Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L.
Abstract
:1. Introduction
2. Results
2.1. Standardization of Protein Isolation Methods
2.2. Proteome Profiling of Macro-Mutant Lines
2.2.1. Number of Differentially Expressed Proteins in the Leaves of the Mutant Lines of Nigella sativa
2.2.2. Identification and Expression Patterns of Differentially Expressed Proteins of Mutant lines
2.2.3. Functional Categorization of Differentially Expressed Proteins
2.2.4. Expression of the Genes Encoding Differentially Expressed Proteins
3. Discussion
4. Materials and Methods
4.1. The Macro-Mutant Lines of Nigella sativa L.
4.2. Analysis of the Proteome Profile of the Macro-Mutant Lines
4.2.1. Standardization of Protocols for Protein Isolation
4.2.2. Quantification of Isolated Protein
4.2.3. Two-Dimensional Gel Electrophoresis
4.2.4. Gel Imaging and Statistical Analysis
4.2.5. In-Gel Reduction
4.2.6. Identification and Functional Categorization of Differentially Expressed Proteins
4.3. Gene-Specific qRT-PCR
4.4. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
EMS | Ethyl methyl sulfonate |
2DE | Two dimensional gel electrophoresis |
MALDI-TOF | Matrix assisted laser desorption/ionization-time of flight |
CBB | Coomassie Brilliant Blue |
RT-PCR | Real-time PCR |
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Protein Isolation Method | Quantity of Protein (mg g−1 FW) | Number of Protein Spots |
---|---|---|
Phenol-based method | 3.86 ± 0.113a | 259 ± 10a |
Tris-buffer-based method | 2.94 ± 0.216c | 211 ± 13b |
TCA-acetone based method | 3.38 ± 0.142b | 214 ± 17b |
S.N. | Control/Mutants * | Number of Protein Spots |
---|---|---|
1. | Control | 241 |
2. | HY1 | 258 |
3. | HY3 | 238 |
4. | MHY2 | 222 |
5. | MHY4 | 260 |
6. | NY3 | 249 |
7. | LY1 | 219 |
8. | LY3 | 255 |
9. | VLY2 | 251 |
10. | VLY3 | 220 |
Patterns of Expression | Mutants Lines of Nigella sativa L. | ||||||||
---|---|---|---|---|---|---|---|---|---|
HY1 | HY3 | MHY2 | MHY4 | NY3 | LY1 | LY3 | VLY2 | VLY3 | |
Down-regulated | 8 | 12 | 14 | 10 | 12 | 12 | 12 | 11 | 12 |
Up-regulated | 13 | 8 | 7 | 14 | 8 | 9 | 13 | 8 | 9 |
Appearance of a new protein | 3 | 2 | 1 |
Spot No. | Protein View | MASCOT Score | Nominal Mass (Da) | pI | Sequence Match (%) | Protein Name | Protein Function | Sub-Cellular Location | Pathway/Biological Process | Accession No. |
---|---|---|---|---|---|---|---|---|---|---|
1011 | RBL_ERYCG | 148 | 50841 | 6.22 | 65 | Ribulose bisphosphate carboxylase large chain | Carboxylation of D-ribulose 1,5-bisphosphate | Chloroplast | Photosynthesis | Q33438 |
3401 | RCA_MAIZE | 119 | 48079 | 6.20 | 53 | Ribulose bisphosphate carboxylase/oxygenase activase | Activation of Rubisco | Chloroplast stroma | Photosynthesis | Q9ZT00 |
4201 | ATPG_TOBAC | 90 | 41706 | 8.1 | 38 | ATP synthase gamma chain, chloroplastic | Produces ATP from ADP in the presence of a proton gradient across the membrane. | Chloroplast thylakoid membrane | ATP synthesis, hydrogen ion transport | P29790 |
1201 | PSBO_SPIOL | 50 | 35377 | 5.5 | 30 | Oxygen evolving enhancer protein 1, chloroplastic | It stabilizes the manganese cluster, which is the primary site of water splitting. | Chloroplast thylakoid membrane | Photosynthesis | P12359 |
4301 | P2SAF_ORYSJ | 50 | 45498 | 9.0 | 24 | PSII stability/assembly factor HCF 136, chloroplastic | Essential for PS II biogenesis, required for assembly of an early intermediate in PSII assembly | Chloroplast thylakoid membrane, peripheral membrane protein | Photosynthesis | Q5Z5A8 |
2101 | HIS7A_ARATH | 74 | 29264 | 7.2 | 51 | Imidazole glycerol- phosphate dehydratase 1 | It catalyzes D-erythro-1-(imidazole-4-yl) glycerol 3 phosphate to 3-(imidazole-4-yl)-2-oxopropylphosphate releasing water. | Chloroplast | Amino acid biosynthesis, L-Histidine biosynthesis | Isoform 1: P34047-1 Isoform 2: P34047-2 |
4401 | MDHC1_ARATH | 37 | 35570 | 6.11 | 24 | Malate dehydrogenase 1 | It catalyzes a reversible NAD-dependent dehydrogenase reaction involved in central metabolism and redox homeostasis | Cytoplasm | Carbohydrate metabolic process | P93819 |
4601 | SPSA1_CRAPL | 312 | 119743 | 6.0 | 87 | Sucrose phosphate synthase 1 | It has a role in photosynthetic sucrose synthesis by catalyzing the rate limiting step of sucrose biosynthesis from UDP glucose and fructose-6- phosphate. | Chloroplast thylakoid membrane | Glycan biosynthesis, sucrose biosynthesis | O04932 |
6301 | RK23_CHLUV | 82 | 10046 | 10.2 | 63 | 50S ribosomal protein subunit L23 | Binds to 23S rRNA | Chloroplast | Translation | P56368 |
6801 | 12KD_FRAAN | 77 | 12408 | 9.1 | 64 | Auxin repressed 12.5 kDa protein | Auxin-activated signaling pathway | Auxin signaling pathway | Q05349 | |
6901 | ARP_ARATH | 92 | 60622 | 9.1 | 28 | DNA-(apurinic or apyrimidinic) lyase | It repairs oxidative DNA damages. | Chloroplast | Base-excision repair | P45951 |
7704 | ACCO2_ARATH | 89 | 36183 | 4.98 | 49 | 1-aminocyclo propane-1-carboxylate oxidase 2 | It is involved in ethylene biosynthesis. | Cell wall, ER, cytosol | Ethylene biosynthesis | Q41931 |
2908 | APX1_ARATH | 123 | 27800 | 5.72 | 68 | L-ascorbate peroxidase (APX1) | It plays a key role in hydrogen peroxide removal. | Cytoplasm | Antioxidant defense system | Q05431 |
3658 | SODM1_ARATH | 89 | 25443 | 8.47 | 58 | Manganese superoxide dismutase (MSD1) | It destroys superoxide anion radicals | Mitochondria | Defense response | O81235 |
8054 | G3PC1_ARATH | 100 | 36914 | 6.62 | 45 | Glyceraldehyde 3- phosphate dehydrogenase | It catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate into 3-phospho D-glyceroyl phosphate | Cytoplasm | Glycolytic process | P25858 |
1268 | TBA6_ARATH | 115 | 49537 | 4.93 | 56 | Tubulin alpha-6 chain (TUA6) | Structural constituent of cytoskeleton | Cytoskeleton | Microtubule cytoskeleton organization | P29511 |
7315 | EIF3I_ARATH | 125 | 36388 | 6.50 | 58 | Eukaryotic translation initiation factor 3 subunit 2(eIF3I1/TRIP-1) | Component of eukaryotic initiation factor 3 (eIF-3) | Cytoplasm | Protein synthesis | Q38884 |
2401 | AOC3_ARATH | 66 | 28015 | 9.19 | 43 | Allene oxide cyclase 3 | It is involved in the production of 12-oxo-phytodienoic acid, a precursor of jasmonic acid. | Chloroplast | Jasmonic acid biosynthesis process | Q9LS01 |
3001 | DUT_ARATH | 70 | 17603 | 5.3 | 51 | Deoxyuridine 5′-triphosphate nucleotidol hydrolase | It produces dUMP, the immediate precursor of thymidine nucleotides and decreases the intracellular concentration of Dutp | Cytoplasm | Nucleotide metabolism (pyrimidine metabolism) | Q9STG6 |
3909 | RAN1_ORYS1 | 120 | 25275 | 6.38 | 56 | GTP-binding nuclear protein Ran-1 | It is involved in nucleocyto- plasmic transport. | Nucleus | Protein import into nucleus | P41916 |
4006 | FIE1_ORYSJ | 99 | 52,100 | 7.55 | 44 | Polycomb group protein FIE1 | It is involved in cell differentiation, endosperm development, and seed development. | Nucleus | Differentiation, Transcription, Transcription regulation | Q6ZJW8 |
4518 | PRE1_ARATH | 120 | 10,560 | 9.00 | 79 | Transcription factor PRE1 | It integrates multiple signaling pathways to regulate cell elongation and plant development. | Nucleus | Gibberellic acid mediated signaling pathway | Q9FLE9 |
4900 | V5REB1_NIGSA | 51 | 23010 | 5.67 | 72 | Geranyl diphosphate synthase | It is involved in the isoprenoid biosynthetic process | Cytoplasm | Key gene of monotrepene pathway | V5REB1 |
Protein Name | Expression Pattern of Proteins in Leaves of Control and Mutant Lines of Nigella sativa L. | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Control | HY1 | HY3 | MHY2 | MHY4 | NY3 | LY1 | LY3 | VLY2 | VLY3 | |
Ribulose bisphosphate carboxylase large chain | | |||||||||
Ribulose bisphosphate carboxylase/oxygenase activase | | |||||||||
ATP synthase gamma chain | | |||||||||
Oxygen evolving enhancer protein 1 | | |||||||||
PSII stability/assembly factor HCF 136, chloroplastic | | |||||||||
Imidazole glycerol-phosphate dehydratase 1 | | |||||||||
Malate dehydrogenase 1 | | |||||||||
Sucrose phosphate synthase 1 | | |||||||||
50S ribosomal subunit L23 | | |||||||||
Auxin repressed 12.5 kDa protein | | |||||||||
DNA-(apurinic or apyrimidinic site) lyase | | |||||||||
1-aminocyclopropane-1-carboxylate oxidase 2 | | |||||||||
L-ascorbate peroxidase (APX1) | | |||||||||
Manganese superoxide dismutase (MSD1) | | |||||||||
Glyceraldehyde 3- phosphate dehydrogenase A | | |||||||||
Tubulin alpha-6 chain (TUA6) | | |||||||||
Eukaryotic translation initiation factor 3 subunit 2 (eIF3I1/TRIP-1) | | |||||||||
Allene oxide cyclase 3 | | |||||||||
Deoxyuridine 5′-triphosphate nucleotidol hydrolase | | |||||||||
GTP-binding nuclear protein Ran-1 | | |||||||||
Polycomb group protein FIE1 | | |||||||||
Transcription factor PRE1 | | |||||||||
Geranyl diphosphate synthase | |
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Asif, A.; K. Ansari, M.Y.; Hashem, A.; Tabassum, B.; Abd_Allah, E.F.; Ahmad, A. Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L. Plants 2019, 8, 321. https://doi.org/10.3390/plants8090321
Asif A, K. Ansari MY, Hashem A, Tabassum B, Abd_Allah EF, Ahmad A. Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L. Plants. 2019; 8(9):321. https://doi.org/10.3390/plants8090321
Chicago/Turabian StyleAsif, Ambreen, Mohammad Yunus K. Ansari, Abeer Hashem, Baby Tabassum, Elsayed Fathi Abd_Allah, and Altaf Ahmad. 2019. "Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L." Plants 8, no. 9: 321. https://doi.org/10.3390/plants8090321