Abstract

Results

Comparative analysis of citrate biosynthesis-related enzymes

CS catalyzes the condensation of acetyl-CoA and OAA to form citrate and PEPC catalyzes the β-carboxylation of phosphoenolpyruvate to produce OAA. CS is directly responsible for citrate synthesis, whereas PEPC has been suggested to influence citrate biosynthesis³³. Although one PEPC gene and one CS gene were previously cloned from citrus^17,18,33, we further screened the citrus genome databases and the PCR confirmation indicated that there were at least three PEPC and two CS gene members in the current citrus genome databases (Table S2, Fig. S5A,B). Then, we analyzed their expression levels in both the ‘AL’ and ‘HAL’ fruit juice sacs at 235 days after florescence (DAF) (Fig. 1a,b). The PEPC1 and PEPC2 transcript levels were significantly higher in ‘HAL’ than ‘AL’, whereas the PEPC3 transcript level in ‘HAL’ was markedly lower than that in ‘AL’ (Fig. 1a). In contrast to the three PEPC genes, the transcript levels of the two CS genes were both significantly higher in ‘HAL’ than ‘AL’ (Fig. 1b). We further analyzed their enzyme activities and found that the PEPC (Fig. 1a1) and CS (Fig. 1b1) activities were significantly higher in ‘HAL’ than ‘AL’.

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Figure 1

Gene expression and activity analysis of citrate biosynthesis-related enzymes.

PEPC, OAA, CS, AL and HAL are the abbreviations of phosphoenolpyruvate carboxylase, oxaloacetate, citrate synthase, ‘Anliu’ orange and ‘Hong Anliu’ orange, respectively. (a) and (b) refer to the results of the PEPC and CS gene expression analysis, respectively. a1 and b1 refer to the activities (U·min⁻¹·g⁻¹FW) of the PEPC and CS enzymes, respectively. The asterisks (*) on the bars indicate significant differences (P<0.05) between the AL and HAL fruits in the t-tests (LSD).

Comparative analysis of citrate transport-related genes

The expression profiles of the genes encoding vacuolar H⁺-ATPase (VHA), the vacuolar H⁺-PPase (VHP), CsPH8 encoding the P-type proton pump²³ and CsCit1²⁵ were compared between ‘AL’ and ‘HAL’ (Fig. 2).

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Figure 2

Expression profiles of the genes involved in citrate transport.

(a,b) refer to the expression profiles of the genes encoding the VHA assembly factor (af) or different VHA subunits, respectively (c) refers to the expression profiles of the genes encoding VHP (d) refers to the expression profiles of CsPH8 (Shi et al.²³) and CsCit1 (Shimada et al.²⁵). The asterisks (*) on the bars indicate significant differences (P<0.05) between the AL and HAL fruits in the t-tests (LSD).

VHA consists of a peripheral V1 domain and a membrane-integral V0 domain. VHA contains 13 subunits (VHA-A, VHA-B, VHA-C, VHA-D, VHA-E, VHA-F, VHA-G, and VHA-H for the V1 domain and VHA-a, VHA-c, VHA-c”, VHA-d, and VHA-e for the V0 domain); moreover, there is an assembly factor (af) for VHA assembly³⁴. Through screening the citrus genome databases and PCR confirmation, we found at least one gene encoding af, VHA-A, B, C, D, G, c”, d, and e, two genes encoding VHA-E, F, H, and a, and four genes encoding VHA-c (Table S2, Fig. S5F–H). Transcript analysis indicated that the VHA-af transcript level of ‘HAL’ was half that of ‘AL’ (Fig. 2a). Moreover, the transcript levels of some genes (i.e., VHA-D, VHA-H2, VHA-c4, VHA-d, and VHA-e) were extremely or significantly lower, whereas the transcript levels of VHA-H1, VHA-c1, and VHA-c3 were significantly higher in ‘HAL’ than ‘AL’. Additionally, the transcript levels of other genes encoding the VHA V1 and V0 domains showed no significant difference between the two cultivars (Fig. 2a,b). VHP is another V-type proton pump³⁵. Inquiry of the citrus genome and PCR confirmation indicated the presence of at least four genes (VHP1-4) encoding VHP (Table S1 and Fig. S5I). Transcript analysis showed that the transcript levels of VHP1-4 were significantly lower in ‘HAL’ than ‘AL’ (Fig. 2c). Similar to VHP1-4, the CsPH8 transcript level was also significantly lower in ‘HAL’ than ‘AL’. However, the CsCit1 transcript level had no significant difference between ‘HAL’ and ‘AL’ (Fig. 2d).

Comparative analysis of citrate degradation- or utilization-related genes and/or enzyme activities

Citrate participates in the TCA cycle primarily for energy metabolism in the mitochondria¹¹. In the cell cytoplasm, citrate can use for amino acid or GABA biosynthesis through the production of glutamate^12,28, for the biosynthesis of many secondary metabolites and for gluconeogenesis through the ACL-degradation pathway^10,13,26,36.

Aco, IDH, GS and GAD are the key enzymes involved in citrate catabolism through the Aco-GABA pathway (Fig. 3). A previous study indicated that three genes (Aco1, Aco2 and Aco3) encode Aco in the citrus genome²⁹. Here, transcript analysis showed that the Aco1 transcript level in ‘HAL’ was slightly higher than that of ‘AL’ and the Aco2 and Aco3 transcript levels were more than six-fold higher in ‘HAL’ than in ‘AL’ (Fig. 3a). Enzyme activity analysis showed that the mit-Aco activity was significantly lower (Fig. 3a1) and the cyt-Aco activity was significantly higher (Fig. 3a2) in ‘HAL’ than ‘AL’. IDH is the second key enzyme involved in citrate catabolism. One gene encoding cyt-IDH or NADP-IDH was cloned from citrus by Sadka et al.³⁰. In this study, inquiry of the citrus genome databases and PCR confirmation showed that at least three NAD-IDH (mitochondrial type) and NADP-IDH (cytoplasmic type) genes exist in the citrus genome (Table S1 and Fig. S5C,D). Transcript analysis indicated that the transcript levels of the three NAD-IDH genes (Fig. 3b) and two NADP-IDH genes (NADP-IDH1 and NADP-IDH3, Fig. 3c) were significantly higher in ‘HAL’ than ‘AL’. Moreover, the enzyme activity analysis showed that the activities of myt-IDH (Fig. 3b​b1)1) and cyt-IDH (Fig. 3c​c1)1) were both significantly higher in ‘HAL’ than ‘AL’. GS and GAD are two enzymes that catalyze glutamate in the cytoplasm (Fig. 3). Three genes (GS1-3) encoding GS were confirmed in the citrus genome (Table S1 and Fig. S5B) and two genes (GAD1-2) were previously confirmed by Liu et al.³¹. Transcript analysis indicated that the transcript levels of GS2 (Fig. 3d), GS3 (Fig. 3d) and GAD2 (Fig. 3e) were significantly higher in ‘HAL’ than ‘AL’. Differently, the GS1 transcript level was similar between ‘HAL’ and ‘AL’ (Fig. 3d) and the GAD1 transcript level was significantly lower in ‘HAL’ than ‘AL’ (Fig. 3e). Furthermore, the enzyme activities of GS and GAD showed similar levels between ‘HAL’ and ‘AL’ (Fig. 3d,e).

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Figure 3

Comparison of citrate degradation- and utilization-related genes expression levels and/or their respective enzyme activities.

Aco, IDH, α-KG, GS, GAD, ACL, PEPCK, FBPase, AL and HAL are the abbreviations of aconitase, isocitrate dehydrogenase, α-ketoglutarate, glutamine synthesis, glutamate decarboxylase, ATP-citrate lyase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, ‘Anliu’ orange and ‘Hong Anliu’ orange, respectively. a-f refer to the results of the gene expression analysis, respectively. a1 and a2 refer to the activities (U·min⁻¹·g⁻¹FW) of mitochondrial Aco and cytoplasmic Aco, respectively. b1 and c1 refer to the activities (U·min·g⁻¹FW) of NAD-IDH and NADP-IDH, respectively. d1, e1, and f1 refer to the activities of GS (OD·h⁻¹·g⁻¹FW), GAD (GABA mg·h⁻¹·g⁻¹FW), and ACL (μmol·min⁻¹·g⁻¹FW), respectively. The asterisks (*) on the bars indicate significant differences (P<0.05) between the AL and HAL fruits in the t-tests (LSD).

ACL catalyzes the cleavage of citrate to yield acetyl-CoA and OAA, which involve in the biosynthesis of many secondary metabolites or gluconeogenesis (Fig. 3). Three ACL genes have been confirmed in the citrus genome²⁶. Here, transcript analysis showed that the transcript levels of ACLα1, ACLα2 and ACLβ were more four-fold higher in ‘HAL’ than ‘AL’ (Fig. 3f). Moreover, the ACL enzyme activity in ‘HAL’ was significantly higher than that in ‘AL’ (Fig. 3f1).

The PEPCK and FBPase are the key enzymes involving in gluconeogenesis^11,15. The sequence inquiry and PCR confirmation indicated that at least two genes encoding PEPCK and FBPase exist in the citrus genome (Table S1, Fig. S5A,E). The PEPCK1 transcript level was significantly lower and the PEPCK2 transcript level was significantly higher in ‘HAL’ than ‘AL’ (Fig. 3g). Differently from the PEPCK genes, the transcript levels of the FBPase1 and FBPase2 genes were both significantly higher in ‘HAL’ than ‘AL’ (Fig. 3h).

Discussion

‘HAL’ is a bud mutant of the ‘AL’ sweet orange¹. It is characterized by a comprehensive alteration in metabolites, such as the lycopene, the soluble sugars (sucrose, fructose and glucose), the organic acids and other secondary metabolites^1,2,4. To help investigating the possible reason for the comprehensive change, we analyzed the metabolites in the fruits of the two cultivars at 242 DAF by using LC-Q/TOF-MS technique in the present study. Although we found more than 600 features showing significant difference between the two cultivars under the current analysis conditions (Table S1), only approximately one-tenth of the different features were identified (Table 1). Nevertheless, the profiles of differently identified metabolites between the two cultivars were almost consistent with the report of Pan et al.², implying that the data is believable.

As the carboxylic acid metabolism is the central point of the biosynthesis and/or catabolic networks of other metabolites, we further assessed the significance of citrate metabolism in the comprehensive metabolite alterations in ‘HAL’. Clearly, citrate is significantly lower in the juice sacs of ‘HAL’ than ‘AL’ during fruit development and ripening^1,4, which was further confirmed in the present study (Table 1). However, we found that the transcript levels of citrate biosynthesis-related genes (CS1, CS2, PEPC1, PEPC2) and the enzyme activities of CS and PEPC were significantly higher in ‘HAL’ than ‘AL’ (Fig. 1), indicating that ‘HAL’ has an increased citrate biosynthesis ability compared with ‘AL’. Thus, we can deny the hypothesis that the low accumulation of citrate in ‘HAL’ is possibly due to the reduction of citrate biosynthesis.

It is well known that a partial block of myt-Aco activity is a prerequisite for citrate transport into the cell cytoplasm^16,19. Bogin and Wallace¹⁹ first suggested that the citramalate was possibly the inhibitor of myt-Aco. Degu et al.²⁸ found that spraying citramalate did inhibit Aco activity and increased citrate accumulation in citrus fruit juice sacs. Consistent with the study of Pan et al.², we found that the citramalate or citramalic acid content was significantly higher (1.24-fold) in ‘HAL’ than ‘AL’ (Table 1), moreover, the myt-Aco activity was significantly lower in ‘HAL’ than ‘AL’ (Fig. 3a1). These results implied that more citrate in the mitochondrion transports into the cytoplasm in ‘HAL’ than ‘AL’.

When citrate transports into the cytoplasm, it should utilize immediately or store quickly in the vacuole to keep the cytoplasm neutral for cell activity^15,37. The uptake of citrate into the vacuole depends on the activity of the proton pumps^21,22 and the utilization of citrate depends on the activities of enzymes involved in Aco-GABA pathway¹² and/or ACL-pathway²⁶. Here, we found that some key VHA genes (Fig. 2a,b) [e.g., the VHA assembly factor gene (VHA-af), all the VHP genes (Fig. 2c), and the juice sac-predominant p-type proton pump gene (CsPH8)²³] showed significantly lower expression levels in ‘HAL’ than ‘AL’. On the other hand, the activities of cyt-Aco and NADP-IDH in the Aco-GABA pathway and ACL in the ACL-degradation pathway were significantly higher in ‘HAL’ than ‘AL’ (Fig. 3). These results indicated that in the ‘HAL’, more citrate does not accumulate in the vacuole but utilizes in the cytoplasm because the lower expression levels of proton pump genes reduce the ability of the proton pump to uptake citrate into the vacuole. Moreover, the higher enzyme activities of cyt-Aco, NADP-IDH and ACL increase the ability to utilize citrate.

The TCA cycle is very important for organisms by generating energy and providing various precursors for other biochemical reactions¹¹. The present study showed that the citramalate content increased significantly (Table 1) and the myt-Aco activity reduced clearly (Fig. 3a1), suggesting that the TCA cycle should be seriously blocked in ‘HAL’ compared with ‘AL’. However, we also found that the cyt-Aco (Fig. 3a2) and NADP-IDH (Fig. 3c1) activities increased significantly in ‘HAL’ compared with ‘AL’ and the activities of GS (Fig. 3d1) and GAD (Fig. 3e1) of which both enzymes catalyze glutamate showed similar levels between ‘HAL’ and ‘AL’. These results suggested that the direct products (isocitrate and α-KG) of cyt-Aco and NADP-IDH should be significantly higher in ‘HAL’ than ‘AL’. Nevertheless, they decreased significantly in ‘HAL’ compared with ‘AL’ (Table 1). Therefore, we inferred that more cytoplastic isocitrate and α-KG in ‘HAL’ import into the mitochondrion and participate in the TCA cycle again. The compensation of isocitrate and α-KG from the catalysis of cytoplastic citrate by cyt-Aco and NADP-IDH contributes to the stability of the TCA cycle in ‘HAL’.

Another cytoplastic citrate-degrading pathway is through ACL catalysis, which links the production of many secondary metabolites^13,36. For example, Crifò et al.³² suggested that citrate could utilize for flavonoid biosynthesis through ACL catalysis in blood oranges under cold storage. In the present study, the ACL gene transcript levels (Fig. 3f) and enzyme activity (Fig. 3f1) were significantly higher in ‘HAL’ than ‘AL’. These findings indicated that ACL is more active in the low-acid orange ‘HAL’ than in the normal-acid orange ‘AL’, implying that more citrate is cleaved to produce more OAA and acetyl-CoA. OAA can reenter into the mitochondrion for citrate biosynthesis or use for gluconeogenesis or amino acid biosynthesis^11,15. Here, we found that the transcript levels of three key genes [PEPCK2 (Fig. 3b), FBPase1 and FBPase2 (Fig. 3h)] involving in gluconeogenesis, the intermediate sugar (Glu-6P) and the soluble sugar (Suc, Glu, and Fru) contents were significantly higher in ‘HAL’ than in ‘AL’ (Table 1). These results indicated the OAA from the catalysis of cytoplastic citrate by ACL participates in the gluconeogenesis, which at least accounts partially for the significant increase of the soluble sugars in ‘HAL’. Judging from the Fig. 4, the significant increase of Glu-6P at least contributes partially to the significantly increase of ascorbic acid, histidine (His), pyrimidine or purine in ‘HAL’ compared with ‘AL’ (Table 1). In addition, the contents of serine, glycine and tryptophan were significantly higher in ‘HAL’ than ‘AL’ (Table 1), which is possibly related to the participation of OAA or pyruvate (Fig. 4). On the other hand, another product of acetyl-CoA via ACL catalysis usually utilizes for fatty acid extension or the biosynthesis of isoprenoids and other secondary metabolites^13,32,36, including the biosynthesis of lysine (Lys), methyl jasmonate (MeJA), zeatin, lycopene or ABA (Fig. 4). The present study showed that the content of zeatin was significantly higher but the contents of Lys, MeJA and ABA were significantly lower in ‘HAL’ than ‘AL’ (Table 1), consistent with the previous study of Pan et al.². In addition, we are also sure that lycopene is significantly higher in ‘HAL’ than ‘AL’ because lycopene can be visibly found in ‘HAL’ (Fig. S6) and has been detected by HPLC at a significant level in ‘HAL’^1,4. In this study, consistent with a previous metabolomics analysis ², we failed to identify lycopene in ‘HAL’ possibly due to the lack of a standard reference database for citrus fruits or just using a colomn C18 not a C30 in the LC-MS analysis. Furthermore, we believe that there should have more significantly altered secondary metabolites in ‘HAL’ because 90% of the differential features failed identification in this study (Table S1 and Table 1). This failure is largely due to our basic lack of knowledge of many metabolic pathways in plants and our current inability to identify the majority of metabolites synthesized in the citrus plant.

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Figure 4

Overview of the major metabolic pathways involved in mutant trait formation.

The red circle indicates that the metabolite content, enzyme activity or gene expression level is significantly higher in the mutant cultivar (‘HAL’) than the wild type cultivar (‘AL’). The green circle indicates that the metabolite content, enzyme activity or gene expression level is significantly lower in ‘HAL’ than ‘AL’. The black circle indicates that the difference of other metabolites between ‘HAL’ and ‘AL’ is not clear.

Taken together, this study confirmed that the orange mutant ‘HAL’ has a comprehensive alteration in metabolites except for the increase in lycopene and soluble sugars and the decrease in citrate. Combining the present and previous results^1,2,3,4,6, we established a model to explain the comprehensive alteration in metabolites in the orange mutant ‘HAL’ (Fig. 4). In detail, although the citrate content is very low in ‘HAL’ juice sacs, ‘HAL’ still has higher ability to biosynthesize the citrate comprared with ‘AL’. Moreover, the citramalate content (the inhibitor of mit-Aco) increases significantly and the mit-Aco activity decreases significantly in ‘HAL’, resulting in more synthesized citrate exports into the cytoplasm. Because the transcript levels of some key genes of proton pump decreases significantly in ‘HAL’ compared with ‘AL’, which reduces the ability for the uptake of citrate into the vacuole, more citrate cannot store in the vacuole. The abundant citrate utilize immediately because the activities of cys-Aco, NADP-IDH and ACL increase significantly in the ‘HAL’ compared with the ‘AL’, which keeps the cytoplasmic pH constant. Some citrate-splitting products possibly reenter the mitochondrion to maintain the stability of the TCA cycle rather than participate in the biosynthesis of GABA or glutamine. The others utilize for gluconeogenesis and/or secondary metabolite metabolism, which results in a significant increase in the soluble sugars, ascorbic acid, histidine, pyrimidine, purine, zeatin, and lycopene or a significant decrease in methyl jasmonate and ABA (Fig. 4).

Frankly, the model gives a rough but reasonable explanation for the comprehensive change in metabolites of ‘HAL’ compared with its wild type, ‘AL’. In the model, the decrease of proton pump genes’ expression, namely, the reduction of proton pump ability to promote the citrate storing in the vacuole plays a pivotal role for the comprehensive alteration in metabolites of ‘HAL’. Although the lower ABA content in the ‘HAL’ compared with ‘AL’ is possibly due to the higher accumulation of lycopene in ‘HAL’, which always reduces ABA biosynthesis^1,38, there is a long way to verify this model in the future because of the complexity in the secondary metabolism and the difficulty for gene function identification of perennial plants.

Methods

Additional Information

How to cite this article: Guo, L.-X. et al. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant. Sci. Rep. 6, 29343; 10.1038/srep29343 (2016).

Supplementary Material

Acknowledgments

Footnotes

References