Abstract
Main conclusion
Phosphate deficiency promotes anthocyanin accumulation in Arabidopsis through direct binding of PHR1 to the P1BS motifs on the promoters of F3'H and LDOX and thereby upregulating their expression.
Abstract
Phosphorus is one of the essential elements for plants, and plants mainly absorb inorganic phosphate (Pi) from soil. But Pi deficiency is a common factor limiting plant growth and development. Anthocyanin accumulation in green tissues (such as leaves) is one of the characteristics of many plants in response to Pi starvation. However, little is known about the mechanism by which Pi starvation induces anthocyanin accumulation. Here, we found that the mutation of the gene PHOSPHATE STARVATION RESPONSE1 (PHR1), which encodes a key factor involved in Pi starvation signaling in Arabidopsis, significantly attenuates anthocyanin accumulation under Pi-limiting conditions. Moreover, the expression of several Pi deficiency-upregulated genes that are involved in anthocyanin biosyntheses, such as flavanone 3’-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and production of anthocyanin pigment 1 (PAP1), was significantly lower in the phr1-1 mutant than in the wild type (WT). Both yeast one-hybrid (Y1H) analysis and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that PHR1 can interact with the promoters of F3'H and LDOX, but not DFR and PAP1. By electrophoretic mobility shift assay (EMSA), it was further confirmed that the PHR1-binding sequence (P1BS) motifs located on the F3'H and LDOX promoters are required for the PHR1 bindings. Also, in Arabidopsis protoplasts, PHR1 enhanced the transcriptional activity of the F3'H and LDOX promoters, but these effects were markedly impaired when the P1BS motifs were mutated. Taken together, these results indicate that PHR1 positively regulates Pi starvation-induced anthocyanin accumulation in Arabidopsis, at least in part, by directly binding the P1BS motifs located on the promoters to upregulate the transcription of anthocyanin biosynthetic genes F3'H and LDOX.
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Data availability
All data generated or analyzed during this study are included in this article or the supplementary material. The materials generated during the current study are available from the corresponding author on reasonable request.
Abbreviations
- CHS:
-
Chalcone synthase
- DFR:
-
Dihydroflavonol 4-reductase
- F3’H:
-
Flavanone 3’-hydroxylase
- LDOX:
-
Leucoanthocyanidin dioxygenase
- LP:
-
Low phosphate
- P1BS:
-
PHR1-binding sequence
- PAP1:
-
Production of anthocyanin pigment 1
- PHR1:
-
Phosphate starvation response1
- PHT:
-
Phosphate transporter
- Pi:
-
Inorganic phosphate
- PSI:
-
Phosphate starvation-inducing
- PSR:
-
Pi starvation response
- TF:
-
Transcription factor
- TT8:
-
Transparent testa 8
- TTG1:
-
Transparent testa glabra1
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Acknowledgements
This work was supported by the National Natual Science Foundation of China (31701985 and 31700256), Natural Science Foundation of Fujian Province, China (2021J01092), Science and Technology Innovation Special Fund Project of Fujian Agriculture and Forestry University (CXZX2020045A), and National College Students Innovation and Entrepreneurship Training Program (S202110389046 and X202210389079).
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Supplementary file1 Fig. S1 a Schematic diagram of the T-DNA insert mutant phr1-1. yellow, exon; blue, UTR; black line, intron. b Homozygous of phr1-1 was confirmed by PCR. Fig. S2 a Maps of construction of PHR1-OX and 35S::PHR1-HA. b Transgenic plants were confirmed by PCR. c Transcript levels of PHR1 were tested using RT-qPCR in WT, PHR1-OX, and 35S::PHR1-HA. Values are means ± SE (n = 4). d Protein level of PHR1-HA was tested using Western blot in WT and 35S::PHR1-HA. The results were repeated three times with similar results. Fig. S3 Maps of the vectors pJG-ΔPHR1 and pLacZi-2μ. Fig. S4 a Seven-d-old in 1/2MS media-grown seedlings of WT, phr1-1, and PHR1-OX were treated with LP for the indicated time, then the anthocyanin contents were determined. They were transferred to LP media for 7 d. b Pi contents of the shoots were measured. c The transcript levels of CHS, CHI, UF3GT, TT8, and TTG1 were analyzed. Mean vaues ± SE (n = 4) Fig. S5 Schematic diagram of the promoters (2 kb upstream of the translation start site ‘ATG’) of several anthocyanin biosynthesis-related genes. Red line, P1BS motif. Fig. S6 Inducible expression and purification of MBP alone and MBP-ΔPHR1. T total protein of broken E. coli; S supernatant protein, FT flow through. Fig. S7 Sequencing the reporters and displaying the sequence adjacent to the P1BS. The results showed that the mutations in P1BS motifs were successful. pro, wild promoter; mpro, mutated promoter in the P1BS motif. Arrows indicate sequencing direction. (DOCX 4088 KB)
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Liu, Z., Wu, X., Wang, E. et al. PHR1 positively regulates phosphate starvation-induced anthocyanin accumulation through direct upregulation of genes F3’H and LDOX in Arabidopsis. Planta 256, 42 (2022). https://doi.org/10.1007/s00425-022-03952-w
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DOI: https://doi.org/10.1007/s00425-022-03952-w