A 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of “Zaosu Red” pear (Pyrus pyrifolia White Pear Group): a deletion in the PpBBX24 gene is associated with the red skin of pear

Abstract

Red skin is an important quality trait for pear fruits and is determined by the concentration and composition of anthocyanins. The regulatory mechanism underlying anthocyanin accumulation is a popular topic in fruit research. Red mutants are ideal materials for studying the molecular mechanism of color diversity in pear. Although several red pear mutants have been cultivated and are in production, no exact locus containing the responsible genetic mutation has been identified. In this study, by combining the bulked segregant analysis with whole-genome sequencing, we identified a 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene from the red pear mutant “Zaosu Red”. We further verified that the deletion was present only in the red mutant of “Zaosu” and in its red offspring, which was different from that which occurred in other red pear fruits. This deletion results in a coding frame shift such that there is an early termination of the PpBBX24 gene and loss of key NLS and VP domains from PpBBX24. The lost domains may reduce or alter the normal function of PpBBX24. In addition, we found that the transcript levels of the PpMYB10 and PpHY5 genes in red samples were significantly higher than those in green samples, whereas the results for the normal-type PpBBX24 gene were the opposite. We ultimately revealed that the 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of the “Zaosu Red” pear. This finding of somatic mutational events will be helpful for breeding new red pear cultivars and for understanding the regulatory mechanisms involved in pear skin pigmentation.

References

[1] Huang, C., Yu, B., Teng, Y., Su, J. & Shu, Q. Effects of fruit bagging on coloring and related physiology, and qualities of red Chinese sand pears during fruit maturation. Sci. Hortic. 121, 149-158 (2009).

[2] Sun, Y. et al. Postharvest pigmentation in red Chinese sand pears (Pyrus pyrifolia Nakai) in response to optimum light and temperature. Postharvest Biol. Tec. 91, 64-71 (2014).

[3] Gamble, J., Jaeger, S. R. & Harker, F. R. Preferences in pear appearance and response to novelty among Australian and New Zealand consumers. Postharvest Biol. Technol. 41, 38-47 (2006).

[4] Bao, L., Chen, K. S., Zhang, D., Li, X. & Teng, Y. W. An assessment of genetic variability and relationships within Asian Pears based on AFLP (amplified fragment length polymorphism) markers. Sci. Hortic. 116, 374-380 (2008).

[5] Bai, S. L. et al. Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation. Sci. Rep. 7, 63 (2017).

[6] Bai, S. et al. BBX 16, a B‐box protein, positively regulates light‐induced anthocyanin accumulation by activating MYB 10 in red pear. Plant Biotechnol. J. 17, 1985-1997 (2019).

[7] Yang, Y. N. et al. Expression differences of anthocyanin biosynthesis genes reveal regulation patterns for red pear coloration. Plant Cell Rep. 34, 89-198 (2015).

[8] Qian, M. et al. Analysis of different pigmentation patterns in ‘Mantianhong’ (Pyrus pyrifolia Nakai) and ‘Cascade’ (Pyrus communis L.) under bagging treatment and postharvest UV-B/visible irradiation conditions. Sci. Hortic. 151, 75-82 (2013).

[9] Lespinasse, Y. & Guérif, P. Inheritance of red leaf colour from pear red sports of ‘Doyenne du Comice’, ‘Bartlett’ and ‘Beurre Hardy’. Acta Hortic. 909, 97-102 (2011).

[10] Dondini, L. et al. The inheritance of the red colour character in European pear (Pyrus communis) and its map position in the mutated cultivar ‘Max Red Bartlett’. Plant Breed. 127, 524-526 (2008).

[11] Steyn, W. J., Wand, S. J. E., Holcroft, D. M. & Jacobs, G. Red colour development and loss in pears. Acta Hortic. 671, 79-85 (2005).

[12] Zhang, X. et al. Differential gene expression analysis of Yunnan red pear, Pyrus pyrifolia, during fruit skin coloration. Plant Mol. Biol. Rep. 29, 305-314 (2011).

[13] Zhang, D., Qian, M., Yu, B. & Teng, Y. Effect of fruit maturity on UV-B-induced post-harvest anthocyanin accumulation in red Chinese sand pear. Acta Physiol. Plant 35, 2857-2866 (2013).

[14] Ni, J. et al. Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘Red Zaosu’pear fruits by interacting with MYB114. Plant Mol. Biol. 99, 67-78 (2019).

[15] Ngo, T. & Zhao, Y. Stabilization of anthocyanins on thermally processed red D’Anjou pears through complexation and polymerization. LWT-Food Sci. Technol. 42, 1144-1152 (2009).

[16] Winkel-Shirley, B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126, 485-493 (2001).

[17] Yu, B. et al. Isolation of anthocyanin biosynthetic genes in red Chinese sand pear (Pyrus pyrifolia Nakai) and their expression as affected by organ/tissue, cultivar, bagging and fruit side. Sci. Hortic. 136, 29-37 (2012).

[18] Yang, Y. N. et al. Molecular cloning and gene expression differences of the anthocyanin biosynthesis-related genes in the red/green skin color mutant of pear (Pyrus communis L.). Tree Genet. Genomes 9, 1351-1360 (2013).

[19] Ban, Y. et al. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol. 48, 958-970 (2007).

[20] Xu, W. J., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 20, 176-185 (2015).

[21] Albert, N. W. et al. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J. 65, 771-784 (2011).

[22] Feng, S. Q., Wang, Y. L., Yang, S., Xu, Y. T. & Chen, X. S. Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta 232, 245-255 (2010).

[23] Yao, G. F. et al. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant J. 92, 437-451 (2017).

[24] Tao, R. et al. The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘Red Zaosu’ pear. Planta 248, 37-48 (2018).

[25] Bai, S. et al. Two B‐box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with PpHY5 in the peel of pear fruit. Plant J. 100, 1208-1223 (2019).

[26] Wu, J. et al. Identification of differentially expressed genes related to coloration in red/green mutant pear (Pyrus communis L.). Tree Genet. Genomes 9, 75-83 (2013).

[27] Xue, H. et al. The genetic locus underlying red foliage and fruit skin traits is mapped to the same location in the two pear bud mutants ‘Red Zaosu’ and ‘Max Red Bartlett’. Hereditas 155, 25 (2018).

[28] Li, P., Zhang, Y., Einhorn, T. C. & Cheng, L. Comparison of phenolic metabolism and primary metabolism between green ‘Anjou’ pear and its bud mutation, red ‘Anjou’. Physiol. Plant. 150, 339-354 (2014).

[29] Booi, S., van Dyk, M. M., du Preez, M. G., Rees, D. J. G. & Labuschagné, I. Molecular typing of red and green phenotypes of ‘Bon Rouge’ pear trees, with the use of microsatellites. Acta Hort. 671, 293-297 (2005).

[30] Li, J. et al. A new red pear cultivar ‘Nanhong Li’. Acta Hort. Sin. 38, 1821-1822 (2011).

[31] Khanna, R. et al. The Arabidopsis B-box zinc finger family. Plant Cell 21, 3416-3420 (2009).

[32] Gangappa, S. N. & Botto, J. F. The BBX family of plant transcription factors. Trends Plant Sci. 19, 460-470 (2014).

[33] Wei, C. et al. The Arabidopsis B-box protein BZS1/BBX20 interacts with HY5 and mediates strigolactone regulation of photomorphogenesis. J. Genet. Genom. 43, 555-563 (2016).

[34] Fang, H. et al. The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell Environ. 42, 2090-2104 (2019).

[35] Job, N., Yadukrishnan, P., Bursch, K., Datta, S. & Johansson, H. Two B-box proteins regulate photomorphogenesis by oppositely modulating HY5 through their diverse C-terminal domains. Plant Physiol. 176, 2963-2976 (2018).

[36] Xu, D. et al. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation. Proc. Natl. Acad. Sci. U. S. A. 113, 7655-7660 (2016).

[37] Kim, D. H. et al. A rice B-Box protein, OsBBX14, finely regulates anthocyanin biosynthesis in rice. Int. J. Mol. Sci. 19, 2190 (2018).

[38] Bai, B. et al. OsBBX14 promotes photomorphogenesis in rice by activating OsHY5L1 expression under blue light conditions. Plant Sci. 284, 192-202 (2019).

[39] Bai, S. et al. An apple B-box protein, MdCOL11, is involved in UV-B-and temperature-induced anthocyanin biosynthesis. Planta 240, 1051-1062 (2014).

[40] An, J. P. et al. MdBBX22 regulates UV‐B‐induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT2 for 26S proteasome‐mediated degradation. Plant Biotechnol. J. 17, 2231-2233 (2019).

[41] Wang, C. Q., Sarmast, M. K., Jiang, J. & Dehesh, K. The transcriptional regulator BBX19 promotes hypocotyl growth by facilitating COP1-mediated EARLY FLOWERING3 degradation in Arabidopsis. Plant Cell 27, 1128-1139 (2015).

[42] Holtan, H. E. et al. BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21. Plant Physiol. 156, 2109-2123 (2011).

[43] An, J. P. et al. An apple B-Box protein MdBBX37 modulates anthocyanin biosynthesis and hypocotyl elongation synergistically with MdMYBs and MdHY5. Plant Cell Physiol. 61, 130-143 (2019).

[44] Fang, H. et al. MdCOL4 interaction mediates crosstalk between UV-B and high temperature to control fruit coloration in apple. Plant Cell Physiol. 60, 1055-1066 (2019).

[45] Jiang, L. et al. Arabidopsis STO/BBX24 negatively regulates UV-B signaling by interacting with COP1 and repressing HY5 transcriptional activity. Cell Res. 22, 1046 (2012).

[46] Gangappa, S. N., Holm, M. & Botto, J. F. Molecular interactions of BBX24 and BBX25 with HYH, HY5 HOMOLOG, to modulate Arabidopsis seedling development. Plant Signal. Behav. 8, e25208 (2013).

[47] Holm, M., Hardtke, C. S., Gaudet, R . & Deng, X. W. Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1. EMBO J. 20, 118-127 (2001).

[48] Yan, H. et al. Nuclear localization and interaction with COP1 are required for STO/BBX24 function during photomorphogenesis. Plant Physiol. 156, 1772-1782 (2011).

[49] Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. Next-generation sequencing from bulked segregant analysis identifies a dwarfism gene in watermelon. Sci. Rep. 8, 2908 (2018).

[50] Klein, H. et al. Bulked-segregant analysis coupled to whole genome sequencing (BSA-Seq) for rapid gene cloning in Maize. G3 Genes Genom. Genet. 8, 3583-3592 (2018).

[51] Li, F. et al. Identification of a locus for seed shattering in rice (Oryza sativa L.) by combining bulked segregant analysis with whole-genome sequencing. Mol. Breed. 39, 36 (2019).

[52] Lu, Z. et al. Fine mapping of the temperature-sensitive semi-dwarf (Tssd) locus regulating the internode length in peach (Prunus persica). Mol. Breed. 36, 20 (2016).

[53] Doyle, J. J. & Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus 12, 39-40 (1990).

[54] Ou, C. et al. A de novo genome assembly of the dwarfing pear rootstock Zhongai 1. Sci. Data 6, 281 (2019).

[55] Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).

[56] Reumers, J. et al. Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing. Nat. Biotechnol. 30, 61-68 (2012).

[57] Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly 6, 80-92 (2012).

[58] Hill, J. T. et al. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq. Genome Res. 23, 687-697 (2013).

[59] Fekih, R. et al. MutMap+: genetic mapping and mutant identification without crossing in rice. PLoS One 8, e68529 (2013).

[60] Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357-360 (2015).

[61] Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290-295 (2015).

[62] Florea, L., Song, L. & Salzberg, S. L. Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues. F1000 Res. 2, 188 (2013).