The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak
BMC Genomics volume 23, Article number: 739 (2022)
Here we respond to Zhou (BMC Genomics 21:734, 2020) “Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying peel and pulp color formation” published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou (BMC Genomics 21:734, 2020) are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key flavonoid and anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status.
Betalain pigments are restricted to the Caryophyllales, where they replace the otherwise ubiquitous anthocyanins in several families . However, not all families in the Caryophyllales produce betalains. The complex pigment distribution over evolutionary lineages can be explained by at least four independent origins of the betalain biosynthesis . Interestingly, anthocyanins have not been observed in betalain-pigmented species [1, 3, 4]. Consequently, the theory of mutual exclusion of both pigments was established and repeatedly supported by numerous studies in the last decades [3, 4]. Although co-occurrence of anthocyanins and betalains can be achieved through genetic engineering , simultaneous accumulation of both pigments within a native species has never previously been reported in nature. Zhou et al.  stated that “the anthocyanin coexistence with betalains is unneglectable” in their publication about the pigmentation of pitayas. Here, we outline some reasons why we do not think the study by Zhou et al.,  provides solid evidence for the presence of anthocyanins in betalain-pigmented pitayas.
Patterns of proposed anthocyanin accumulation are weak basis for subsequent correlation with genes expression
Zhou et al., investigated metabolic differences between three pitaya cultivars: red peel/red pulp (RR), yellow peel/white pulp (YW), and green peel/white pulp (GW). They indicate that 70 different anthocyanins are differentially accumulated in the pulps of these cultivars, but Table S9 lists only 14 anthocyanins. Unfortunately, these crucial metabolic analyses were apparently restricted to a single sample per cultivar (Table S9) which prevents any solid conclusions about the quantity of pigments. Nevertheless, we calculated the total amounts of detected anthocyanins per tissue and cultivar based on the data they provide (Table 1). While the total anthocyanin amount in the red peel is substantially higher than the amounts in yellow or green peel, there is only a very small difference between the different pulp samples. The difference between the two white pulps is substantially higher than the difference between one white and the red pulp. There are more anthocyanins reported in the yellow or green peel than in the red pulp. The detection of delphinidin and malvidin glycosides (blue pigments) would imply the presence of a functional flavonoid 3’,5’-hydroxylase (F3’5’H) in all three cultivars. However, Zhou et al., do not mention such an enzyme and our analyses revealed no evidence for the presence of F3’5’H transcripts in pitaya. Zhou et al., report that “metabolites with similar fragment ions were suggested to be the same compounds”. But as we previously outlined this method does not follow best practice . We would expect at least to see the LC-MS/MS spectra and co-elution/fragmentation of the pigments versus authentic reference compounds , especially when reporting the unexpected occurrence of anthocyanins in a betalain-pigmented species.
Transcriptomic analysis indicates likely block in anthocyanidin biosynthesis at the level of DFR and ANS genes
Zhou et al., claim “Our results demonstrated that anthocyanin biosynthesis was one of the significantly enriched pathways”. We do not see any evidence for this statement, because the analysis presented in their Fig. 5 covers only the general phenylpropanoid pathway and selected steps of the flavonoid biosynthesis. Our re-evaluation based on the construction of gene trees for all steps in the pathway (Additional files 1 and 2) indicates several cases of misidentification and missed gene copies, although their annotation is difficult to evaluate as multiple transcripts are reported for each gene class, and clear orthology is not established in absence of a phylogenetic analysis. Important genes of the anthocyanin biosynthesis like DFR and ANS are missing. For example, DFR (Cluster-16519.0) and ANS (Cluster-7001.0) are both present in the transcriptome assembly, but were not presented by Zhou et al. Zhou et al., write “The comparative pitaya transcriptome showed the differential regulation of the anthocyanin pathway and genes controlling almost every single step in the pathway were differentially regulated”. However, two very important anthocyanin biosynthesis genes, DFR and ANS, are not presented in Fig. 5 of Zhou et al. . Since expression of DFR and ANS would be crucial for the formation of anthocyanins, we performed a re-analysis of the flavonoid biosynthesis including genes for the reactions leading to anthocyanidins (Fig. 1). Our analysis is based on a transcriptome assembly of RR (red fruits) and indicates a block in the anthocyanin biosynthesis at DFR and ANS (Fig. 1). We would expect to see high transcript abundance of all genes necessary for anthocyanin formation if anthocyanins would be substantially contributing to the red colour, especially at the intensity they report. We also included LAR and ANR in our analysis because the enzymes encoded by these genes are responsible for proanthocyanidin biosynthesis. Anthocyanins and proanthocyanins have shared precursors and could be considered as competing pathways. The presence of ANR transcript at high levels indicates that the low levels of DFR and ANS transcripts might be involved in the proanthocyanidin biosynthesis rather than the anthocyanin biosynthesis. In other words, proanthocyanidin production could be an explanation for the recovery of DFR, ANS, LAR, and ANR transcripts. However, it is important to emphasize that the transcript abundances of DFR and ANS are in any case extremely low (average TPM < 1), and inconsistent with high levels of anthocyanins. These low transcript abundances for DFR and ANS align well with previous reports about the absence of anthocyanin in betalain-pigmented species [8,9,10].
In conclusion we feel that the biochemical evidence for anthocyanins lacks appropriate standards. Furthermore, the investigation of core anthocyanin biosynthesis genes via RNA-Seq does not provide insights into the accumulation of anthocyanins, because there is no clear difference in bulk anthocyanin content between differently pigmented pitaya varieties e.g. red vs. white. Finally, there is little correlation between the levels of transcription of anthocyanin synthesis genes, and proposed levels of anthocyanins. But there is clear evidence of highly reduced DFR and ANS expression, which is not consistent with meaningful levels of anthocyanins. Altogether we suggest that the evidence of anthocyanins in pitaya remains weak, despite claims to the contrary.
The applied methods are almost identical to our previous analysis of a very similar data set .
RNAseq datasets of different cultivars were retrieved from the Sequence Read Archive via fastq-dump . Trimming and adapter removal based on a set of all available Illumina adapters were performed via Trimmomatic v0.39  using SLIDINGWINDOW:4:15 LEADING:5 TRAILING:5 MINLEN:50 TOPHRED33. A customized Python script was used to rename the surviving read pairs prior to the transcriptome assembly. Clean read pairs were subjected to Trinity v2.4.0  for de novo transcriptome assembly using a k-mer size of 25. Short contigs below 200 bp were discarded. Previously described Python scripts  and BUSCO v3  were applied for the calculation of assembly statistics for evaluation. Assembly quality was assessed based on continuity and completeness. Although assemblies were generated for all three species, the assembly generated on the basis of the data sets of Hylocereus undatus (SRR11603186-SRR11603191) was used for all down-stream analyses.
Prediction of encoded peptides was performed using a previously described approach to identify and retain the longest predicted peptide per contig . Genes involved in the flavonoid biosynthesis were identified via KIPEs  using the peptide mode. Phylogenetic trees with pitaya candidate sequences and previously characterized sequences  were constructed with FastTree v2  (WAG + CAT model) based on alignments constructed via MAFFT v7  and cleaned with pxclsq  to achieve a minimal occupancy of 0.1 for all alignment columns.
Transcript abundance quantification
Quantification of transcript abundance was performed with kallisto v0.44.0  using the RNAseq reads and our Hylocereus undatus transcriptome assembly . Customized Python scripts (pitaya2_exp_plots_summary.py and pitaya2_exp_plots_tissue.py) were applied to summarize and visualize expression values in a comparative way as previously described .
Timoneda A, Feng T, Sheehan H, Walker-Hale N, Pucker B, Lopez‐Nieves S, et al. The evolution of betalain biosynthesis in Caryophyllales. New Phytol. 2019;224:71–85.
Sheehan H, Feng T, Walker-Hale N, Lopez‐Nieves S, Pucker B, Guo R, et al. Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales. New Phytol. 2020;227:914–29.
Stafford HA. Anthocyanins and betalains: evolution of the mutually exclusive pathways. Plant Sci. 1994;101:91–8.
Clement JS, Mabry TJ. Pigment Evolution in the Caryophyllales: a Systematic Overview*. Bot Acta. 1996;109:360–7.
Polturak G, Grossman N, Vela-Corcia D, Dong Y, Nudel A, Pliner M, et al. Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals. PNAS. 2017;114:9062–7.
Zhou Z, Gao H, Ming J, Ding Z, Lin X, Zhan R. Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying Peel and pulp color formation. BMC Genomics. 2020;21:734.
Pucker B, Singh HB, Kumari M, Khan MI, Brockington SF. The report of anthocyanins in the betalain-pigmented genus Hylocereus is not well evidenced and is not a strong basis to refute the mutual exclusion paradigm. BMC Plant Biol. 2021;21:297.
Shimada S, Takahashi K, Sato Y, Sakuta M. Dihydroflavonol 4-reductasecDNA from non-Anthocyanin-Producing Species in the Caryophyllales. Plant Cell Physiol. 2004;45:1290–8.
Shimada S, Inoue YT, Sakuta M. Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species. Plant J. 2005;44:950–9.
Shimada S, Otsuki H, Sakuta M. Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales. J Exp Bot. 2007;58:957–67.
NCBI. sra-tools C NCBI - National Center for Biotechnology Information/NLM/NIH; 2020. https://github.com/ncbi/sra-tools. Accessed 8 Oct 2020.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol. 2011;29:644–52.
Haak M, Vinke S, Keller W, Droste J, Rückert C, Kalinowski J, et al. High Quality de Novo Transcriptome Assembly of Croton tiglium. Front Mol Biosci. 2018;5. https://doi.org/10.3389/fmolb.2018.00062.
Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–2.
Pucker B, Reiher F, Schilbert HM. Automatic Identification of Players in the Flavonoid Biosynthesis with Application on the Biomedicinal Plant Croton tiglium. Plants. 2020;9:1103.
Price MN, Dehal PS, Arkin AP. FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments. PLoS ONE. 2010;5:e9490.
Katoh K, Standley DM. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol. 2013;30:772–80.
Brown JW, Walker JF, Smith SA. Phyx: phylogenetic tools for unix. Bioinformatics. 2017;33:1886–8.
Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34:525–7.
Pucker B, Brockington S. Pitaya transcriptome assemblies and investigation of transcript abundances II. 2021. https://pub.uni-bielefeld.de/record/2956788. Accessed 17 Aug 2021.
We thank the Center for Biotechnology (CeBiTec) at Bielefeld University for providing an environment to perform the computational analyses. We thank Nathanael Walker-Hale for useful discussion.
BP is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 436841671. SFB is funded by BBSRC High Value Chemicals from Plants Network & NERC-NSF-DEB RG88096.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Phylogenetic trees of genes in the flavonoid biosynthesis. Identified candidate sequences are highlighted in red.
About this article
Cite this article
Pucker, B., Brockington, S.F. The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak. BMC Genomics 23, 739 (2022). https://doi.org/10.1186/s12864-022-08947-1
- Mutual pigment exclusion