Characterisation of a maternally inherited chlorophyll deficiency mutation
The chlorophyll-deficient mutant (cdm) used herein was screened by EMS treatment of isolated microspores and proved to be a case of maternal inheritance [31]. All cdm plants exhibited pale-yellow leaves, reduced growth, and severely altered chloroplast ultrastructure (Fig. 1). Compared with the wildtype ‘FT’, cdm leaves had significantly lower net rates of photosynthesis (Pn), stomatal conductance (gs), and transpiration (E) and intercellular CO2 concentrations (Ci) (Table 1). Our results indicate that the cdm affects chlorophyll synthesis and chloroplast development, thereby affecting photosynthetic efficiency.
Table 1
Photosynthetic characteristics of ‘FT’ and cdm at the seedling stage
Variey | Net photosynthetic rate (Pn) (µmol·m− 2s− 1) | Stomatal conductance (gs) (µmol·m− 2s− 1) | Intercellular CO2 concentration (Ci) (µmol·m− 2s− 1) | Transpiration rate (E) (µmol·m− 2s− 1) |
18.52 ± 0.45* | 0.21 ± 0.01* | 5.59 ± 0.05* | 378 ± 7.89* |
cdm | 11.21 ± 0.03 | 0.13 ± 0.45 | 3.01 ± 0.41 | 311 ± 5.83 |
Note: Mean and standard error (SE) values were calculated from ten independent replicates, *significantly different at a level of 0.05 by t-test. |
Quantitative Identification And Analysis Of Leaf Proteins
To investigate DEPs associated with the maternally inherited cdm, proteomic analysis of cdm and ‘FT’ was performed. Six samples—including three independent replicates, cdm-1, cdm-2, cdm-3, FT-1, FT-2, and FT-3—were labelled by iTRAQ tag, and 672,463 spectra were generated, 120,475 of which matched known peptides. The distribution of peptide-sequence coverage and charge statistics are shown in Figure S1. Protein coverage was highest at 0–5%, followed by 5–10%, and lowest at 35–40%. Peptide length concentrated at 7–17 amino acid residues, with most identifiable proteins containing more than 11 peptides. In terms of mass and PI distribution, good coverage (0–2.3% of total proteins in each molecular group) was obtained for a wide range of molecular weights (9–289 kDa), while PI varied from 3.8–12.2. A total of 6,245 proteins were identified in the Brassica_rapa_20100830.fasta library (Additional file 2: Table S2).
We next annotated 5,396 proteins using gene ontology (GO) analysis, 3,417 of which were annotated as ‘cellular component’, distributed between ‘cell constituent’ (90.4%), ‘cytoplasm’ (73.5%), ‘organelle’ (65.5%), ‘chloroplast’ (23.4%), ‘nucleus’ (15%), ‘chloroplast stroma’ (9.1%), ‘cell membrane’ (40.4%), ‘thylakoid’ (7.8%), and ‘photosystem’ (1.8%). Additionally, 4,036 proteins were annotated as ‘biological processes’, significantly enriched for ‘cell process’ (73.5%), ‘metabolic process’ (72%), ‘single-organism process’ (46.6%), ‘cellular biosynthetic process’ (32.8%), ‘response to stimuli’ (18.8%), ‘gene expression’ (18.3%), ‘translation’ (13.2%), and ‘photosynthesis’ (3.7%). Lastly, 4,737 proteins were annotated as ‘molecular function’, significantly enriched for ‘catalytic activity’ (66.9%), ‘binding’ (60.4%), ‘hydrolase activity’ (22.7%), ‘transferase activity’ (20.9%), ‘oxidoreductase activity’ (14.2%), ‘structural molecule activity’ (10.0%), ‘transporter activity’ (6.3%), ‘peptidase activity’ (4.9%), and ‘chlorophyll binding’ (0.3%) (Additional file 3: Table S3).
In addition, a Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis revealed 3,896 proteins enriched in 129 pathways including, ‘metabolic pathway’ (42.1%), ‘secondary metabolite biosynthesis’ (24.8%), ‘ribosome synthesis’ (10.6%), ‘carbon metabolism’ (8.3%), ‘amino acid biosynthesis’ (6.5%), ‘protein processing in endoplasmic reticulum’ (5.8%), ‘endocytosis’ (5.3%), ‘RNA transport’ (5.2%), ‘spliceosome’ (4.1%), ‘plant-pathogen interaction’ (4.1%), ‘oxidative phosphorylation’ (3.9%), ‘amino sugar and nucleotide sugar metabolism’ (3.5%), ‘carbon fixation in photosynthetic organisms’ (3.0%), ‘ribosome biogenesis in eukaryotes’ (2.7%), ‘acetaldehyde glyoxylate and dicarboxylate metabolism’ (2.6%), ‘glycine, serine, and threonine metabolism’ (2.4%), ‘glycolysis/gluconeogenesis’ (2.4%), ‘photosynthesis’ (2.1%), ‘fructose and mannose metabolism’ (1.9%), ‘pentose phosphate pathway’ (1.8%), ‘peroxisome’ (1.7%), ‘glyceride metabolism’ (‘glycerolipid metabolism’, 1.0%), ‘photosynthesis-antenna protein’ (0.8%), among other metabolic pathways (Additional file 4: Table S4).
Differences in leaf protein composition in cdm as compared to ‘FT’ leaves maternally inherited chlorophyll-deficient mutation
Comparative analysis of protein expression in cdm and ‘FT’ leaves revealed 540 DEPs with an expression difference greater than 1.2-fold (p < 0.05). In cdm (relative to ‘FT’) 233 (43.1%) DEPs were upregulated and 307 (56.9%) downregulated. Furthermore, 170 DEPs were detected in cdm using MRM, of which 71 were upregulated and 75 downregulated. Further, MRM data agreed with iTRAQ data (Fig. 2, Additional file 5: Table S5).
DEPs were classified by GO analysis into three groups—cellular components, biological processes, and molecular functions. GO enrichment analysis annotated 310 proteins in different cellular components, including ‘cell part’ (90.3%), ‘cytoplasm’ (73.5%), ‘organelle’ (71.9%), ‘cell membrane’ (45.5%), ‘chloroplast’ (36.8%), ‘nucleus’ (12.9%), ‘chloroplast stroma’ (10.0%), ‘thylakoid’ (19.4%), and ‘photosystem’ (9.7%). Based on biological process properties, 337 proteins were classified into ‘cellular process’ (81.0%), ‘metabolic process’ (80.1%), ‘single-organism process’ (40.0%), ‘cellular biosynthetic process’ (31.2%), ‘gene expression’ (22.6%), ‘translation’ (17.5%), and ‘photosynthesis’ (17.2%). Furthermore, 400 DEPs were categorised in the ‘molecular function’ category, and were enriched for ‘catalytic activity’ (53.5%), ‘binding’ (65.6%), ‘hydrolase activity’ (17.8%), ‘transferase activity’ (13.3%), ‘oxidoreductase activity’ (14.0%), ‘structural molecule activity’ (13.8%), ‘transporter activity’ (8.0%), ‘peptidase activity’ (2.8%), and ‘chlorophyll binding’ (2.8%) (Additional file 6: Table S6).
The 540 DEPs were further analysed using KEGG pathway, allowing us to determine that 333 of these proteins were enriched in 94 metabolic pathways. Significantly enriched pathways were sorted according to the number of proteins, including ‘metabolic pathway’ (48.0%), ‘biosynthesis of secondary metabolites’ (20.1%), ‘ribosome synthesis’ (17.1%), ‘carbon metabolism’ (13.5%), ‘photosynthesis’ (10.5%), ‘protein processing in endoplasmic reticulum’ (7.2%), ‘glyoxylate and dicarboxylate metabolism’ (6.6%), ‘carbon fixation in photosynthetic organisms’ (6.3%), ‘photosynthesis-antenna proteins’ (6.0%), ‘RNA transport’ (5.1%), ‘amino acid biosynthesis’ (4.2%), ‘glycine, serine, and threonine metabolism’ (3.6%), ‘amino sugar and nucleotide sugar metabolism’ (3.6%), ‘glycolysis/gluconeogenesis’ (3.3%), ‘pentose phosphate pathway’ (3.0%), ‘peroxisome metabolism’ (3.0%), ‘fructose and mannose metabolism’ (2.7%), ‘oxidative phosphorylation’ (2.7%), ‘ribosome biogenesis in eukaryotes’ (2.7%), and ‘glycerolipid metabolism’ (2.4%) (Additional file 7: Table S7). Comparative analysis of DEPs between cdm and ‘FT’ improves our understanding of chlorophyll deficiency mechanisms in Chinese cabbage.
Characterisation of downregulated expression proteins (DRPs) in cdm and their role in the cdm phenotype
Enrichment analyses between cdm and ‘FT’ allowed for the annotation of 255 and 194 DRPs using GO and KEGG pathway analyses, respectively (Additional file 8: Table S8, Additional file 9: Table S9). The 255 GO DRPs were significantly enriched in the ‘cell-component’ categories, including ‘thylakoid’ (p = 3.49e-58), ‘photosynthetic membrane’ (p = 4.73e-55), ‘chloroplast’ (p = 1.44e-53), ‘photosystem’ (p = 1.33e-42), ‘photosystem Ⅱ’ (p = 1.57e-19), and ‘photosystem Ⅰ’ (p = 3.32e-26). In terms of ‘biological processes’, DRPs were strongly enriched in ‘photosynthesis’ (p = 2.67e-75), ‘photosynthetic reaction centre’ (p = 2.41e-40), ‘generation of precursor metabolites’ (p = 8.15e-34), ‘photosynthetic light harvesting’ (p = 4.41e-29), ‘metabolic process’ (p = 3.30e-27), ‘protein chromophore linkage’ (p = 1.63e-12), ‘photorespiration’ (p = 3.77e-12), ‘carbon fixation’ (p = 3.27e-11). In ‘molecular functions’, DRPs were significantly enriched in ‘chlorophyll binding’ (p = 2.37e-19), ‘catalytic activity’ (p = 3.01e-11), ‘tetrapyrrole binding’ (p = 1.23e-10), and ‘oxidoreductase activity’ (p = 1.76e-09) (Fig. 3a).
The 194 DRPs identified by KEGG pathway analysis were strongly enriched in ‘photosynthesis’ (p = 5.04e-47), ‘metabolic pathways’ (p = 7.51e-40), ‘photosynthesis-antenna proteins’ (p = 9.06e-33), ‘carbon metabolism’ (p = 5.82e-26), ‘glyoxylate and dicarboxylate metabolism’ (p = 4.21e-19), ‘carbon fixation in photosynthetic organisms’ (p = 5.88e-17), ‘biosynthesis of secondary metabolites’ (p = 4.22e-07), ‘pentose phosphate pathway’ (p = 4.19e-06), ‘glycolysis/gluconeogenesis’ (p = 6.53e-06), and ‘glycine, serine, and threonine metabolism’ (p = 6.85e-06) (Fig. 3c).
Compared to ‘FT’ plants, photosynthesis is impaired in cdm [31]. Consistently, 58 (29.9%) of the DRPs were enriched in the biological process of photosynthesis. Most of these proteins were enriched in the same metabolic pathway, including ‘photosystem Ⅱ’ (PSBB, PSBC, PSBE, PSBO, PSBP-1, PSBQ, and PSBR) and ‘photosystem Ⅰ’ in chloroplast proteins, as well as PETA and PETC, which were involved in light and electron transport. Expression of 17 light-harvesting proteins (LHCA1, LHCA2, LHCA3, LHCA4, LHCA6, LHCB1, CAB1, CAB2, LHCB1B1, LHCB1B2, LHCB2.1, LHCB2.3, LHCB3, LHCB4, LHCB4.2, LHCB5, and LHCB6) and 14 proteins associated with carbon cycle metabolism was significantly reduced (Table 2). The DRPs associated with impaired photosynthetic capacity may be related to the chlorophyll deficiency of cdm leaves. Moreover, we observed that mutants exhibited a more obvious yellowing and slow growth in the greenhouse during winter. Further, a low-temperature treatment was performed at 4 ℃. After 24-h cold treatment, it was found that cdm plants became more yellow than those grown at 26℃ (Fig. 4). In contrast, no differences were observed between ‘FT’ plants grown at 4℃ and 26℃. Consistent with these findings, we identified a class of DRPs annotated as ‘cold-stress response’ (1.42e-04) (Table 3).
Table 2
Identified DRPs associated with chlorophyll deficiency
Annotation | Gene-ID | Homologous in A. thaliana | iTRAQ-ratio | MRM- ratio |
Photosynthesis | | | | |
Photosystem II 47 kDa protein (PSBB) | Bra040977 | ATCG00680 | 0.68 | 0.50 |
photosystem II reaction center protein C (PSBC) | Bra041123 | ATCG00280 | 0.53 | |
photosystem II reaction center protein E (PSBE) | Bra041107 | ATCG00580 | 0.48 | 0.26 |
Photosystem II manganese-stabilizing protein (PSBO) | Bra037164 | AT5G66570 | 0.64 | 0.64 |
photosystem II subunit P-1 (PSBP-1) | Bra015520 | AT1G06680 | 0.78 | |
photosystem II subunit Q-2 (PSBQ) | Bra029563 | AT4G05180 | 0.70 | 0.50 |
photosystem II subunit R (PSBR) | Bra008392 | AT1G79040 | 0.64 | |
Photosystem I P700 chlorophyll a apoprotein A1 (PSAA) | Bra041122 | ATCG00350 | 0.58 | 0.35 |
photosystem I subunit D-1 (PSAD-1) | Bra036240 | AT4G02770 | 0.73 | 0.57 |
photosystem I subunit E-2 (PSAE-2) | Bra010350 | AT2G20260 | 0.74 | 0.50 |
photosystem I subunit F (PSAF) | Bra038418 | AT1G31330 | 0.61 | |
photosystem I subunit G (PSAG) | Bra030843 | AT1G55670 | 0.46 | |
photosystem I subunit H2 (PSAH2) | Bra014317 | AT1G52230 | 0.62 | |
photosystem I subunit K (PSAK) | Bra010774 | AT1G30380 | 0.51 | |
photosystem I subunit L (PSAL) | Bra032672 | AT4G12800 | 0.50 | |
photosystem I reaction center subunit PSI-N, chloroplast, putative / PSI-N, putative (PSAN) | Bra037761 | AT5G64040 | 0.60 | |
photosynthetic electron transfer A (PETA) | Bra041106 | ATCG00540 | 0.60 | |
photosynthetic electron transfer C (PETC) | Bra000837 | AT4G03280 | 0.61 | 0.30 |
photosynthetic electron transfer C (PETC) | Bra034200 | AT4G03280 | 0.67 | 0.52 |
thylakoid lumenal 29.8 kDa protein | Bra015696 | AT1G77090 | 0.81 | |
ferredoxin-NADP (+)-oxidoreductase 2 (FNR2) | Bra012203 | AT1G20020 | 0.83 | |
oxygen evolving enhancer 3 (PsbQ) family protein (PQL1) | Bra019675 | AT1G14150 | 0.49 | |
Chlorophyll A-B binding family protein (NPQ4) | Bra014024 | AT1G44575 | 0.66 | |
Antenna proteins (light-harvesting chlorophyll protein complex) |
Light-harvesting complex I chlorophyll a/b binding protein 1 (LHCA1) | Bra003198 | AT3G54890 | 0.63 | |
Light-harvesting complex I chlorophyll a/b binding protein2 (LHCA2) | Bra003451 | AT3G61470 | 0.72 | |
Light-harvesting complex I chlorophyll a/b binding protein 3 (LHCA3) | Bra031427 | AT1G61520 | 0.80 | |
Light-harvesting complex I chlorophyll a/b binding protein 4 (LHCA4) | Bra018144 | AT3G47470 | 0.74 | 0.68 |
Light-harvesting complex I chlorophyll a/b binding protein 6 (LHCA6) | Bra016522 | AT1G19150 | 0.83 | |
Light-harvesting complex II chlorophyll a/b binding protein 1 (LhCB1) | Bra005425 | AT2G34430 | 0.50 | |
Light-harvesting complex II chlorophyll a/b binding protein 1.1 (CAB2) | Bra010807 | AT1G29920 | 0.72 | |
Light-harvesting complex II chlorophyll a/b binding protein 1.3 (CAB1) | Bra030182 | AT1G29930 | 0.70 | 0.65 |
Light-harvesting complex II chlorophyll a/b binding protein 1.4 (LHCB1B1) | Bra005425 | AT2G34430 | 0.50 | |
Light-harvesting complex II chlorophyll a/b binding protein 1.5 (LHCB1B2) | Bra021909 | AT2G34420 | 0.74 | |
Light-harvesting complex II chlorophyll a/b binding protein 2.1 (LHCB2.1) | Bra013183 | AT2G05100 | 0.68 | |
Light-harvesting complex II chlorophyll a/b binding protein 2.3 (LHCB2.3) | Bra039070 | AT3G27690 | 0.69 | |
Light-harvesting complex II chlorophyll a/b binding protein 3 (LHCB3) | Bra002999 | AT5G54270 | 0.63 | 0.81 |
Light-harvesting complex II chlorophyll a/b binding protein 4 (LHCB4) | Bra004989 | AT2G40100 | 0.65 | 0.64 |
Light-harvesting complex II chlorophyll a/b binding protein 4.2 (LHCB4.2) | Bra029732 | AT3G08940 | 0.70 | 0.53 |
Light-harvesting complex II chlorophyll a/b binding protein5 (LHCB5) | Bra037913 | AT4G10340 | 0.75 | 0.78 |
Light-harvesting complex II chlorophyll a/b binding protein6 (LHCB6) | Bra026099 | AT1G15820 | 0.57 | |
Carbon fixation in photosynthetic organisms |
Ribulose-bisphosphate carboxylases (RBCL) | Bra028087 | ATCG00490 | 0.55 | 0.29 |
phosphoglycerate kinase 1 (PGK1) | Bra001470 | AT3G12780 | 0.81 | 0.56 |
Aldolase superfamily protein (ASP) | Bra026426 | AT4G26530 | 0.78 | 0.42 |
Fructose-bisphosphate aldolase (FBA) | Bra010717 | AT4G38970 | 0.83 | 0.73 |
Peroxisomal NAD-malate dehydrogenase (PMDH2) | Bra009397 | AT5G09660 | 0.75 | 0.79 |
Alanine-2-oxoglutarate aminotransferase 2 (AOAT2) | Bra016202 | AT1G70580 | 0.82 | |
Phosphoribulokinase (PRK) | Bra023235 | AT1G32060 | 0.78 | |
Glyceraldehyde-3-phosphate dehydrogenase B subunit (GAPB) | Bra034927 | AT1G42970 | O.83 | 0.53 |
high cyclic electron flow 1 (HCEF1) | Bra007041 | AT3G54050 | 0.82 | 0.83 |
glyceraldehyde 3-phosphate dehydrogenase A subunit (GAPA) | Bra025219 | AT3G26650 | 0.83 | |
glyceraldehyde 3-phosphate dehydrogenase A subunit 2 (GAPA2) | Bra026948 | AT1G12900 | 0.74 | 0.48 |
Fructose-1,6-bisphosphatase (FBP) | Bra014005 | AT1G43670 | 0.79 | 0.48 |
ribulose bisphosphate carboxylase small chain 3B (AST3B) | Bra025431 | AT5G38410 | 0.51 | 0.32 |
Sedoheptulose-bisphosphatase (SBPASE) | Bra014720 | AT3G55800 | 0.81 | |
Table 3
Identified DRPs in response to cold stress
Annotation | Gene-ID | Homologous in A. thaliana | iTRAQ- ratio | MRM- ratio |
catalase 2 (CAT2) | Bra017693 | AT4G35090 | 0.62 | 0.39 |
phosphoglycerate kinase 1 (PGK1) | Bra001470 | AT3G12780 | 0.81 | 0.56 |
glyceraldehyde-3-phosphate dehydrogenase B subunit (GABP) | Bra034927 | AT1G42970 | 0.83 | 0.53 |
Phosphoglucomutase (PGM) | Bra028278 | AT5G51820 | 0.79 | 0.79 |
Fibrillin (FIB) | Bra029481 | AT4G04020 | 0.66 | 0.65 |
chloroplast beta-amylase (CT-BMY) | Bra012676 | AT4G17090 | 0.75 | |
Cold regulated 314 thylakoid membrane 2 (COR314-TM2) | Bra032315 | AT1G29390 | 0.73 | |
Characterisation of upregulated expression proteins (URPs) in cdm |
Enrichment analysis of URPs revealed 194 and 139 proteins annotated for metabolic pathways using GO terms and KEGG pathway, respectively (Additional file 10: Table S10, Additional file 11: Table S11). The 194 URPs were significantly enriched in ‘translation’ (p = 4.27e-37), ‘organic nitrogen synthesis’ (p = 7.15e-33), ‘organic nitrogen metabolism’ (p = 8.92e-30), ‘cellular protein metabolism’ (p = 9.79e-23), ‘gene expression’ (p = 2.4e-17), and ‘protein folding’ (p = 1.94e-16) (Fig. 3b). |
In turn, KEGG pathway analysis of the 139 URPs showed that these proteins were enriched in seven metabolic pathways including, ‘ribosome assembling’ (p = 3.79e-38), ‘protein processing in endoplasmic reticulum’ (p = 2.99e-04), ‘ribosome biogenesis in eukaryotes’ (p = 3.15e-03), ‘spliceosome’ (p = 3.51e-03), ‘carbon metabolism’ (p = 6.91e-03), ‘RNA transport’ (p = 0.033), and ‘glucosinolate biosynthesis’ (p = 0.038) (Fig. 3d). Of these, 4 were related to methylation, 2 to transcription and translation termination, 11 to gene/protein splicing, and 7 to RNA degradation. These data provide useful information for studying the molecular mechanisms underlying chlorophyll deficiency in Chinese cabbage (Table 4). |
Table 4
Identified URPs associated with chlorophyll deficiency
Annotation | Gene-ID | Homologous in A. thaliana | iTRAQ- ratio | MRM- ratio |
Methylation |
Fibrillarin 2 (FIB2) | Bra013905 | AT4G25630 | 1.28 | |
Methyltransferases | Bra025336 | AT3G28460 | 1.64 | |
Fibrillarin 2 (FIB2) | Bra010455 | AT4G25630 | 2.38 | |
Sun family protein (NOL1) | Bra039376 | AT3G13180 | 2.30 | |
Translational, translation termination |
Mediator complex, subunit Med10 | Bra012447 | AT1G26665 | 1.28 | |
Rho termination factor | Bra022830 | AT1G06190 | 2.19 | |
Spliceosome | | | | |
Mitochondrial HSO70-2 (MTHSC70-2) | Bra006027 | AT5G09590 | 1.28 | |
UBP1-associated protein 2A (UBA2A) | Bra007277 | AT3G56860.5 | 1.32 | |
RNA-binding family protein (RRM) | Bra009581 | AT5G02530.2 | 1.36 | |
Mitochondrial heat shock protein 70 − 1 (MTHSC70-1) | Bra010620 | AT4G37910 | 1.23 | 1.68 |
Small nuclear ribonucleoprotein family protein | Bra015768 | AT1G20580 | 1.39 | 1.96 |
Chloroplast heat shock protein 70 − 1 (CPHSC70-1) | Bra019231 | AT4G24280 | 1.20 | |
Heat shock cognate protein 70 − 1 (HSC70-1) | Bra018725 | AT5G02500 | 1.22 | |
RNA-binding family protein (RRM) | Bra020273 | AT5G59950 | 1.25 | |
Proline-rich spliceosome-associated family protein (PSP) | Bra020904 | AT4G21660 | 1.27 | |
Mitochondrial HSO70-2 (MTHSC70-2) | Bra028628 | AT5G09590 | 1.24 | |
P-loop containing nucleoside triphosphate hydrolases superfamily protein | Bra030869 | AT2G28600 | 1.44 | |
RNA degradation | | | | |
Cpn60 chaperonin family protein (TCP-1) | Bra001507 | AT3G13470 | 1.27 | |
Chaperonin 60 beta (CPN60B) | Bra011919 | AT1G55490.2 | 1.37 | |
Chaperonin-60alpha (CPN60A) | Bra011985 | AT2G28000 | 1.36 | 1.22 |
DEA(D/H)-box RNA helicase family protein | Bra018747 | AT1G48650 | 1.47 | |
RNA-binding family protein (ATRBP45C) | Bra019078 | AT4G27000 | 1.21 | 1.85 |
Cpn60 chaperonin family protein (TCP-1) | Bra028922 | AT5G56500 | 1.22 | 2.25 |
Chaperonin 60 beta (CPN60B) | Bra030858 | AT1G55490.2 | 1.44 | |
Differential Abundance Analysis Of Proteins Encoded By Chloroplast Genes
Previously, we identified a missense mutation in RPS4 that affected one of the proteins found in the small subunit of the chloroplast ribosome. Proteins directly bound to the small subunit of the chloroplast ribosome are responsible for the synthesis of proteins encoded by chloroplast genes [31]. We found that 14 of the 6,245 identified proteins were encoded by chloroplast genes, of which, 12 were DRPs (Table 5).
Table 5
Identification of proteins encoded by chloroplast genes in Brassica rapa
Annotation | Gene-ID | Homologous in A. thaliana | iTRAQ- ratio | MRM- ratio |
Ribulose-bisphosphate carboxylases (RBCL) | Bra028087 | ATCG00490 | 0.55 | 0.29 |
Photosystem II reaction center protein B (PSBB) | Bra040977 | ATCG00680 | 0.68 | 0.50 |
Ribosomal protein L14 (RPL14) | Bra040980 | ATCG00780 | 0.81 | 0.89 |
Chloroplast ribosomal protein S3 (RPS3) | Bra040981 | ATCG00800 | 0.74 | 0.60 |
Ribosomal protein L2 (RPL2) | Bra040982 | ATCG01310 | 0.65 | |
Phosphoglucomutase (PETA) | Bra041106 | ATCG00540 | 0.60 | |
photosystem II reaction center protein E (PSBE) | Bra041107 | ATCG00580 | 0.48 | 0.26 |
Ribulose-bisphosphate carboxylases (RBCL) | Bra041116 | ATCG00490 | 0.29 | |
ATP synthase subunit alpha (ATPA) | Bra041120 | ATCG00120 | 0.60 | 0.47 |
Photosystem I, PsaA/PsaB protein (PSAA) | Bra041122 | ATCG00350 | 0.58 | 0.35 |
Photosystem II reaction center protein C (PSBC) | Bra041123 | ATCG00280 | 0.53 | |
Ribosomal protein S2 (RPS2) | Bra041038 | ATCG00160 | 0.68 | |
RNA polymerase subunit alpha (RPOA) | Bra040978 | ATCG00740 | 1.12 | |
Unfolded protein bindin (YCF4) | Bra041105 | ATCG00520 | 0.95 | |
Information on proteins encoded by chloroplast genes in Brassica is limited. However, the chloroplast genome of Chinese cabbage is very similar to that of A. thaliana. Indeed, RPS4 orthologues in A. thaliana and Chinese cabbage share 96.5% amino acid sequence identity (Additional file 13: Figure S2). Protein data files in MGF format were used to map to the Arabidopsis database (TAIR 10-pep-20101214), and 2,346 proteins were identified, of which, 47 were encoded by chloroplast genes. Overall, 29 proteins had differential expression greater than 1.2-fold (p < 0.05). Of these, 26 were downregulated and 3 were upregulated in cdm. MRM was performed again, and 23 proteins were detected from these DEPs, 21 of which were downregulated and 2 upregulated (Table 6). The MRM test and iTRAQ data are in agreement.
Table 6
Identification of chloroplast gene encoded differential proteins in Arabidopsis
Gene-ID | Annotation | P-Value | iTRAQ- ratio | MRM- ratio |
DRPs |
ATCG00020 | photosystem II reaction center protein A (PSBA) | 0 | 0.62 | 0.49 |
ATCG00120 | ATP synthase subunit alpha (ATPA) | 0 | 0.60 | 0.44 |
ATCG00130 | ATPase, F0 complex, subunit B/B', bacterial/chloroplast (ATPF) | 6.60e-75 | 0.64 | 0.30 |
ATCG00140 | ATP synthase subunit C family protein (ATPH) | 1.28e-26 | 0.37 | |
ATCG00150 | ATPase, F0 complex, subunit A protein (ATPI) | 8.42e-23 | 0.59 | |
ATCG00160 | Ribosomal protein S2 (RPS2) | 5.52e-55 | 0.75 | 0.71 |
ATCG00270 | photosystem II reaction center protein D (PSBD) | 1.4e-252 | 0.61 | 0.43 |
ATCG00280 | photosystem II reaction center protein C (PSBC) | 0 | 0.64 | 0.42 |
ATCG00340 | Photosystem I, PsaA/PsaB protein (PSAB) | 1.5e-210 | 0.60 | 0.40 |
ATCG00350 | Photosystem I, PsaA/PsaB protein (PSAA) | 1.2e-142 | 0.56 | 0.36 |
ATCG00380 | Chloroplast ribosomal protein S4 (RPS4) | 1.3e-147 | 0.68 | 0.63 |
ATCG00420 | NADH dehydrogenase subunit J (NDHJ) | 1.27e-07 | 0.57 | |
ATCG00470 | ATP synthase epsilon chain (ATPE) | 1.44e-89 | 0.64 | 0.27 |
ATCG00480 | ATP synthase subunit beta (ATPB) | 0 | 0.64 | 0.50 |
ATCG00490 | Ribulose-bisphosphate carboxylases (RBCL) | 0 | 0.49 | 0.29 |
ATCG00540 | Photosynthetic electron transfer A (PETA) | 5.8e-233 | 0.60 | 0.40 |
ATCG00580 | Photosystem II reaction center protein E (PSBE) | 4.07e-50 | 0.59 | 0.46 |
ATCG00600 | photosynthetic electron transport chain (PETG) | 6.74e-05 | 0.58 | |
ATCG00680 | photosystem II reaction center protein B (PSBB) | 0 | 0.66 | 0.51 |
ATCG00710 | photosystem II reaction center protein H (PSBH) | 3.39e-38 | 0.55 | 0.45 |
ATCG00720 | photosynthetic electron transfer B (PETB) | 1.37e-72 | 0.53 | 0.26 |
ATCG00730 | Photosynthetic electron transfer D (PETD) | 5.03e-19 | 0.39 | 0.33 |
ATCG00770 | Ribosomal protein S8 (RPS8) | 1.73e-42 | 0.74 | 0.77 |
ATCG00800 | Structural constituent of ribosome (RPS3) | 4.12e-94 | 0.71 | 0.69 |
ATCG01060 | Photosynthetic electron transport in photosystem I (PSAC) | 1.82e-101 | 0.62 | 0.40 |
ATCG01100 | NADH dehydrogenase family protein (NDHA) | 1.25e-10 | 0.46 | |
URPs |
ATCG00180 | DNA-directed RNA polymerase family protein (RPOC1) | 5.74e-24 | 1.25 | 2.66 |
ATCG00650 | Ribosomal protein S18 (RPS18) | 8.13e-06 | 1.36 | |
ATCG00740 | RNA polymerase subunit alpha (RPOA) | 2.81e-29 | 1.27 | 2.23 |
Transcriptional Expression Analysis Of Deps
The expression patterns of some photosynthesis-related genes (Bra000837, Bra008392, Bra010350, Bra010774, Bra012203, Bra014024, Bra014317, Bra015520, Bra015696, Bra019675, Bra029563, Bra030843, Bra032672, Bra034200, Bra036240, Bra037164, Bra037761, and Bra038418) were analysed by quantitative real-time polymerase chain reaction (qRT-PCR) (Fig. 5). Expression levels of these genes were consistent with our findings from iTRAQ and MRM. The consistency between these various approaches demonstrates that the results obtained are reliable.