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Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
Agricultural Sciences in China
August 2011
2011, 10(8): 1206-1212
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange PAN Zhi-yong and DENG Xiu-xin National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P.R.China
Abstract The red-flesh mutant Hong Anliu sweet orange is of high nutritional value due to its lycopene accumulation. Our previous studies on this mutant fruits suggested that photosynthesis and oxidative stress could promote the formation of mutation trait. However, leaf rather than fruit is the major part for some important biological processes such as photosynthesis. In this study, we analyzed the proteomic alteration in leaves of the red-flesh mutant Hong Anliu vs. its wild type (WT). Ten differentially expressed proteins were identified, of which two were involved in photosynthesis, three in oxidative stress, two in defense, and three in metabolism. The high up-regulation of photosynthetic proteins proved the hypothesis that enhanced photosynthesis could provide and transport more substrates into mutant fruits for carotenoid biosynthesis. Similar to the previous results in fruits, anti-oxidative proteins were highly up-regulated in leaves, suggesting the whole plant of Hong Anliu suffered from enhanced oxidative stress. Proteins involved in defense and metabolism were also identified, and their possible roles in the mutation were discussed. Key words: sweet orange, lycopene accumulation, proteomic, photosynthesis, oxidative stress
INTRODUCTION Carotenoids are organic pigments widely occurring in plant tissues and they are responsible for the the coloration of flowers and fruits, a common feature important for attracting insects to pollinate flowers and animals to spread seeds (Bartley and Scolnik 1995). Carotenoids especially lycopene have high nutritional value. Accumulating studies have shown that people consuming diets rich in carotenoids are healthier and have lower mortality from a number of chronic illnesses (Bartley and Scolnik 1995; Clinton 1998; Diplock et al. 1998). Citrus fruits are rich in carotenoids, and reported with more than 115 species of carotenoids (Rouseff and Raley 1996). Red-flesh mutants with lycopene accumulation in citrus fruits have been found in grapefruits (Citrus paradisi Macf.), pummelos (Citrus grandis Received 28 September, 2010
Osbeck) (Saunt 2000) and oranges (Citrus sinensis L. Osbeck), i.e., Shara (Monselise and Halevy 1961), Cara Cara (Saunt 2000) and Hong Anliu (Liu et al. 2007). The composition and content of carotenoids in these citrus red-flesh mutants have been categorized (Lee 2001; Xu et al. 2006; Liu et al. 2007). The analysis of carotenoid profile coupled with carotenogenic and isoprenoid genes expression in Cara Cara revealed that the altered carotenoid composition conducted to a positive feedback on carotenoid biosynthesis during fruit development and maturation (Alquezar et al. 2008). Additionally, increased isoprenoid precursors in carotenoid biosynthesis might be associated with the redflesh phenotype of Cara Cara sweet orange (Alquezar et al. 2008). We identified a red-flesh mutant Hong Anliu sweet orange and found that lycopene content in this mutant was 1 000-fold higher than that in its wild type (WT)
Accepted 30 December, 2010
Correspondence DENG Xiu-xin, Professor, Tel: +86-27-87286906, Fax: +86-27-87280016, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60111-9
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
(Liu et al. 2007). The lycopene accumulation was suggested to result from the increased expression of upstream carotenogenic genes and the reduced expression of downstream genes in lycopene biosynthesis (Liu et al. 2007). Further comprehensive transcriptomic studies, including suppression subtraction hybridization (SSH) combined with cDNA microarray and massively parallel signature sequencing technology (MPSS), provided important clues for understanding the formation of mutation traits in Hong Anliu (Liu et al. 2009; Xu et al. 2009). For instance, enhanced photosynthesis was implicated to provide more substrates for carotenoid biosynthesis and the downstream carotenogenic genes were partially impaired. A parallel proteomic study discovered that enhanced oxidative stress promoted lycopene accumulation in the mutant fruits (Pan et al. 2009). It should be pointed out that all those studies were conducted only on fruits. As some important carotenoidrelated biological processes, such as photosynthesis, are mainly occurred in leaves rather than fruits, here a comparative proteomic study in leaves was carried out between Hong Anliu and its WT. Ten differentially expressed proteins were identified, including proteins involved in photosynthesis, oxidative stress, defense and metabolism. This study emphasized the important contribution of photosynthesis and oxidative stress to lycopene accumulation in the red-flesh mutant.
MATERIALS AND METHODS Plant materials The red-flesh mutant Hong Anliu and its WT were cultivated at the Institute of Citrus Research located in Guilin, Guangxi Province, China. Both of them were of the same age, grown in the same orchard, and subjected to standard cultivation protocol. The fully expanded leaves sprouted in current year were sampled from three different trees from each genotype. The leaves were immediately frozen in liquid nitrogen and kept at -80°C until use.
Protein extraction Proteins were extracted using TCA (trichloroacetic acid) combined with phenol as previously described (Wang
1207
et al. 2010). Protein concentration was determined with the Bio-Rad Protein Assay Kit (USA) based on the Bradford method using BSA as standard. Three independent protein extractions were performed.
Two-dimensional electrophoresis (2-DE) analysis 2-DE was performed according to Pan et al. (2009). Proteins were visualized according to Giovanni et al. (2004) with modification. Briefly, gels were fixed in a solution of 40% ethanol and 10% acetic acid for 30 min, washed with MilliQ water for 3×10 min, stained overnight in a CBB G250 solution (0.12% G250, 10% phosphate acid, 10% (NH4)2SO4, 20% methanol), and destained with MilliQ water until the background was clean. Gel images were analyzed according to a previous study (Pan et al. 2009), in which Student’s t-test (P<0.05) was chosen to find out differentially expressed proteins.
Tryptic digestion and protein identification Protein tryptic digestion and identification were carried out using a previous protocol (Pan et al. 2009). MS/ MS data from MALDI-TOF/TOF analysis was submitted to MASCOT identification. National Center for Biotechnology (NCBInr 20080221) non-redundant protein database (6 122 577 sequences; 2 096 230 148 residues) was searched against. The search was performed taking green plants as taxonomy, which contained 473 596 sequences. The other parameters were enzyme of trypsin; one missed cleavage; fixed modifications of carbamidomethyl (C); variable modifications of oxidation (Met). Peptide tolerance of 100 ppm; fragment mass tolerance of ±0.3 Da; peptide charge of 1+ were selected. Only significant hits, as defined by the MASCOT probability analysis (P<0.05), were accepted. Protein score >40 was considered credible.
RESULTS AND DISCUSSION Identification and functional annotation of differentially expressed proteins 2-DE combined with MALDI-TOF-TOF MS was em-
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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ployed to investigate the leaf proteomic variation represented by the red-flesh mutant Hong Anliu (Fig. 1). Statistical analysis of relative quantity data of each protein spot in Hong Anliu and WT was performed with Student’s t-test. Seventeen protein spots were significantly differentially expressed (P<0.05), of which 13 were up-regulated and 4 were down-regulated. The differentially expressed proteins were excised from CBB G250 stained gels and submitted to MALDI-TOF-TOF MS. Using an interrogation of the NCBI non-redundant green plant database with the Mascot search engine, 10 proteins were identified with confidence identification (Mascot score >40, Table). The detailed MS data of the identified proteins were listed in Appendix A, including spot no., protein name, species, accession no., matched peptide count, protein score, and matched peptide sequence. These 10 identified proteins and their expression pattern were enlarged in Fig. 2.
Fig. 1 2-DE profiles of the leaf proteome from Hong Anliu and WT.
It is worth to mention that the observed molecular weight (MW) and isoelectric point (pI) showed some deviation from theoretical MW and pI (Table). This phenomenon might be due to the inherent deviation between experimental and theoretical values, protein posttranslational modification, or partial protein degradation. For instance, the observed MW is smaller than theoretical MW in rubisco activase (spot 2), which might result from partial degradation of the subunits. Both spots 3 and 4 were identified as the same protein Cu/ Zn superoxide dismutase with the same MW but different pI, suggesting they underwent post-translational modification. There might be also the cases in glyceraldehyde-3-phosphate dehydrogenase (spot 8), mitochondrial substrate carrier family protein (spot 9) and
PAN Zhi-yong et al.
Fig. 2 The enlarged views of proteins differentially expressed between leaves of Hong Anliu and WT.
cysteine synthase (spot 10). The 10 identified proteins included 9 with annotation and one (spot 5) without. Blastp search of this protein (spot 5) revealed it showed 63% identities with a putative iron/ascorbate-dependent oxidoreductase (gi|33115140). This protein (gi|33115140) together with other 9 proteins was taken into functional categories. Based on the metabolic and functional features as described in KEGG pathways, GO annotation and related literatures, two identified proteins were predicted to be involved in photosynthesis (spots 1-2), three in oxidative stress (spots 3-5), two in defense (spots 67), and three in metabolism (spots 8-10). Each group of the differentially expressed proteins was discussed according to their potential roles in the red-flesh mutation.
Photosynthesis related proteins Two proteins, photosystem II (PSII) protein (spot 1) and rubisco activase (spot 2), were involved in photosynthesis. PSII is the pigment protein complex embedded in the thylakoid membrane of higher plants, algae, and cyanobacteria that harnesses solar energy to drive the photosynthetic water-splitting reaction (Buchanan et al. 2004); rubisco activase participates in photosynthesis by activating the Rubisco (ribulose-1, 5-biphosphate-carboxylase/oxygenase) which catalyzes CO2 assimilation by the carboxylase/oxygenase reaction of ribulose-1,5-biphosphate (RuBP) (Salvucci and Ogren 1996). The significant up-regulation of photo-
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
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Table Identification of differentially expressed proteins between Hong Anliu and WT1) Pep. count
Protein score
Theoretical MW (kDa)/pI
Observed MW (kDa)/pI
Photosystem II protein 33 kDa Spinacia oleracea
7
248
33.0/5.0
32.8/5.1
gi|12620881
Rubisco activase
Gossypium hirsutum
4
44
47.9/5.5
25.6/5.5
3
gi|2274917
Cu/Zn superoxide dismutase
Citrus sinensis
4
146
12.7/5.8
12.7/5.3
4
gi|2274917
Cu/Zn superoxide dismutase
Citrus sinensis
5
497
12.7/5.8
12.7/6.4
5
gi|115466716
Os06g0176700
Oryza sativa
9
48
23.9/5.0
25.6/5.7
Spot no.
Accession no.
1
gi|224916
2
Protein name
Species
Expression pattern1)
(japonica cultivar-group) 6
gi|23496447
Acidic class II chitinase
Citrus jambhiri
2
70
32.1/5.1
39.0/4.8
7
gi|1220144
Chitinase
Citrus sinensis
2
61
31.9/5.0
39.0/4.9
8
gi|120665
Glyceraldehyde-3-phosphate
Nicotiana tabacum
5
106
47.4/8.8
50.0/6.3
Arabidopsis thaliana
11
54
33.9/9.9
31.5/6.0
Arabidopsis thaliana
7
60
41.6/8.1
41.8/5.5
dehydrogenase 9
gi|15240954
Mitochondrial substrate carrier family protein
10 1)
gi|15224351
Cysteine synthase
Protein relative expression in WT (open squares) and Hong Anliu (filled squares) is provided. Columns and bars represent the means and SE (n=3), respectively.
synthetic proteins in the mutant leaves (Table, Fig. 2) well consistent with our previous transcriptomic study that genes involved in photosynthesis exhibited an upregulated pattern in the red-flesh mutant fruits (Xu et al. 2009). Similarly, in a lycopene-accumulated hp-2 tomato mutant, genes encoding proteins of photosynthetic apparatus were up-regulated at most fruit development stages (Kolotilin et al. 2007). Although the previous studies on citrus and tomato fruits implied photosynthesis would promote carotenoid biosynthesis, the fruit actually is not a major part for photosynthesis. Our result that photosynthetic proteins were highly up-regulated in leaves of the red-flesh mutant vs. its WT, proved that photosynthesis was enhanced in the red-flesh mutant. The determination of photosynthetic rate and stomatal conductance also revealed that photosynthesis was indeed enhanced in the red-flesh mutant (Xu et al. 2009), thus the current proteomic study provided a molecular evidence for the close relationship between photosynthesis and carotenoid biosynthesis. Photosynthesis produces sugars which participate in glycolysis leading to pyruvate. The resultant pyruvate is an important intermediate providing substrates for methylerythritol phosphate (MEP) pathway followed
by carotenoid biosynthesis. Therefore enhanced photosynthesis would provide more substrates for carotenoid biosynthesis. It is worth to note that the leaf proteomics together with previous fruits transcriptomics suggested that both leaves and fruits could provide substrates for carotenoid biosynthesis. However, leaves were believed to provide and transport major substrates into fruits. For the special accumulation of lycopene, it seems to just occur in fruits by the partial impairment of downstream carotenogenic genes of lycopene biosynthesis (Liu et al. 2007; Xu et al. 2009).
Anti-oxidative proteins Both spots 3 and 4 were identified as Cu/Zn superoxide dismutase (Table). Cu/Zn superoxide dismutase is a member of superoxide dismutase (SOD) that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. It is an important antioxidant defense in nearly all cells exposed to oxygen (Fridovich 1995). Over-expression of Cu/Zn superoxide dismutase increased resistance to oxidative stress in tobacco (Gupta et al. 1993), potato (Perl et al. 1993), rice (Prashanth et al. 2008), and alfalfa (McKersie et al. 2000). An-
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other protein Os06g0176700 (spot 5), a homology of iron/ascorbate-dependent oxidoreductase, is also involved in oxidative stress. All the anti-oxidative proteins (spots 3-5) were significantly up-regulated in the mutant leaves (Table, Fig. 2), suggesting that the whole plant of mutant suffered from enhanced oxidative stress. However, the leaf and fruit would cooperate to resist oxidative stress via promoting lycopene accumulation.
Defense-related proteins Both spots 6 and 7 were identified as members of chitinase, and they were significantly up-regulated (Table, Fig. 2). Chitinase is a class of pathogenesisrelated proteins and could prevent fungal development by hydrolyzing fungal cell wall (Schlumbaum et al. 1986). Chitinase could also protect plant against bacteria and insect attack (Collinge et al. 1993; Zhao and Chye 1999). The up-regulation of chitinase supported our previous speculation that certain unknown stress was enhanced in the mutant (Pan et al. 2009).
Metabolism The sweet orange red-flesh mutant exhibited a pleiotropic mutation conferring not only abnormal accumulation of lycopene, but also low citric acid and high sucrose (Liu et al. 2007). Extensive molecular pathways such as photosynthesis, carotenoid biosynthesis, citric acid cycle and glycolysis were affected by the mutation (Liu et al. 2009; Xu et al. 2009), among which genes involved in glycolysis were generally up-regulated in the mutant. It is reasonable that enhanced glycolysis benefits carotenoid biosynthesis since it provides the substrate pyruvate for MEP pathway followed by carotenoid biosynthesis. It seems contradictory that glyceraldehyde-3-phosphate dehydrogenase (GAPDH, spot 8) was down-regulated as GAPDH is an important enzyme in glycolysis by catalyzing glyceraldehyde 3phosphate (GADP) to 1,3-bisphosphoglycerate (1, 3BPG). Studies in mammals found that GAPDH plays broad roles in many biological processes, covering the functions in membrane-bound and transport (Ercolani et al. 1992; Robbins et al. 1995), phosphotransferase (Engel et al. 1998), apoptosis (Tsuchiya et al. 2004), and histone expression (Zheng et al. 2003). Thus,
PAN Zhi-yong et al.
GAPDH may also act multiple functions in plants, and it is not just sole protein in glycolysis. Therefore, it is difficult to assess its role in a single pathway by protein expression pattern. The mitochondrial substrate carrier family protein (spot 9, Table) spans the mitochondrial lipid bilayer and catalyzes the transport of metabolites across the inner mitochondrial membrane (Palmieri 1994). The down-regulation of mitochondrial substrate carrier protein would reduce the substrate transport between mitochondrial and cytosol and thus reduce the metabolic efficiency in mitochondrial metabolisms such as citric acid cycle, which might be associated with the trait of low citric acid in the mutant. Spot 10 (Table) was identified as cysteine synthase. This enzyme participates in three metabolic pathways: cysteine, selenoamino acid and sulfur metabolism. Its up-regulation suggested that these metabolisms were affected by the mutation. The major role of cysteine synthase seems to resist oxidative stress because cysteine serves as a precursor for the synthesis of glutathione (GSH) which functions as a donor of reducing equivalents for reactive oxygen species (ROS) scavenging (Youssefian et al. 2001; Mittler et al. 2004). The up-regulation of cysteine synthase indicated that oxidative stress was enhanced in the mutant which was consistent with the idea mentioned above.
CONCLUSION This study provided important clues that enhanced photosynthesis and oxidative stress would promote the formation of red-flesh trait in the Cara Cara sweet orange. The results well verified and complemented our previous transcriptomic and proteomic data and were helpful in better understanding the mechanism of red-flesh mutation in sweet orange.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2011CB100601) and the National Natural Science Foundation of China (30830078, 30921002). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/appendix
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
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© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
August 2011
2011, 10(8): 1206-1212
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange PAN Zhi-yong and DENG Xiu-xin National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P.R.China
Abstract The red-flesh mutant Hong Anliu sweet orange is of high nutritional value due to its lycopene accumulation. Our previous studies on this mutant fruits suggested that photosynthesis and oxidative stress could promote the formation of mutation trait. However, leaf rather than fruit is the major part for some important biological processes such as photosynthesis. In this study, we analyzed the proteomic alteration in leaves of the red-flesh mutant Hong Anliu vs. its wild type (WT). Ten differentially expressed proteins were identified, of which two were involved in photosynthesis, three in oxidative stress, two in defense, and three in metabolism. The high up-regulation of photosynthetic proteins proved the hypothesis that enhanced photosynthesis could provide and transport more substrates into mutant fruits for carotenoid biosynthesis. Similar to the previous results in fruits, anti-oxidative proteins were highly up-regulated in leaves, suggesting the whole plant of Hong Anliu suffered from enhanced oxidative stress. Proteins involved in defense and metabolism were also identified, and their possible roles in the mutation were discussed. Key words: sweet orange, lycopene accumulation, proteomic, photosynthesis, oxidative stress
INTRODUCTION Carotenoids are organic pigments widely occurring in plant tissues and they are responsible for the the coloration of flowers and fruits, a common feature important for attracting insects to pollinate flowers and animals to spread seeds (Bartley and Scolnik 1995). Carotenoids especially lycopene have high nutritional value. Accumulating studies have shown that people consuming diets rich in carotenoids are healthier and have lower mortality from a number of chronic illnesses (Bartley and Scolnik 1995; Clinton 1998; Diplock et al. 1998). Citrus fruits are rich in carotenoids, and reported with more than 115 species of carotenoids (Rouseff and Raley 1996). Red-flesh mutants with lycopene accumulation in citrus fruits have been found in grapefruits (Citrus paradisi Macf.), pummelos (Citrus grandis Received 28 September, 2010
Osbeck) (Saunt 2000) and oranges (Citrus sinensis L. Osbeck), i.e., Shara (Monselise and Halevy 1961), Cara Cara (Saunt 2000) and Hong Anliu (Liu et al. 2007). The composition and content of carotenoids in these citrus red-flesh mutants have been categorized (Lee 2001; Xu et al. 2006; Liu et al. 2007). The analysis of carotenoid profile coupled with carotenogenic and isoprenoid genes expression in Cara Cara revealed that the altered carotenoid composition conducted to a positive feedback on carotenoid biosynthesis during fruit development and maturation (Alquezar et al. 2008). Additionally, increased isoprenoid precursors in carotenoid biosynthesis might be associated with the redflesh phenotype of Cara Cara sweet orange (Alquezar et al. 2008). We identified a red-flesh mutant Hong Anliu sweet orange and found that lycopene content in this mutant was 1 000-fold higher than that in its wild type (WT)
Accepted 30 December, 2010
Correspondence DENG Xiu-xin, Professor, Tel: +86-27-87286906, Fax: +86-27-87280016, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60111-9
Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
(Liu et al. 2007). The lycopene accumulation was suggested to result from the increased expression of upstream carotenogenic genes and the reduced expression of downstream genes in lycopene biosynthesis (Liu et al. 2007). Further comprehensive transcriptomic studies, including suppression subtraction hybridization (SSH) combined with cDNA microarray and massively parallel signature sequencing technology (MPSS), provided important clues for understanding the formation of mutation traits in Hong Anliu (Liu et al. 2009; Xu et al. 2009). For instance, enhanced photosynthesis was implicated to provide more substrates for carotenoid biosynthesis and the downstream carotenogenic genes were partially impaired. A parallel proteomic study discovered that enhanced oxidative stress promoted lycopene accumulation in the mutant fruits (Pan et al. 2009). It should be pointed out that all those studies were conducted only on fruits. As some important carotenoidrelated biological processes, such as photosynthesis, are mainly occurred in leaves rather than fruits, here a comparative proteomic study in leaves was carried out between Hong Anliu and its WT. Ten differentially expressed proteins were identified, including proteins involved in photosynthesis, oxidative stress, defense and metabolism. This study emphasized the important contribution of photosynthesis and oxidative stress to lycopene accumulation in the red-flesh mutant.
MATERIALS AND METHODS Plant materials The red-flesh mutant Hong Anliu and its WT were cultivated at the Institute of Citrus Research located in Guilin, Guangxi Province, China. Both of them were of the same age, grown in the same orchard, and subjected to standard cultivation protocol. The fully expanded leaves sprouted in current year were sampled from three different trees from each genotype. The leaves were immediately frozen in liquid nitrogen and kept at -80°C until use.
Protein extraction Proteins were extracted using TCA (trichloroacetic acid) combined with phenol as previously described (Wang
1207
et al. 2010). Protein concentration was determined with the Bio-Rad Protein Assay Kit (USA) based on the Bradford method using BSA as standard. Three independent protein extractions were performed.
Two-dimensional electrophoresis (2-DE) analysis 2-DE was performed according to Pan et al. (2009). Proteins were visualized according to Giovanni et al. (2004) with modification. Briefly, gels were fixed in a solution of 40% ethanol and 10% acetic acid for 30 min, washed with MilliQ water for 3×10 min, stained overnight in a CBB G250 solution (0.12% G250, 10% phosphate acid, 10% (NH4)2SO4, 20% methanol), and destained with MilliQ water until the background was clean. Gel images were analyzed according to a previous study (Pan et al. 2009), in which Student’s t-test (P<0.05) was chosen to find out differentially expressed proteins.
Tryptic digestion and protein identification Protein tryptic digestion and identification were carried out using a previous protocol (Pan et al. 2009). MS/ MS data from MALDI-TOF/TOF analysis was submitted to MASCOT identification. National Center for Biotechnology (NCBInr 20080221) non-redundant protein database (6 122 577 sequences; 2 096 230 148 residues) was searched against. The search was performed taking green plants as taxonomy, which contained 473 596 sequences. The other parameters were enzyme of trypsin; one missed cleavage; fixed modifications of carbamidomethyl (C); variable modifications of oxidation (Met). Peptide tolerance of 100 ppm; fragment mass tolerance of ±0.3 Da; peptide charge of 1+ were selected. Only significant hits, as defined by the MASCOT probability analysis (P<0.05), were accepted. Protein score >40 was considered credible.
RESULTS AND DISCUSSION Identification and functional annotation of differentially expressed proteins 2-DE combined with MALDI-TOF-TOF MS was em-
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ployed to investigate the leaf proteomic variation represented by the red-flesh mutant Hong Anliu (Fig. 1). Statistical analysis of relative quantity data of each protein spot in Hong Anliu and WT was performed with Student’s t-test. Seventeen protein spots were significantly differentially expressed (P<0.05), of which 13 were up-regulated and 4 were down-regulated. The differentially expressed proteins were excised from CBB G250 stained gels and submitted to MALDI-TOF-TOF MS. Using an interrogation of the NCBI non-redundant green plant database with the Mascot search engine, 10 proteins were identified with confidence identification (Mascot score >40, Table). The detailed MS data of the identified proteins were listed in Appendix A, including spot no., protein name, species, accession no., matched peptide count, protein score, and matched peptide sequence. These 10 identified proteins and their expression pattern were enlarged in Fig. 2.
Fig. 1 2-DE profiles of the leaf proteome from Hong Anliu and WT.
It is worth to mention that the observed molecular weight (MW) and isoelectric point (pI) showed some deviation from theoretical MW and pI (Table). This phenomenon might be due to the inherent deviation between experimental and theoretical values, protein posttranslational modification, or partial protein degradation. For instance, the observed MW is smaller than theoretical MW in rubisco activase (spot 2), which might result from partial degradation of the subunits. Both spots 3 and 4 were identified as the same protein Cu/ Zn superoxide dismutase with the same MW but different pI, suggesting they underwent post-translational modification. There might be also the cases in glyceraldehyde-3-phosphate dehydrogenase (spot 8), mitochondrial substrate carrier family protein (spot 9) and
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Fig. 2 The enlarged views of proteins differentially expressed between leaves of Hong Anliu and WT.
cysteine synthase (spot 10). The 10 identified proteins included 9 with annotation and one (spot 5) without. Blastp search of this protein (spot 5) revealed it showed 63% identities with a putative iron/ascorbate-dependent oxidoreductase (gi|33115140). This protein (gi|33115140) together with other 9 proteins was taken into functional categories. Based on the metabolic and functional features as described in KEGG pathways, GO annotation and related literatures, two identified proteins were predicted to be involved in photosynthesis (spots 1-2), three in oxidative stress (spots 3-5), two in defense (spots 67), and three in metabolism (spots 8-10). Each group of the differentially expressed proteins was discussed according to their potential roles in the red-flesh mutation.
Photosynthesis related proteins Two proteins, photosystem II (PSII) protein (spot 1) and rubisco activase (spot 2), were involved in photosynthesis. PSII is the pigment protein complex embedded in the thylakoid membrane of higher plants, algae, and cyanobacteria that harnesses solar energy to drive the photosynthetic water-splitting reaction (Buchanan et al. 2004); rubisco activase participates in photosynthesis by activating the Rubisco (ribulose-1, 5-biphosphate-carboxylase/oxygenase) which catalyzes CO2 assimilation by the carboxylase/oxygenase reaction of ribulose-1,5-biphosphate (RuBP) (Salvucci and Ogren 1996). The significant up-regulation of photo-
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Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
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Table Identification of differentially expressed proteins between Hong Anliu and WT1) Pep. count
Protein score
Theoretical MW (kDa)/pI
Observed MW (kDa)/pI
Photosystem II protein 33 kDa Spinacia oleracea
7
248
33.0/5.0
32.8/5.1
gi|12620881
Rubisco activase
Gossypium hirsutum
4
44
47.9/5.5
25.6/5.5
3
gi|2274917
Cu/Zn superoxide dismutase
Citrus sinensis
4
146
12.7/5.8
12.7/5.3
4
gi|2274917
Cu/Zn superoxide dismutase
Citrus sinensis
5
497
12.7/5.8
12.7/6.4
5
gi|115466716
Os06g0176700
Oryza sativa
9
48
23.9/5.0
25.6/5.7
Spot no.
Accession no.
1
gi|224916
2
Protein name
Species
Expression pattern1)
(japonica cultivar-group) 6
gi|23496447
Acidic class II chitinase
Citrus jambhiri
2
70
32.1/5.1
39.0/4.8
7
gi|1220144
Chitinase
Citrus sinensis
2
61
31.9/5.0
39.0/4.9
8
gi|120665
Glyceraldehyde-3-phosphate
Nicotiana tabacum
5
106
47.4/8.8
50.0/6.3
Arabidopsis thaliana
11
54
33.9/9.9
31.5/6.0
Arabidopsis thaliana
7
60
41.6/8.1
41.8/5.5
dehydrogenase 9
gi|15240954
Mitochondrial substrate carrier family protein
10 1)
gi|15224351
Cysteine synthase
Protein relative expression in WT (open squares) and Hong Anliu (filled squares) is provided. Columns and bars represent the means and SE (n=3), respectively.
synthetic proteins in the mutant leaves (Table, Fig. 2) well consistent with our previous transcriptomic study that genes involved in photosynthesis exhibited an upregulated pattern in the red-flesh mutant fruits (Xu et al. 2009). Similarly, in a lycopene-accumulated hp-2 tomato mutant, genes encoding proteins of photosynthetic apparatus were up-regulated at most fruit development stages (Kolotilin et al. 2007). Although the previous studies on citrus and tomato fruits implied photosynthesis would promote carotenoid biosynthesis, the fruit actually is not a major part for photosynthesis. Our result that photosynthetic proteins were highly up-regulated in leaves of the red-flesh mutant vs. its WT, proved that photosynthesis was enhanced in the red-flesh mutant. The determination of photosynthetic rate and stomatal conductance also revealed that photosynthesis was indeed enhanced in the red-flesh mutant (Xu et al. 2009), thus the current proteomic study provided a molecular evidence for the close relationship between photosynthesis and carotenoid biosynthesis. Photosynthesis produces sugars which participate in glycolysis leading to pyruvate. The resultant pyruvate is an important intermediate providing substrates for methylerythritol phosphate (MEP) pathway followed
by carotenoid biosynthesis. Therefore enhanced photosynthesis would provide more substrates for carotenoid biosynthesis. It is worth to note that the leaf proteomics together with previous fruits transcriptomics suggested that both leaves and fruits could provide substrates for carotenoid biosynthesis. However, leaves were believed to provide and transport major substrates into fruits. For the special accumulation of lycopene, it seems to just occur in fruits by the partial impairment of downstream carotenogenic genes of lycopene biosynthesis (Liu et al. 2007; Xu et al. 2009).
Anti-oxidative proteins Both spots 3 and 4 were identified as Cu/Zn superoxide dismutase (Table). Cu/Zn superoxide dismutase is a member of superoxide dismutase (SOD) that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. It is an important antioxidant defense in nearly all cells exposed to oxygen (Fridovich 1995). Over-expression of Cu/Zn superoxide dismutase increased resistance to oxidative stress in tobacco (Gupta et al. 1993), potato (Perl et al. 1993), rice (Prashanth et al. 2008), and alfalfa (McKersie et al. 2000). An-
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1210
other protein Os06g0176700 (spot 5), a homology of iron/ascorbate-dependent oxidoreductase, is also involved in oxidative stress. All the anti-oxidative proteins (spots 3-5) were significantly up-regulated in the mutant leaves (Table, Fig. 2), suggesting that the whole plant of mutant suffered from enhanced oxidative stress. However, the leaf and fruit would cooperate to resist oxidative stress via promoting lycopene accumulation.
Defense-related proteins Both spots 6 and 7 were identified as members of chitinase, and they were significantly up-regulated (Table, Fig. 2). Chitinase is a class of pathogenesisrelated proteins and could prevent fungal development by hydrolyzing fungal cell wall (Schlumbaum et al. 1986). Chitinase could also protect plant against bacteria and insect attack (Collinge et al. 1993; Zhao and Chye 1999). The up-regulation of chitinase supported our previous speculation that certain unknown stress was enhanced in the mutant (Pan et al. 2009).
Metabolism The sweet orange red-flesh mutant exhibited a pleiotropic mutation conferring not only abnormal accumulation of lycopene, but also low citric acid and high sucrose (Liu et al. 2007). Extensive molecular pathways such as photosynthesis, carotenoid biosynthesis, citric acid cycle and glycolysis were affected by the mutation (Liu et al. 2009; Xu et al. 2009), among which genes involved in glycolysis were generally up-regulated in the mutant. It is reasonable that enhanced glycolysis benefits carotenoid biosynthesis since it provides the substrate pyruvate for MEP pathway followed by carotenoid biosynthesis. It seems contradictory that glyceraldehyde-3-phosphate dehydrogenase (GAPDH, spot 8) was down-regulated as GAPDH is an important enzyme in glycolysis by catalyzing glyceraldehyde 3phosphate (GADP) to 1,3-bisphosphoglycerate (1, 3BPG). Studies in mammals found that GAPDH plays broad roles in many biological processes, covering the functions in membrane-bound and transport (Ercolani et al. 1992; Robbins et al. 1995), phosphotransferase (Engel et al. 1998), apoptosis (Tsuchiya et al. 2004), and histone expression (Zheng et al. 2003). Thus,
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GAPDH may also act multiple functions in plants, and it is not just sole protein in glycolysis. Therefore, it is difficult to assess its role in a single pathway by protein expression pattern. The mitochondrial substrate carrier family protein (spot 9, Table) spans the mitochondrial lipid bilayer and catalyzes the transport of metabolites across the inner mitochondrial membrane (Palmieri 1994). The down-regulation of mitochondrial substrate carrier protein would reduce the substrate transport between mitochondrial and cytosol and thus reduce the metabolic efficiency in mitochondrial metabolisms such as citric acid cycle, which might be associated with the trait of low citric acid in the mutant. Spot 10 (Table) was identified as cysteine synthase. This enzyme participates in three metabolic pathways: cysteine, selenoamino acid and sulfur metabolism. Its up-regulation suggested that these metabolisms were affected by the mutation. The major role of cysteine synthase seems to resist oxidative stress because cysteine serves as a precursor for the synthesis of glutathione (GSH) which functions as a donor of reducing equivalents for reactive oxygen species (ROS) scavenging (Youssefian et al. 2001; Mittler et al. 2004). The up-regulation of cysteine synthase indicated that oxidative stress was enhanced in the mutant which was consistent with the idea mentioned above.
CONCLUSION This study provided important clues that enhanced photosynthesis and oxidative stress would promote the formation of red-flesh trait in the Cara Cara sweet orange. The results well verified and complemented our previous transcriptomic and proteomic data and were helpful in better understanding the mechanism of red-flesh mutation in sweet orange.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2011CB100601) and the National Natural Science Foundation of China (30830078, 30921002). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/appendix
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Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange
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