Changes in the content of starch, malate and soluble sugar in the Wh parts of chimeric leaves
Plant leaf albino is an obvious and common chlorophyll deficient mutation, which affects plant growth by changing physiological and biochemical levels [17]. The chimeric leaves in Ananas comosus var. bracteatus are composed of the normal green parts and albino white parts. Compared with the Gr parts, the Wh parts had higher starch content and lower soluble sugar content (P < 0.05; Fig. 1a and b). Some study showed that lower photosynthetic rate is due to accumulated starch content and decreased soluble sugar content [18]. In addition, the Wh parts had higher malate content (P < 0.05; Fig. 1c). Malate is the initial product of CO2 fixation in CAM plant, and also is the respiratory substrate for ATP production in mitochondria [19]. Our results suggested that photosynthetic activity and respiratory property were altered between the two parts.
The proteome profile was altered in the Wh parts of chimeric leaves
Compared to the Gr parts, 805 proteins were upregulated and 457 proteins were downregulated in the Wh parts of chimeric leaves using a 1.5-fold threshold (P < 0.05; Additional file 3: Table S1). Many of the upregulated proteins were enriched in the spliceosome, ribosome, mRNA surveillane pathway and RNA degradation (Additional file 4: Figure S3). Therefore, the different manner of gene regulation might exist between the Wh and Gr parts of chimeric leaves. Whereas a large portion of downregulated protein were highly enriched in photosynthesis, glycolysis, oxidative phosphorylation and citrate cycle (Additional file 4: Figure S3). These results suggested that the function of photosynthesis and energy metabolism might be suppressed in the Wh parts of chimeric leaves. This is accordance with our comparative proteomic data studied previously [13]. Furthermore, the overlap in differentially expressed proteins and proteins with differentially expressed lysine succinylation sites was studied. There were 51 proteins with consistent changes between succinylation levels and protein abundance, whereas 30 proteins demonstrated opposing changes (Additional file 5: Table S2).
The level of succinylation in the Wh parts of chimeric leaves was increased
To obtain an overview of the extent of lysine succinylation in chimeric leaves of Ananas comosus var. bracteatus, we performed Western blot analysis using lysine succinylation-specific pan-antibodies. Lysine succinylation was observed on a great many of proteins with varying molecular masses in both green and white leaf samples (Fig. 2). These results suggested that lysine succinylation was abundant in chimeric leaves of Ananas comosus var. bracteatus. Notably, succinylation level in the Wh parts of chimeric leaves was significantly higher than that of the Gr parts in Western blot. In order to analyze the succinylation level in situ, immunohistochemistry analysis of the freehand sections of the Wh and Gr parts of chimeric leaves were carried out. Compared to negative control (Fig. 3c, d), both the Wh parts (Fig. 3a) and Gr parts (Fig. 3b) of chimeric leaves possessed brown positive signal. Furthermore, the staining of lysine succinylation in the Wh parts of chimeric leaves was stronger than that of the Gr parts. These results indicated that the succinylome level in the Wh parts of chimeric leaves was increased.
Proteome-wide analysis of lysine-succinylated peptides and proteins in Ananas comosus var. bracteatus
The protein succinylation in the Gr and Wh parts of chimeric leaves was revealed by combining with anti-succinyllysine antibody-dependent enrichment and high-resolution liquid chromatographytandem mass spectrometry (LC-MS/MS). We checked the mass error of all the identified peptides to assess the accuracy of MS data. As shown in Fig. 4a, the mass error of all the identified peptides was near zero, which indicates that the reliability of the MS data fit the requirement. With regard to peptide length, most peptides were distributed between 8 and 16, which suggests that sample preparation met the standards (Fig. 4b). And succinylome quantitative data distribution was shown in Fig. 4c.
After LC-MS/MS analysis and database search, a total of 855 succinylated sites in 335 proteins were identified, and 593 succinylated sites in 237 proteins were accurately quantified. Compared to the Gr parts, 232 (61.1%) sites in 128 proteins were quantified as upregulated targets, and 148 (38.9%) sites in 70 proteins were quantified as downregulated targets in the Wh parts of chimeric leaves using a 1.5-fold threshold (P < 0.05; Fig. 5a; Additional file 6: Table S3). These results showed that global succinylation level was increased in the Wh parts of chimeric leaves. This is accordance with the Western blot and immunohistochemistry analysis results.
Previous studies showed that various succinylated proteins have been identified in bacteria [9], fungi [20], protozoans [21] and mammalian cells [14, 22]. However, only nine succinylome studies have been reported in plants. The number of succinylated proteins in rice [5] and tea [16] is almost eight times and six times more than that in Ananas comosus var. bracteatus, respectively. But the number of succinylated proteins in Ananas comosus var. bracteatus was much higher than that in strawberry stigmata [23], common wheat [24], rice seeds [25], tomato [26], Taxus×media [27], Brachypodium distachyon [28], Dendrobium officinale [29]. In physiological level, different species and tissues may possess differential profile of succinylation. In technical level, sample preparation, method, number of proteins in the databases varied among researches may result in the different succinylated profile. Notably, 5 succinylation sites were found on histone proteins in Ananas comosus var. bracteatus, including 2 sites on H2B.1, 2 sites on H3.3 and 1 site on H4. Lysine succinyltion found in histone represents an evolutionarily conserved histone mark in eukaryotic [6]. And modification at different locations or different PTMs at the same histone site can be associated with very different transcriptional programs [6].
The number of succinylated sites in the identified proteins was counted in this study (Fig. 5b). Of the succinylated proteins, 54.6% (183/335) had only one succinylated site, 14.3% (48/335) possessed two succinylated sites, 9.3% contained three succinylated sites, and the remaining were modified on four or more lysine residues. Each succinylated protein had 2.55 (855/335) succinylated sites on average. Notably, ribulose bisphosphate carboxylase (RuBisCO) large chain, which is the protein with the most succinylated sites in chimeric leaves of Ananas comosus var. bracteatu, possessed 15 succinylated sites. Similarly, the large chain of RuBisCO is also extensively succinylated in rice leaves, containing 16 independent succinyl-lysine residues [5].
Functional annotation and subcellular localization of the succinylated proteins
Using Gene Ontology (GO) functional classification analysis, the potential role of succinylation in chimeric leaves of Ananas comosus var. bracteatus was studied. In biological process (Fig. 6a), the three largest groups of succinylated proteins were involved in metabolic process (35%), followed by cellular process (27%) and single-organism process (26%). This is accordance with other plants [25, 26, 28], suggesting that this distribution pattern is not novel at all. In cellular component (Fig. 6b), most succinylated proteins were located in the cell (41%), macromolecular complex (21%), membrane (20%) and organelle (17%). In molecular function (Fig. 6c), we found that the largest group of succinylated proteins (49%) was related to catalytic activities, suggesting that the succinylation enzyme may affect biological processes. The second largest group (36%) possesses binding activities, which means succinylation may work in DNA transcription and PPIs. So, in conclusion, lysine succinylation may affect multiple biological processes in chimeric leaves of Ananas comosus var. bracteatus by changing the molecular functions of proteins in diverse cellular components.
The subcellular localizations of the identified proteins were also predicted. Generally, succinylation is highly concentrated in mitochondria because the succinyl-CoA and succinate formed via the CAC and odd numbered fatty acid oxidation primarily accumulates in the mitochondrial matrix [3]. For example, 70% of succinylated proteins mainly exist in the mitochondria in mouse liver cells [22]. In addition to non-enzymatic succinylation by succinyl CoA, succinylation can be mediated by in an α-ketoglutarate-dependent manner [3]. The oxoglutarate dehydrogenase (OGDH), which is a component of the α-ketoglutarate dehydrogenase (KGDH) complex, can serve as a succinyltransferase [3]. Some study indicated that the α-KGDH complex is much greater effective than succinyl-CoA owing to the catalysis of the OGDH [30]. In this study, most succinylated proteins were located in the chloroplast, cytoplasm, mitochondria and nucleus, accounting for 47, 23, 16 and 7% of all the identified proteins, respectively (Fig. 6d). It revealed that lysine succinylation can exist in outside of mitochondria. One possibility is that a functional α-KGDH complex exist in outside of mitochondria. Some study indicated that the component and activity of α-KGDH complex can be readily measured in cytosolic fractions [29]. And experiments have shown that α-KGDH complex can be localized in the nucleus [31]. But whether it is localized in the chloroplast has not been experimentally proven. A second, but unlikely, possibility is that succinyl-CoA is transported from the mitochondria. A third possibility is that an alternative succinyltransferase depending on α-ketoglutarate manner exists in outside of mitochondria. But other explanations are possible. Notably, the number of succinylated chloroplast proteins was much higher than that of succinylated mitochondrial proteins in this study. This is accordance with other plants [24, 27]. The detection of succinylation sites is biased to occur on more abundant proteins [22]. Therefore, a larger number of succinylation sites can be identified on chloroplast proteins that accounted for a large proportion of total protein in plants.
Analysis of succinylated lysine sequence motifs
The frequency of different amino acids around the succinylated lysine from − 10 to + 10 was measured, which can investigate the nature of succinylated sites in chimeric leaves of Ananas comosus var. bracteatus. The frequency of lysine (K) at + 5 was highest (Fig. 7a). Using the motif-x program, the sequence motifs in all the identified peptides were identified. Three conserved motifs were identified from 855 succinylated sites, namely, Ksu(X9) K, Ksu(X7) K and Ksu(X4) K (Ksu indicates the succinylated lysine, and X indicates a random amino acid residue) (Fig. 7b), and these motifs exhibited different abundances (Fig. 7c). Among these motifs, Ksu(X4) K and Ksu(X7) K were previously identified in other plant species [16, 18, 19, 21,22,23,24,25,26,27,28, 32]. Notably, Ksu(X7) K was also observed in the marine bacterium [33], indicating that some motifs might be conservative between plant and bacteria.
The succinylome profile was changed in the Wh parts of chimeric leaves
To explore the role of succinylation in the formation and growth of chimeric leaves in Ananas comosus var. bracteatus, we analyzed the proteins which possess differentially expressed lysine succinylation sites between the Wh and Gr parts of chimeric leaves using GO annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis (P<0.05; Fig. 8). Briefly, in molecular function enrichment analysis, proteins with upregulated Ksu sites in the Wh parts were associated with antioxidant activity and isomerase activity. For example, the intensity of all Ksu sites in catalase-1 and superoxide dismutase [Cu-Zn] was markedly increased in the Wh parts. It is possible that a higher level of succinylation maintains cellular redox homeostasis in the Wh parts of chimeric leaves through altering the activities of antioxidant enzymes. Conversely, proteins with downregulated Ksu sites in the Wh parts were associated with oxidoreductase activity and binding activity. In detail, these proteins with downregulated Ksu sites mainly are core enzyme and coenzyme in the CAC and mitochondrial electron transport chain (ETC). In cellular component enrichment analysis, we found that proteins with upregulated Ksu sites in the Wh parts were highly located at mitochondria. Mitochondria is power house of eukaryotic cells, which can fuel metabolism with ATP to maintain the movement and growth of organism [34]. Conversely, proteins with downregulated Ksu sites in the Wh parts were enriched in the ATP synthase complex. ATP synthase is a key enzyme in photophosphorylation and oxidative phosphorylation, affecting the production of ATP required for cell life activities. These results suggested photosynthetic activity and respiratory properties were altered in the Wh parts of chimeric leaves. It might result from the downregulated succinylation of proteins associated with ATP synthase complex. In the biological process enrichment analysis, differentially changed succinylated proteins were enriched in 23 processes, particularly processes involved in metabolism and energy generation. KEGG pathway enrichment analysis of proteins whose succinylation level changed was carried out. The protein-processing pathways in the peroxisome, fatty acid degradation, alpha-linolenic metabolism, fatty acid metabolism, fructose and mannose metabolism, and plant MAPK signaling pathway were enriched among the proteins with upregulated Ksu sites in the Wh parts. Whereas upregulated proteins in the Wh parts were mainly enriched in spliceosome and ribosome through previous study [13]. Proteins with downregulated Ksu sites in the Wh parts were enriched in pathways involving the CAC, carbon metabolism, glyoxylate metabolism, dicarboxylate metabolism, pyruvate metabolism, and 1-oxocarboxylic acid metabolism. And previous study has shown that lots of downregulated proteins in the Wh parts were enriched in photosynthesis and respiration [13], which indicated that protein abundant and succinylation level may work together to regulate photosynthesis and respiration in chimeric leaves. These results suggested that the changed succinylation level may be a candidate regulator to metabolism- and energy-related processes of chimeric leaves in Ananas comosus var. bracteatus.
Succinylated proteins involved in CAM photosynthesis in the Gr and Wh parts of chimeric leaves
Photosynthesis, which provides chemical energy for maintaining plant life, plays important roles in plant metabolic processes [35]. In this study, the Wh parts of chimeric leaves had higher starch content and lower soluble sugar content, which suggested the Wh parts take a low photosynthetic rate and may act as a photosynthetic product sink. Previous studies have shown that lysine succinylation is prevalent in various photosynthetic organisms [8]. In this study, six succinylated proteins involved in photosynthesis were identified only in the Gr parts of chimeric leaves, including three subunits in photosystem I (PsaC, PsaD, PsaF), two subunits in photosystem II (PsbB, PsbP) and light-harvesting complex II chlorophyll a/b binding protein 2 (Lhcb2). Furthermore, succinylation level on cytochrome b6-f complex iron-sulfur subunit (PetC) was downregulated and that on photosystem II oxygen-evolving enhancer protein 1 (PsbO) was upregulated in the Wh parts of chimeric leaves. In detail, the succinylation level of the K162 site in PsbO was upregulated about eight times. PsbO is the largest extrinsic subunit of PSII, which plays critical roles in oxygen evolution reaction [36]. In cyanobacterium, PsbO succinylation can hinder correct interactions between PsbO and other PSII subunits by the conformation changes in the head domain of PsbO, leading to a negative regulation of oxygen evolution [8]. However, the Ksu sites in PsbO of Ananas comosus var. bracteatus differed from cyanobacterium, it may work differently in regulating photosynthesis. The increase (decrease) of succinylation level of the light harvesting proteins, PSI and PSII proteins may work in the decrease of the photosynthetic rate in the Wh parts of the chimeric leaves.
Pineapple is a typical CAM plant. The hallmark of CAM photosynthesis is the conversion of CO2 into malate at night. In this study, some enzymes involved catalyzing the conversion of CO2 into malate were identified as succinylated proteins, including phosphoenolpyruvate carboxylase (PEPC), NAD-dependent malic enzyme (NADP-ME) and malate dehydrogenase (MDH1) (Fig. 9). In detail, succinylation level in PEPC and NADP-ME were upregulated in the Wh parts of chimeric leaves. But MDH1 not only possessed an upregulated site but also a downregulated site in the Wh parts. Enzymatic assays showed that the activity of NADP-ME in the Wh parts of chimeric leaves was significantly higher than that in the Gr parts (P < 0.05; Additional file 7: Figure S4), but the Wh and Gr parts of chimeric leaves had no significant difference in the activity of PEPC and MDH1 (Additional file 7: Figure S4). NADP-ME in the CAM plants plays a key role in photosynthesis by providing CO2 for fixation in the Calvin cycle. These findings suggested that the increased succinylation level in NADP-ME may lead to more CO2 provided by Wh parts. And the CO2 provided by the Wh parts might be used in the Gr parts, leading to maintain the growth of chimeric leaves of Ananas comosus var. bracteatus.
The Calvin cycle is one of the CO2 assimilation pathways, and the hallmark of Calvin cycle is the conversion CO2 into carbohydrates [37]. In this study, seven succinylated enzymes were found in Calvin cycle (Fig. 9). Furthermore, four enzymatic proteins with downregulated Ksu sites were found in the Wh parts of chimeric leaves, such as the RuBisCO large chain and small chain, phosphoglycerate kinase (PGK), transketolase (TK). Importantly, RuBisCO possessed 15 and 5 succinylated sites in the large chain and small chain, respectively. RuBisCO catalyzes the limiting step of photosynthetic capacity, which plays central roles in Calvin cycle [38]. In this study, the activity of RuBisCO is inversely proportional to succinylation level of RuBisCO (P < 0.05; Additional file 7: Figure S4). Similarly, deacetylated RuBisCO can increase the activity of RuBisCO in Arabidopsis [39]. These results suggested lower level of succinylation in RuBisCO may lead to lower photosynthetic capacity in the Wh parts of chimeric leaves, through increasing activity of RuBisCO to adding content of starch. Conversely, the succinylation level of all modification sites in fructose-bisphosphate aldolase (ALDO), sedoheptulose-1,7-bisphosphatase (SBPase), glyceraldehyde-3-phosphate dehydrogenase 2 (GAPD2), and phosphoribulokinase (PRK) were upregulated in the Wh parts of chimeric leaves. Interestingly, the protein levels of GAPD2, SBPase and ALDO were downregulated in the Wh parts of chimeric leaves, indicating that the increased succinylation levels were not simply due to the increased protein levels. Our results suggested that the increase (decrease) of succinylation level of certain protein involved in the Calvin cycle may lead to higher starch content and lower soluble sugar content in the Wh parts of chimeric leaves. Therefore, the Wh parts of chimeric leaves may act as photosynthetic product sink and carbon source to enhance the photosynthetic rate of chimeric leaves.
Succinylated enzymes involved in glycolysis, CAC and pyruvate metabolism in the Gr and Wh parts of chimeric leaves
The respiratory, including glycolysis, ETC and CAC, plays crucial roles in organism survival by supplying energy to various cellular functions [40]. Most glycolytic enzymes that catalyzes the conversion of glucose to pyruvate were identified as succinylated proteins in bacteria, mammals and plants [29]. In Ananas comosus var. bracteatus, five succinylated proteins were quantified in both the Wh and Gr parts of chimeric leaves (Fig. 10). Among these proteins, the succinylation level of all modification sites in 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (GPGP), enolase (ENO), GAPD2, and ALDO were upregulated in the Wh parts of chimeric leaves. Only the succinylation level of PGK was downregulated in the Wh parts of chimeric leaves. The increased acetylation level in bacteria GAPD promoted glycolysis but suppressed gluconeogenesis, indicating that acetylation level of GAPD can control the direction of glycolysis versus gluconeogenesis [41]. However, the function of succinylation in GAPD remains unknown.
The conversion of pyruvate to acetyl-CoA was catalyzed by pyruvate dehydrogenase complex (PDHC) [42]. Three components of PDHC were succinylated in both Wh and Gr parts of chimeric leaves, namely, E1 component alpha subunit (PHDA), E1 component beta subunit (PDHB), dihydrolipoyl transacetylase (DLAT) and dihydrolipoyl dehydrogenase (DLD). And the downregulated sites were identified in the Wh parts (Fig. 10).
In Ananas comosus var. bracteatus, ten enzymes in the CAC were succinylated in both Wh and Gr parts of chimeric leaves, and most of these enzymes had more than one Ksu site (Fig. 10). Among these proteins, the succinylation level of all modification sites in aconitate hydratase (ACO), succinyl-CoA synthetases (LSC1 and LSC2), and succinate dehydrogenase (SDHA) were downregulated in the Wh parts of chimeric leaves. Conversely, isocitrate dehydrogenase (IDH1) and dihydrolipoamide succinyltransferase (DLST) possessed downregulated sites in the Wh parts of chimeric leaves. In addition, citrate synthase (CS), OGDH and malate dehydrogenase (MDH1) not only had downregulated sites but also upregulated sites in the Wh parts of chimeric leaves. IDH1 is the rate-limited enzyme in the CAC [43]. In this study, two Ksu sites (K64 and K217) on IDH1were identified. Using mutagenesis-based analysis, succinylation can directly affect IDH1 activity in E. coli [4].
In addition to regulating CAC, α-KGDH complex also mediates succinylation either by enzymatic manner or by non-enzymatic manner [3]. In this study, E1K (OGDH), and E2K (DLST) of α-KGDH complex were succinylated. These results suggested that the different succinylated level in α-KGDH complex between the Wh and Gr parts of chimeric leaves might become a crucial regulation point. It not only regulates respiration pathway but also the entire cellular energy metabolism in chimeric leaves of Ananas comosus var. bracteatus.
Succinylated enzymes involved in photorespiration in the Gr and Wh parts of chimeric leaves
Photorespiration is a complex metabolic process in which green plants consume O2 under light and release CO2. In our study, the succinylation level of three succinylated enzymatic proteins were upregulated in the Wh parts of chimeric leaves, including peroxisomal (S)-2-hydroxy-acid oxidase (GLO), glycerate dehydrogenase (HPR1), and serine-glyoxylate aminotransferase (SGAT) (Fig. 11). Conversely, the succinylation level of all modification sites in serine hydroxymethyltransferase (SHMT), the large chain and small chain of RuBisCO were downregulated in the Wh parts of chimeric leaves. Studies have demonstrated that photorespiration not only involves in primary metabolism, including energy metabolism, amino acid synthesis and redox signaling, but also participates in plant resistance to biotic and abiotic stress [44,45,46]. Therefore, the lysine succinylation level may affect photorespiration capacity and ultimately lead to a difference in resistance between the Gr and the Wh parts of chimeric leaves. In addition, phosphorylation, acetylation and ubiquitination were identified in the photorespiration pathway in other plants [47,48,49]. However, as with succinylation, how other PTMs regulate the functions of enzymes in the photorespiration pathway has not been reported.
