Two B‐box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit

Abstract

Light is indispensable for the accumulation of anthocyanin in the peel of red pear fruit (Pyrus pyrifolia Nakai). ELONGATED HYPOCOTYL 5 (HY5) is considered to be a critical regulator for induction of anthocyanin biosynthesis, but detailed characterization of its regulatory mechanism is needed. In this study, multiple genetic and biochemical approaches were applied to identify the roles of P. pyrifolia HY5 (PpHY5) and two B-box (BBX) proteins, PpBBX18 and PpBBX21, in the transcriptional regulation of PpMYB10. The functions of the two BBX proteins were analyzed in overexpression lines using pear calli-based approaches. On its own PpHY5 was unable to activate downstream genes. The two BBX proteins, PpBBX18 and PpBBX21, physically interacted with PpHY5 and antagonistically regulated anthocyanin biosynthesis in Arabidopsis and pear. PpBBX18 formed a heterodimer with PpHY5 via two B-box domains, in which PpHY5 bound to the G-box motif of PpMYB10 and PpBBX18 provided the trans-acting activity, thus inducing transcription of PpMYB10. PpBBX21 interacted with PpHY5 and PpBBX18 and hampered formation of the PpHY5-PpBBX18 active transcription activator complex, and subsequently repressed anthocyanin biosynthesis. The present results demonstrate the fine-tuned regulation of anthocyanin biosynthesis via transcriptional regulation of PpMYB10 by PpHY5-associated proteins and provide insights into light-induced anthocyanin biosynthesis.

ABA‐responsive ABRE‐BINDING FACTOR3 activatesDAM3expression to promote bud dormancy in Asian pear

“…Proteins were expressed in Escherichia coli strain BL21 and purified with Ni‐NTA Sefinose Resin (Settled Resin) (Sangon Biotech, China) and Econo‐Pac® Disposable Chromatography Columns (Bio‐Rad). The electrophoretic mobility shift assay (EMSA) was performed as previously described (Bai et al, ), with minor modifications. Oligonucleotide probes were synthesized and labelled with biotin at the 3′ end (Genebio, China).…”

“…The yeast two‐hybrid (Y2H) assays were conducted as described by Bai, Tao, Yin, et al (). Briefly, the complete ABF3 and ABF2 CDSs were cloned into the AD vector as the prey.…”

B‐box proteins: Pivotal players in light‐mediated development in plants

“…MdHY5 activates the expression of MdBBX33/MdCOL11, the product of which controls the expression of MdMYBA to promote lightinduced anthocyanin accumulation (Bai et al 2014). In addition, PpBBX16, PpBBX18 and PpBBX21 mediate anthocyanin biosynthesis via PpHY5 in pear (Bai et al 2019c(Bai et al , 2019d. Both PpBBX16 and PpBBX18 interact with PpHY5 and enhance its biochemical activity; PpHY5 subsequently associates with the G-box ciselement within the promoters of PpMYB10 and PpBBX18 and activates the transcription of the former to induce anthocyanin accumulation (Bai et al 2019c(Bai et al , 2019d.…”

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

“…In this study, the candidate PpBBX24 associated with the red characteristic of "Zaosu Red" is a B-box protein and belongs to a class of zinc finger transcription factors that contain one or more B-box domains at the N-terminus involved in mediating protein-protein interactions, of which 32 members have been identified in Arabidopsis 31,32 . It has been verified that several plant BBXs play important regulatory roles in photomorphogenesis and anthocyanin accumulation, among which AtBBX20 33 , MdBBX20 34 , AtBBX21 35,36 , PpBBX16 6 , PpBBX18 25 , OsBBX14 37,38 , and MdBBX22 39,40 act as positive regulators, whereas AtBBX19 41 , PpBBX21 23 , AtBBX32 42 , MdBBX37 43 , MdCOL4 (BBX24) 44 , AtBBX24 45 , and AtBBX25 35,46 act as negative regulators.…”

“…Recently, Tao et al 24 revealed that PpHY5 participated in anthocyanin biosynthesis induced by blue light in red pear. Bai et al 6,25 found that PpBBX16 cooperated with PpHY5 and positively regulated light-induced anthocyanin accumulation by activating MYB10 in red pear; PpBBX18 and PpBBX21 antagonistically regulate anthocyanin biosynthesis via competitive association with PpHY5 in the peel of pear fruits. Ni et al 14 reported that the ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in red pear fruits by interacting with MYB114, and Wu et al 26 indicated that PyMADS18 is likely to be involved in anthocyanin accumulation and the regulation of anthocyanin synthesis in the early development of pear fruits.…”