- and y-ions derived from the peptide ions.
in a metal pulverizer immersed in liquid nitrogen. The proteins in these tissues were precipitated in precooled 10% TCA and 10 mM DTT in acetone for 4 h at -20 °C, followed by centrifugation at 20 000g for 15 min at 4 °C. After several washing steps, the precipitates were dried in a SpeedVac and resuspended in the lysis buffer containing 7 M urea, 2 M thiourea, 4% NP-40, 10 mM DTT, 0.5% ampholyte (pH 3.59.5), 1 mM PMSF, 2 mM EDTA, and 20 mM Tris-HCl, pH 8.5, followed by sonication for 5 min and centrifugation at 50 000g for 30 min at 15 °C. The supernatants were stored at -20 °C until use. Considering the importance of the quantitative comparison of 2-DE images, the quantities of the extracted proteins from the plant tissues were carefully measured using 2-D Quant Kit (Amersham Biosciences). Two-Dimensional Gel Electrophoresis (2-DE). The proteins, 350 µg/gel for leaf proteins and 300 µg/gel for embryo, were mixed with the rehydration buffer containing 8 M urea, 2% (w/ v) CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer (pH 4-7 or pH 3-10), and 0.002% bromophenol blue and rehydrated overnight
with the IPG strips (18 cm with a linear gradient of pH 4-7 for leaf and pH 3-10 for embryo). The isoelectric focusing was carried out for 50 kVh using IPGphor (Amersham Biosciences) at 20 °C. Subsequently, the IPG strips were treated with reduction and alkylation in SDS-PAGE running buffer at equilibrium. These strips were then loaded and run on 12% acrylamide Laemmli gels (26 × 20 cm) using the Ettan DALT II system (Amersham Biosciences) with a programmable power control, 0.5 h at 2.5 W per gel, then at 15 W per gel until the dye front reached the gel bottom. The separated proteins were visualized using silver staining. Image Acquisition and Data Analysis. The 2-DE images were acquired with UMAX Powerlook 2100XL (Maxium Technologies, Inc.) in a transmission mode. The image analysis was performed with a combination of software computation, Phoretix 2D (Amersham Biosciences), and manual visualization, in which all the 2-DE images were globally analyzed by the software and the identified differentially expressed spots were manually re-checked. To have the comparable data for quanJournal of Proteome Research • Vol. 6, No. 4, 2007 1357
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Figure 6. The comparison of MALDI-TOF MS spectra for the 2-DE spots from the leaves of RB207 and E. crusgalli. (A) Spot no. 914 from RB207 leaf and spot no. 866 from E. crusgalli leaf, in which no. 914 was identified as probenazole-induced protein; (B) spot no. 927 from RB207 leaf and spot no. 869 from E. crusgalli leaf, in which no. 927 was identified as root-specific, pathogenesisrelated protein.
titative analysis, several key parameters in the image analysis were fixed as constant values, that is, sensitivity at 9500 and 9430, operator size at 75 and 61, noise factor at 5 and 5, and background at 210 and 28444, for leaf and embryo 2-DE images, respectively. The average spot intensity was normalized to the total spot volume with a multiplication factor of 100. The comparison of their 2-DE images was conduced in three steps. First, several differentially expressed spots with the infinite changes in spot volume between R207 and RB207 were defined. If there was a unique spot in RB207, this spot was further examined for its presence on the 2-DE images of E. crusgalli tisues. If this spot was shared by the two tissues, this partially indicated that it might be an expression product directly from the E. crusgalli gene. Second, the image analysis proceeded with the identification of the quantitative changes in spot volume between R207 and RB207. In this analysis, the intensities of 2-DE spots from R207 were normalized as a reference, and a 3-fold difference in spot volume was set as a significant threshold. Finally, when the qualitative and quantitative difference data were integrated, conclusions could be drawn as to how many different 2-DE spots indeed presented between the two rice tissues. Mass Spectrometric Analysis for Protein Identification. The differentially expressed 2-DE spots were carefully excised and dehydrated with acetonitrile. These spots were reduced with 10 mM DTT at 56 °C for 1 h and alkylated with 55 mM iodoacetamide in the dark at room temperature for 45 min. Finally, the gel pieces were thoroughly washed with 25 mM ammonium bicarbonate, water/acetonitrile (50/50), and acetonitrile and completely dried in a SpeedVac. The proteins 1358
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were digested in 5 µL of modified trypsin solution (1 ng/µL in 25 mM ammonium bicarbonate) for 6 h at 37 °C. The digestion reaction was stopped with the addition of 1 µL of 5% TFA. The digestions were dropped onto the AnchorChip (Bruker Daltonics, Bremen, Germany) followed by mixing matrix solution consisting of R-cyano-4-hydroxycinnamic acid (4 mg/mL) in 70% acetonitrile with 0.1% TFA. The dried chips were subjected to a Bruker AutoFlex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). The mass spectrometer was operated under 19 kV accelerating voltage in the reflectron mode and the m/z range from 600 to 4000. The monoisotopic peptide masses obtained from MALDI-TOF MS were analyzed by m/z software. Mass spectra were internally calibrated with peptides arising from trypsin autoproteolysis at m/z ) 842.509 and m/z ) 2211.105 to reach a typical mass measurement accuracy of 100 ppm. Database searches were conducted using MASCOT software 1.9 (Matrix Science, London, U.K.) against the NCBInr Oryza sativa (rice) and Viridiplantae (green plants) protein database. To evaluate the false positives of the MASCOT search results, the reverse database for rice proteins was constructed using RSF program generated from Beijing Proteome Research Center. In this database, all rice proteins had their amino acid sequences reversed but retained consistent orders for bi-amino acid neighbors to arginine or lysine. Some digestive products from 2-DE spots were analyzed by liquid chromatography iontrap tandem mass spectrometry (LC-MS/MS) using a LCQ DecaXP ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LC-MS/MS data were searched with SEQUEST algorithm for protein identification against the NCBInr Oryza sativa (rice) and Viridiplantae (green plants) protein database. Statistical Analysis. The average values of the parallel experiments were given as the mean ( SD. The comparison of differences among the groups was carried out by Student’s t test. Significance was defined as p < 0.05.
Results Comparison of the Agronomic Characteristics between the Receptor and Its Variant. The agronomic traits, mainly in plant height, leaf size, and seed quality, from three species, E. crusgalli, R207, and RB207, are summarized in Table 1. Taking the traits together, the RB207 rice exhibited several differences in physiological features, such as plant height, leaf length, and spikelet number, from its original strain. This prompted us to hypothesize that the transferred DNA fragments of E. crusgalli could have direct or indirect impacts onto rice gene expression in RB207, which are able to cause the phenotypic changes. The differential proteomic analysis, thus, can monitor these impacts. Two-Dimensional Electrophoresis Analysis of the Protein Extractions from the Plant Leaves and Embryos. On the basis of the 2-DE images shown in Figures 1 and 2, the proteins from the three plant leaves and embryos were well-resolved, at least for the relatively abundant and soluble proteins in E. crusgalli, R207, and RB207. The total of 2-DE spots stained by silver from these plant leaves and embryos were statistically counted. In leaves, there were 953 ( 15 (n ) 4), 1084 ( 11 (n ) 4), and 1091 ( 12 (n ) 4), for E. crusgalli, R207, and RB207, respectively. In embryos, there were 986 ( 3 (n ) 4), 884 ( 10 (n ) 4), and 893 ( 15 (n ) 4), for E. crusgalli, R207, and RB207, respectively. The overlapped analysis revealed that the 2-DE patterns of E. crusgalli leaf and embryo proteins were very different from those of rice counterparts. However, the 2-DE images obtained from the two rice leaves and embryos were quite comparable.
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Seeking Transformation Markers Table 2. Identification of the Leaf Proteins (a) Identification of the Leaf Proteins Unique in R207
spot no.
GI no.a
Mr calc.b/obs.c
pI calc./obs.
296 324
gi|50510015 gi|50911777
54020/40949.38 45179/39858.11
8.01/6.201 5.68/4.892
score
sequence coverage
matched rate
source
protein name
12/23 13/21
rice rice
alanine aminotransferase phosphoribulokinase precursor
Metabolic Protein 139 24 124 24
(b) Identification of the Leaf Proteins Unique in RB207 spot no.
GI no.
Mr calc./obs.
111
gi|15823775
51821/50300.48
5.43/5.738
26 385 385
gi|50924021 gi|8926334 gi|50934469
60920/65593.96 27400/36058.29 37715/36058.29
4.72/4.77 6.73/5.166 6.71/5.166
429 448
gi|55700925 gi|2429286
34781/34937.94 33299/34330.98
5.32/5.238 5.77/5.683
927
gi|38678114
17004/17016.81
4.88/5.001
675
gi|21742895
27184/28495.66
503
gi|50920071
39713/32963.57
pI calc./obs.
score
sequence coverage
matched rate
source
protein name
27/43
rice
UDP-glucose pyrophosphorylase
8/21 22/47 23/47
rice rice rice
Calreticulin, Calreticulin family Putative tyrosine phosphatase Putative SHOOT1 protein
14/25 10/17
rice rice
peroxidase 29 precursor peroxidase
12/20
rice
root specific pathogenesis-related protein 10
rice
tRNA binding domain
rice
pirin-like protein
Metabolic Protein 330 66 Signal Transduction 77 18 249 80 238 66 Antioxidant Protein 130 34 93 29 Stress-Induced Protein 150 78
Translation Related Protein 5.63/4.938 104 30 8/21 Other 8.42/5.998 75 27 10/29
(c) Identification of the Different Leaf Proteins between R207 and RB207: Increased in RB207 spot no.
GI no.
Mr calc./obs.
pI calc./obs.
score
sequence coverage
matched rate
protein name
source
Metabolic Protein 43 17/36 rice 39 15/21 rice
change fold
chelatase subunit Fructose-bisphosphate aldolase, chloroplast precursor (ALDP) inorganic pyrophosphatase Predicted OJ1767_D02.15-2 gene product Lipocalin, Lipocalin/cytosolic fatty-acid binding protein family Hydrolase, haloacid dehalogenase-like hydrolase
3.88 ( 0.21 6.47 ( 0.42
peroxidase R-soluble NSF attachment protein TPA: class III peroxidase 29 precursor
3.38 ( 0.10 4.50 ( 0.15 4.50 ( 0.15 5.65 ( 0.59
rice
precursor chloroplastic glutamine synthetase ribulose-1,5-bisphosphate carboxylase activase 33 kDa oxygen evolvingprotein of photosystem II Photosystem I reaction center subunit IV
7.08 ( 0.89
rice
ATPase R subunit, 3′-partial
4.18 ( 0.79
283 373
gi|50901006 45123/39623.47 5.51/5.052 gi|78099750 42208/36429.71 6.38/5.314
166 217
488 488
gi|50913151 31762/33218.2 gi|51964418 20457/33218.2
5.8/4.967 4.61/4.967
234 92
48 44
18/35 9/35
rice rice
517
gi|50928883 53260/32451.33 9.61/4.936
112
27
12/29
rice
537
gi|38347448 39811/31980.55 6.75/4.96
177
46
19/33
rice
416 426 426
gi|2429286 33299/35094.63 5.77/5.683 gi|50943481 32750/34803.54 5.04/5.065 gi|55700925 34781/34803.54 5.32/5.065
149 133 72
Antioxidant Protein 54 15/42 rice 40 11/27 rice 32 7/27 rice
182
gi|19387272 49770/44086.12 6.18/5.185
146
Photosynthetic Protein 39 15/40 rice
182
gi|13569643 21738/44086.12 4.78/5.185
97
52
9/40
rice
478
gi|34914480 35068/33427.63 6.1/5.086
231
57
19/40
rice
778
gi|50936537 15537/23756.96 9.64/5.664
111
42
621
gi|20143564 29355/30116.29 5.21/5.307
141
51
7/18 ATPase 14/42
3.38 ( 0.25 3.38 ( 0.25 3.10 ( 0.40 9.99 ( 0.47
5.65 ( 0.59 3.07 ( 0.31
(d) Identification of the Different Leaf Proteins between R207 and RB207: Decreased in RB207 GI no.
Mr calc./obs.
pI calc./obs.
score
8 38
gi|50906765 gi|77554291
73573/80120.59 61150/61112.65
5.09/5.157 5.12/4.827
93 224
Chaperone 17 7/9 31 17/22
2 5 54
gi|51090904 gi|51090904 gi|52076758
112427/102767 112427/91217 61907/58083.83
6.35/6.334 6.35/6.226 6.01/6.298
67 114 76
Metabolic Protein 9 8/16 9 10/12 15 8/21
395
gi|51979042
42594/35589.69
9/6.243
117
spot no.
sequence coverage
23
matched rate
10/16
protein name
change fold
rice rice
dnaK-type molecular chaperone BiP rubisco subunit binding-protein R subunit precursor
-5.24 ( 0.31 -9.60 ( 1.46
rice rice rice
glycine dehydrogenase glycine dehydrogenase pyrophosphate-dependent phosphofructokinase β subunit MDH_glycosomal_mitochondrial, malate dehydrogenases (MDH) glycosomal and mitochondrial
-6.50 ( 0.65 -5.13 ( 0.52 -6.08 ( 0.84
source
rice
-7.34 ( 0.37
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Table 2. (Continued) (d) Identification of the Different Leaf Proteins between R207 and RB207: Decreased in RB207 spot no.
GI no.
Mr calc./obs.
pI calc./obs.
score
34 37
gi|34915052 gi|50931091
66671/62617.91 73580/61154.5
6.83/6.19 6.9/4.794
124 81
84 88
gi|31433181 gi|114521
55202/52660.79 55687/52329.3
5.95/6.158 5.95/6.119
72 252
6
gi|50926886
82451/88542.08
5.69/5.026
98
a
sequence coverage
matched rate
protein name
change fold
ferredoxin-nitrite reductase tyrosine-phosphorylated nuclear protein
-4.47 ( 0.56 -8.47 ( 1.29
ATPase alpha subunit ATP synthase alpha chain
-3.48 ( 0.03 -4.00 ( 0.25
OSJNBa0091D06.15
-4.27 ( 0.24
source
Photosynthetic Protein 27 12/21 rice 18 9/24 rice ATPase 21 8/25 rice 38 22/38 rice Unknown Protein 16 8/11 rice
GI number of NCBI database. b Calc. is a value on the database. c Obs. is a value on the experiment.
According to the strategy of image analysis mentioned above, two spots, nos. 914 and 927, which were found on the 2-DE gels of RB207 but were different from R207 leaves, also presented similar locations to another two spots, nos. 866 and 869, on the 2-DE gels of E. crusgalli leaves (Figure 3A). The further comparison of 2-DE images between R207 and RB207 demonstrated that 35 spots on the 2-DE of RB207 leaves had the increased spot volumes, including 21 spots that only appeared in RB207, and 37 spots of R207 had the increased spot volumes, including 17 spots that only appeared in R207. Two typical images with significant changes in spot volume are depicted in Figure 3B,C, in which, as compared with the 2-DE images of RB207 leaves, the spot volumes of nos 3, 8, and 10 from R207 increased 6.5-, 5.1-, and 5.7-fold, respectively, and the spot volume of no. 641 from R207 decreased 4.2-fold. For these 2-DE images of plant embryos, as compared with R207, 20 spots on the 2-DE gels of RB207 embryo were found with infinite changes in spot volume; however, none of them were matched with the 2-DE spots of E. crusgall embryos. Furthermore, indeed, several 2-DE spots were defined with quantitative changes in spot volume between R207 and RB207. A total of 26 spots from RB207 embryo displayed the increased spot volumes, including unique 20 spots; and a total of 27 spots from R207 embryo exhibited the increased spots volumes, including 19 unique spots. Two typical images of 2-DE from rice embryos, indicating significant changes of spot volumes, are depicted in Figure 4, in which spot no. 740 from R207 embryo is intensified 3.5-fold versus RB207, and spot no. 187 from R207 embryo is reduced 5.3-fold versus RB207. Identification of the Differentially Expressed 2-DE Spots by Mass Spectrometry. Identification of the differentially expressed 2-DE spots was carried out mainly by MALDI-TOF MS, but some spots were verified by LC-MS/MS, if the information of amino acid sequence was required. Figure 5 illustrates the identification result for spot no. 962 present only in R207 by MALDI-TOF MS and LC-MS/MS. By MALDI-TOF MS, 8 peptide fragments matched to the BRI1-KD interacting protein 114 (gi|42733490), whereas the spectrum of LC-MS/ MS detected 2 partial amino acid sequences that matched to the BRI1-KD interacting protein 114. In the rice leaves, a total of 72 spots with the significant changes in spot volume were excised and digested for protein identification by MALDI-TOF MS. The mass spectrometry identified 55 spots as rice proteins, an approximately 76% identification rate (Supporting Information Table 1). These identified proteins are categorized into 10 functional groups, such as chaperone, metabolic, photosynthetic, antioxidant, proton pump related, signal transduction, translation related, ATPase, storage, and stress-induced protein. 1360
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In the rice embryos, 53 spots with the significant changes in spot volume were excised and digested for protein identification by MALDI-TOF MS. The mass spectrometry identified 41 spots as rice proteins, an approximately 77% identification rate (Supporting Information Table 2). These identified proteins are categorized into 11 functional groups, such as storage, development, metabolism, embryo, chaperone, antioxidant, stress-induced, proton-pump, anion channel, transcription factor, and translocase protein. As mentioned above, two spots on the 2-DE gels of RB207 leaves, nos. 914 and 927, were interesting because they were absent from 2-DE gels of R207 leaves but held similar 2-DE locations on the gels of E. crusgalli leaves. The MALDI-TOF MS spectra of these spots from RB207 and E. crusgalli are shown in Figure 6. The spot no. 914 gave six typical peptide mass signals at 1269.606, 1621.681, 1273.557, 1768.807, 2409.179, and 2425.149, leading to define a rice protein, probenazoleinduced protein, using MASCOT algorithm. The MALDI-TOF MS spectrum of spot no. 866 from E. crusgalli leaf was totally different from that of no. 914, even though several peptide mass signals were found at 1416.647, 1650.884, 1799.917, and 1896.958. Hence, nos. 914 and 866 were unlikely to be the same protein (Figure 6A). The similar situation was observed in the other two spots, no. 927 (RB207) and no. 869 (E. crusgalli) (Figure 6B). In evidence, these data upon 2-DE image and mass spectrometry were unable to support the hypothesis that the transferred E. crusgalli genomic DNA fragments could encode and express their own gene products in the transgenic rice. Analysis of the Differentially Expressed Proteins from Rice Leaf and Embryo. The Supporting Information Tables 1 and 2 summarize the identified proteins that have significantly different spot volumes between R207 and RB207. Many spots, either with infinite changes or with relatively quantitative changes in spot volume, were identified as the modification forms of the same proteins. For instance, dehydroascorbate reductase (gi|5168334) in rice leaves exhibited a series of spots on the 2-DE images, some unique in R207 (nos. 695, 713, and 714), and the others unique in RB207 (nos. 682 and 694). This protein was also found in multiple forms in rice embryos, two unique isoforms (nos. 635 and 636) only in R207 and other two unique isoforms (nos. 591 and 594) only in RB207. The factors involved in the post-translational modifications in rice proteins are very complicated and variable. To simplify the proteomic analysis, these modified forms are temporally disregarded from our analysis, and the Tables 2 and 3 only give the identification proteins without considering those isospots, either in R207 or in RB207. The 11 differentially expressed, unique proteins in rice leaves shown in Table 2a,b show very diverse functions. These
research articles
Seeking Transformation Markers Table 3. Identification of the Embryo Proteins (a) Identification of the Embryo Proteins Unique in R207 spot no.
GI no.a
Mr calc.b/obs.c
260
gi|38347227
41636/42151.88
261
gi|50919843
41835/42010.03
627
gi|50944819
22836/27213.57
584 629
gi|50509467 gi|57900426
26038/29151.3 22812/27306.04
pI calc./obs.
5.66/5.932
sequence coverage
score
matched rate
source
protein name
8/24
rice
PrpB, PEP phosphonomutase and related enzymes
13/28
rice
embryo-specific protein
8/23
rice
p23 co-chaperone
11/32 7/22
rice rice
glutathione-S-transferase thioredoxin family Trp26-like protein
Metabolic Protein 81 27
Embryo Specific Protein 8.26/7.792 128 25 Chaperone 4.33/4.353 87 36 Antioxidant Protein 5.01/5.291 135 36 4.96/5.119 80 44
(b) Identification of the Embryo Proteins Unique in RB207 spot no.
GI no.
Mr calc./obs.
pI calc./obs.
sequence coverage
score
matched rate
31
gi|27476086
70688/67391.49
5.46/5.725
Chaperone 193 38
16
gi|50929381
70026/82618.11
6.23/6.689
Metabolic Protein 100 19
40
gi|34909414
59601/62405.64
6.16/5.571
129
120 120 125 125 126 126 127 127 306 162
gi|15823775 gi|33113259 gi|15823775 gi|780372 gi|33113259 gi|15823775 gi|780372 gi|15823775 gi|50921241 gi|50931063
51821/48881.09 48285/48881.09 51821/48915.74 48299/48915.74 48285/48741.71 51821/48741.71 48299/48865.85 51821/48865.85 37149/40544.64 49436/46291.12
5.43/6.007 5.41/6.007 5.43/6.146 5.42/6.146 5.41/6.055 5.43/6.055 5.42/6.19 5.43/6.19 5.9/5.794 5.37/5.735
326
gi|17979213
29032/39406.17
4.74/5.683
281 71 145 50 150 33 101 29 126 44 74 26 124 47 75 26 93 29 90 25 Antioxidant Protein 72 21
712
gi|50902088
18132/17186.48
6.1/6.358
Stress-Induced Protein 122 56 11/32
712
gi|50940763
58352/17186.48
8.72/6.358
724
gi|42733490
16703/16366.32
136
gi|20043077
49681/48073.26
19
source
protein name
20/39
rice
heat shock 70 kDa protein, mitochondrial precursor
9/16
rice
10/14
rice
27/64 19/65 13/27 10/27 15/41 9/40 16/61 10/44 9/19 8/19
rice rice rice rice rice rice rice rice rice rice
MoeA, Molybdopterin biosynthesis enzyme NADP dependent malic enzyme UDP-glucose pyrophosphorylase enolase UDP-glucose pyrophosphorylase enolase enolase UDP-glucose pyrophosphorylase enolase UDP-glucose pyrophosphorylase Arginase, Arginase family ankyrin protein
6/14
Solanum tuberosum
Embryo Specific Protein 78 16 9/32 Proton Pump Related Protein 5.79/6.014 92 32 6/16 Unknown Protein 7.03/6.19 70 17 7/18
14-3-3 protein isoform 16R
rice
Usp, Universal stress protein family
rice
early embryogenesis protein
rice
BRI1-KD interacting protein 114
rice
Hypothetical protein
(c) Identification of the Different Embryo Proteins between R207 and RB207: Increased in RB207 spot no.
GI no.
Mr calc./obs.
pI calc./obs.
score
sequence coverage
matched rate
184
gi|51854319
58301/45276.38
6.34/6.063
152
Metabolic Protein 36 12/19
rice
glucose-1-phosphate adenylylransferase
5.35 ( 0.34
521
gi|50932487
29584/31375.56
8.56/9.267
82
Anion Channel Protein 35 8/23
rice
voltage-dependent anion-selective channel (VDAC) protein
3.50 ( 0.36
source
protein name
change fold
(d) Identification of the Different Embryo Proteins between R207 and RB207: Decreased in RB207 spot no.
GI no.
Mr calc./obs.
pI calc./obs.
151
gi|55297200
44409/46906.18
5.58/5.871
620
gi|77552993
16359/25127.3
5.36/5.778
a
score
sequence coverage
matched rate
Metabolic Protein 126 32 12/31 Transcription Factor 187 59 13/36
source
protein name
change fold
rice
glycine cleavage T protein-like
-4.31 ( 1.10
rice
Hypothetical protein LOC-Os 2G05210
-5.59 ( 0.66
GI number of NCBI database. b Calc. is a value on the database. c Obs. is a value on the experiment.
proteins involved in signal transduction and antioxidant, such as calreticulin, tyrosine phosphatase, and peroxidase, may be responsible for the modification of leaf functions. Moreover, most proteins identified in rice leaves (63%) with the relatively
quantitative changes in spot volume belong to the enzymes participating in metabolism and photosynthesis. Importantly, several enzymes that are the key components in photorespiratory pathways were down-regulated or up-regulated in RB207 Journal of Proteome Research • Vol. 6, No. 4, 2007 1361
research articles leaves, such as malate dehydrogenase and glutamine synthase. In contrast to differentially expressed proteins in leaves between R207 and RB207, there were a few rice embryo proteins with relatively quantitative changes, but most of them with infinite changes in spot volume. Metabolism-related enzymes, such as NADP-dependent malic enzyme, UDP-glucose pyrophosphorylase, enolase, and arginase were major unique differentially expressed proteins in RB207 embryo.
Discussion On the basis of the significant phenotypic changes in the transgenic rice, RB207, we have established a series of proteomic investigations to explore the molecular markers associated with the transgenic process. As shown in Table 1, the leaf area and seed weight in RB207 are significantly different from its origin, R207; thus, the two rice tissues were selected for the proteomic comparison. If the DNA fragments of E. crusgalli, integrated into the rice genome via an unknown mechanism, could express their own gene products in the target rice, the following two premises must be reached: (1) the inserted DNA fragments can retain the intact structures of E. crusgalli genes, including gene upstream and encoding regions, and (2) the regulatory factors of the transcription machinery in rice can recognize the upstream sequences of the inserted genes. Obviously, the comparison results upon 2-DE and mass spectrometry described above demonstrate that none of the E. crusgalli proteins were identified in the two tissues of RB207; therefore, this seemed not to match the two primary conditions above. On the other hand, another possibility should not be excluded based upon the current observation, because the integrated E. crusgalli DNA fragments are able to somehow exert some impact on rice gene expression, even though the inserts do not code for any genes in the transgenic plants. The technique of AFLP introduced by Zobeau and Vos does not rely on prior genomic sequence but provides simultaneous amplification of many restriction fragments for evaluation of genetic diversity, variety identification, mapping, and trait loci quatitation.14 With AFLP, 32 primer pairs were designed for amplification of genes from R207, RB207, and E. crusgalli, and the PCR reactions generated 2132, 2155, and 2988 bands for the three materials, respectively. Compared to the PCR products of R207, RB207 had 105 additional and 82 missing bands,12 whereas, 30 new DNA bands which appeared in RB207 were confirmed to be from E. crusgalli. Hence, Xing et al. anticipated that the addition of new bands could contribute to improvement in agronomic traits of the receptors of E. crusgalli genes.12 From the proteomic data above, some differentially expressed 2-DE spots between R207 and RB207 were found either in rice leaves (72 spots) or in rice embryos (53 spots). Corresponding to these changes in agronomic traits, the differentially expressed proteins involved in photosynthesis as well as metabolism in RB207 leaves and in glycogenesis in RB207 embryo were verified. To some extent, the proteomic data was in agreement with the conclusions of AFLP analysis, that is, the transgenic rice, RB207, exhibited some changes at the molecular level of DNA or of protein. Since the photorespiration in C3 plant has a low efficiency of CO2 assimilation, recently, significant progress has been achieved in the overexpression of the key enzymes of C4-type biochemistry in transgenic plants.15 Hausler et al. reported that multiple C4-cycle genes, such as PEPC, NADP malic enzyme (ME), and PEP phosphatase (PEPP), were overexpressed in transgenic potato and tobacco.16 The most pronounced at1362
Journal of Proteome Research • Vol. 6, No. 4, 2007
Zhao et al.
tenuating effects on photorespiration were observed with PEPC/NADP-ME double transformation of potato plant and with PEPC single transformation of tobacco plant. However, in all these efforts to introduce C4-cycle genes into C3 plant with transgenic approaches, these transformed genes were not specifically unique in C4 pathways, but also existed in C3 plants.17 The trait changes from C3 to C4 plant may be mainly dependent upon the alterations in enzyme activities as well as in tissue specificities. Now the question is whether some C4cycle genes are regulated in their expression or biological activity in the E. crusgalli transgenic rice. As shown in Table 2, over 60% of differentially expressed proteins between R207 and RB207 are the enzymes participating in photosynthesis and metabolism. NADP-dependent malate dehydrogenase (MDH) was found with low expression in RB207 leaves. Usually, this enzyme catalyzes the conversion of oxaloacetate to malate, and plays a central role in shuttling excessive redox equivalents from chloroplast into the cytosol.18 In leaves of C3 plants, MDH can prevent the reduction of stroma once the rate of electron transport and NADPH generation are over CO2 assimilation. Rondeau et al. compared the MDH family members in C3 and C4 plants and concluded that two types of MDH existed and performed different functions.19 Glutamine synthetase (GS), a key enzyme in ammonia assimilation in plants, was confirmed with increased expression in RB207 leaves. This enzyme is mainly located in rice leaves, present in the mesophyll and parenchyma sheath cells.20 Li et al. observed that GS inhibition resulted in the light inactivation of tobacco PEPC-PK in tobacco leaves (C3 plant), suggesting GS could positively regulate PEPCrelated pathways and improve the photosynthetic performance in rice.21 In summary, the comparison of proteomic data reinforces the hypothesis that the trait changes in RB207 could be induced by the gene transfer of E. crusgalli. However, the proteomic evidence still lacks enough authority to conclude whether the insertion of E. crusgalli DNA fragments indeed disturbs rice gene expression and leads to functional changes. Further investigation, such as quantitative analysis of differential transcriptomes and proteomes between R207 and RB207, and the determination of specific activity of C4-cycle enzymes in RB207, will extend the current knowledge of gene transfer induced by spike-stalk injection in plants.
Acknowledgment. This work was supported by 973 Grant (2006CB910105) from Department of Science and Technology, China, and Grant (KSCX2-YW-N-026) from Chinese Academy of Sciences. Supporting Information Available: Tables summarizing the identified proteins with significant differentially expressed spot volumes between R207 and RB207. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Yuan, L. Hybrid rice breeding for super high yield. Hybrid Rice 1997, 63, 1-6. (2) Giri, C.C.; Vijaya, L.G. Production of transgenic rice with agronomically useful genes: an assessment. Biotechnol. Adv. 2000, 18 (8), 653-683. (3) Spreitzer, R. J.; Salvucci, M. E. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 2002, 53, 449-475. (4) Matsuoka, M.; Furbank, R. T.; Fukayama, H.; Miyao, M. Molecular engineering of C4 photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 297-314.
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Seeking Transformation Markers (5) Nomura, M.; Higuchi, T.; Ishida, Y., Ohta, S.; Komari, T.; Imaizumi, N.; Miyao-Tokutomi, M.; Matsuoka, M.; Tajima, S. Differential expression pattern of C4 bundle sheath expression genes in rice, a C3 plant. Plant Cell. Physiol. 2005, 46(5), 754-761. (6) Zhao, B.; Jia, J.; Wang, Q.; Wang, B.; Yuan, L. Comparison of the nucleotide sequence of the specific DNA fragments between the variation line and its donor. Hybrid Rice 2001, 87, 46-49. (7) De la Pena, A.; Lorz, H.; Schell, J. Transgenic rye plant obtained by injecting DNA into young floral tillers. Nature 1987, 325, 274276. (8) Miao, J.; Zhao, M.; Li, W. Molecular evidences for total DNA transformation from Leymus racemouses to wheat. Acta Genet. Sin. 2000, 27, 621-627. (9) Hu, C.; Wang, L. In planta soybean transformation technologies developed in China: procedure, confirmation and field performance. Dev. Biol. Plant, 1999, 35, 417-420. (10) Chen, W.; Chiu, C.; Liu, Y.; Lee, T.; Cheng, J.; Lin, C.; Wu, Y.; Chang, H. Gene transfer via pollen-type pathway for antifusarium wilt in watermelon. Biochem. Mol. Biol. Int. 1998, 46, 1201-1209. (11) Zhao, B.; Jia, J.; Yang, H.; Li, C.; Zhan, Q.; Wang, B.; Zhou, K.; Yuan, L. RAPD analysis of new rice strains developed through the method of spike-stalk-injection DNA from wild relative. Acta Agron. Sin. 2000, 26, 424-430. (12) Xing, Q.; Zhao, B.; Xu, K.; Yang, H.; Liu, X.; Wang, S.; Jin, D.; Yuan, L.; Wang, B. Test of agronomic characteristics and amplified fragment length polymorphism analysis of new rice germplasm developed from transformation of genomic DNA of distant relatives. Plant Mol. Biol. Rep. 2004, 22, 155-164. (13) Agrawal, G. K.; Rakwal, R. Rice proteomics: a cornerstone for cereal food crop proteomes. Mass Spectrom. Rev. 2006, 25 (1), 1-53.
(14) Zabeau, M.; Vos, P. Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Application No. 92402629 (Publ No. 0534858A1), 1993. (15) Jeanneau, M.; Vidal, J.; Gousset-Dupont, A.; Lebouteiller, B.; Hodges, M.; Gerentes, D.; Perez, P. Manipulating PEPC levels in plants. J. Exp. Bot. 2002, 53 (376), 1837-1845. (16) Hausler, R. E.; Rademacher, T.; Li, J.; Lipka, V.; Fischer, K. L.; Schubert, S.; Kreuzaler, F.; Hirsch, H. J. Single and double overexpression of C(4)-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants. J. Exp. Bot. 2001, 52 (362), 1785-1803. (17) Hausler, R. E.; Hirsch, H. J.; Kreuzaler, F.; Peterhansel, C. Overexpression of C(4)-cycle enzymes in transgenic C(3) plants: a biotechnological approach to improve C(3)-photosynthesis. J. Exp. Bot. 2002, 53(369), 591-607. (18) Musrati, R.A.; Kollarova, M.; Mernik, N.; Mikulasova, D. Malate dehydrogenase: distribution, function and properties. Gen. Physiol. Biophys. 1998, 17 (3), 193-210. (19) Rondeau, P.; Rouch, C.; Besnard, G. NADP-malate dehydrogenase gene evolution in Andropogoneae (Poaceae): gene duplication followed by sub-functionalization. Ann. Bot. (London) 2005, 96 (7), 1307-1314. (20) Yamaya, T.; Obara, M.; Nakajima, H.; Sasaki, S.; Hayakawa, T.; Sato, T. Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. J. Exp. Bot. 2002, 53 (370), 917925. (21) Li, B.; Zhang, X.; Chollet, R. Phosphoenolpyruvate carboxylase kinase in tobacco leaves is activated by light in a similar but not identical way as in maize. Plant Physiol. 1996, 111 (2), 497-505.
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