The ethylene-responsive transcription factor PpERF9 represses PpRAP2.4 and PpMYB114 via histone deacetylation to inhibit anthocyanin biosynthesis in pear.

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica) and plum (Prunus spp.). By contrast, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism remains unclear. In this study, we identified and characterized an ethylene-induced ETHYLENE RESPONSE FACTOR (ERF) transcription factor, PpETHYLENE RESPONSE FACTOR9 (PpERF9), which functions as a transcriptional repressor. Our analyses indicated PpERF9 can directly inhibit expression of the MYB transcription factor gene PpMYB114 by binding to its promoter. Additionally, PpERF9 inhibits the expression of the transcription factor gene PpRELATED TO APETALA2.4 (PpRAP2.4), which activates PpMYB114 expression, by binding to its promoter, thus forming a PpERF9-PpRAP2.4-PpMYB114 regulatory circuit. Furthermore, PpERF9 interacts with the co-repressor PpTOPLESS1 (PpTPL1) via EAR motifs to form a complex that removes the acetyl group on histone H3 and maintains low levels of acetylated H3 in the PpMYB114 and PpRAP2.4 promoter regions. The resulting suppressed expression of these 2 genes leads to decreased anthocyanin biosynthesis in pear. Collectively, these results indicate that ethylene inhibits anthocyanin biosynthesis by a mechanism that involves PpERF9-PpTPL1 complex-mediated histone deacetylation of PpMYB114 and PpRAP2.4. The data presented herein will be useful for clarifying the relationship between chromatin status and hormone signaling, with implications for plant biology research.

Ethylene-activated PpERF105 induces the expression of the repressor-type R2R3-MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit.

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica), strawberry (Fragaria × ananassa), and plum (Prunus spp.). However, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism has not been characterized. In this study, ethylene induced the expression of PpERF105, which encodes a transcription factor. PpERF105 functioned as a transcriptional activator, but it inhibited anthocyanin biosynthesis in pear. A transcriptome analysis revealed that PpERF105 activated the expression of PpMYB140, which encodes an R2R3-MYB transcriptional repressor. Moreover, PpMYB140 directly inhibited the expression of anthocyanin-related structural genes. It also competed with PpMYB114 for the binding to bHLH3, ultimately resulting in the formation of the MYB140-bHLH-WDR complex rather than the conventional MBW complex, thereby further inhibiting anthocyanin biosynthesis. Furthermore, PpMYB140 prevented the overaccumulation of anthocyanins in the absence of ethylene. Collectively, our study data indicate that ethylene-induced PpERF105 inhibits anthocyanin biosynthesis by upregulating PpMYB140 expression. Our findings may be useful for elucidating the molecular basis of the ethylene-mediated inhibition of anthocyanin biosynthesis in fruit.

Light-Induced Basic/Helix-Loop-Helix64 Enhances Anthocyanin Biosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-Mediated Degradation in Pear.

Light is indispensable for the anthocyanin accumulation of red pear (Pyrus pyrifolia). Anthocyanin biosynthesis is catalyzed by a series of enzymes encoded by structural genes, which are regulated by a MYB-basic/helix-loop-helix-WD repeat (MYB-bHLH-WDR [MBW]) complex. The bHLH proteins of subgroup (SG) IIIf are believed to be involved in the regulation of anthocyanin accumulation. In this study, we revealed that pear PpbHLH64, which belongs to SGIIIb, positively regulates anthocyanin biosynthesis and is regulated by light at the transcriptional and posttranslational levels. Specifically, an exposure to light induced PpbHLH64 expression and anthocyanin accumulation in pear fruit and calli. Under light conditions, pear calli overexpressing PpbHLH64 exhibited enhanced red coloration, whereas the anthocyanin accumulation decreased in the PpbHLH64-RNA interference calli. Additionally, the transient overexpression of PpbHLH64 in pear fruit peel increased anthocyanin accumulation, whereas the virus-induced gene silencing of PpbHLH64 had the opposite effect. Further analyses indicated that PpbHLH64 is a transcriptional activator that directly binds to the promoter of UDP-GLUCOSE:FLAVONOID 3-O-GLYCOSYLTRANFERASE to upregulate expression. Moreover, PpbHLH64 interacted with PpMYB10, but not with PpMYB114, to form an MBW complex that significantly induces the accumulation of anthocyanins. Furthermore, PpbHLH64 was targeted by CONSTITUTIVE PHOTOMORPHOGENIC1 in darkness for subsequent degradation by the 26S proteasome. A genetic analysis indicated that PpbHLH64 functions downstream of B-BOX18, a component of the light signal transduction pathway. However, we were unable to detect the direct interaction between PpbHLH64 and PpBBX18. The characterization of PpbHLH64 in this study highlights the importance of SGIIIb bHLH proteins for light-induced anthocyanin accumulation.

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.

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.

R2R3-MYB transcription factor PpMYB17 positively regulates flavonoid biosynthesis in pear fruit.

MAIN CONCLUSION: PpMYB17 positively regulates flavonoid biosynthesis in pear fruit by activating PpCHS, PpCHI, PpF3H, and PpFLS in the flavonoid biosynthesis pathway independently of bHLH or WD40 cofactors in the MBW complex. Flavonoids are important secondary metabolites in plants. The flavonoid biosynthesis pathway is regulated by various transcription factors, with MYB transcription factors considered to be the key regulators. However, the regulation of flavonoid biosynthesis in the pear fruit has not been fully characterized. The R2R3-MYB transcription factor PpMYB17 was isolated from 'Red Zaosu' pear fruit and functionally characterized. An exposure to light upregulated PpMYB17 expression in the pear fruit. A phylogenetic analysis indicated PpMYB17 is related to the flavonol regulators. A subcellular localization assay suggested that PpMYB17 is a nuclear protein. Overexpression of PpMYB17 increased the flavonoid content of pear calli and Arabidopsis via the upregulated expression of structural genes in the flavonoid biosynthesis pathway, especially FLS. The LC-MS/MS analysis revealed most of the differentially accumulated flavonols, flavanones, flavones, isoflavones, and anthocyanins were significantly more abundant in PpMYB17-overexpressing calli than in wild-type calli. Moreover, PpMYB17 did not interact with PpbHLH3, PpbHLH33, or PpWD40 in a yeast system. Dual-luciferase assays demonstrated that PpMYB17 strongly activates the promoters of PpCHS, PpCHI, PpF3H, PpFLS, and PpUFGT which are key downstream genes in the flavonoid biosynthesis pathway, independently of the PpbHLH3 cofactor. These gene expression changes may enhance flavonoid biosynthesis in pear fruit. The data presented may be useful for further elucidating the flavonoid biosynthesis regulatory network, potentially leading to the development of new pear cultivars that produce fruits with increased flavonoid contents.

Ethylene mediates the branching of the jasmonate-induced flavonoid biosynthesis pathway by suppressing anthocyanin biosynthesis in red Chinese pear fruits.

Flavonoid accumulation in most fruits is enhanced by ethylene and jasmonate. However, little is known about the hormone functions related to red pear fruit coloration or their combined effects and potential underlying mechanisms. Various treatments were used to investigate the flavonoid metabolite profile and pear transcriptome to verify the effects of ethylene and jasmonate on flavonoid biosynthesis in red pear fruits as well as the mechanism behind this. Ethylene inhibits anthocyanin biosynthesis in red Chinese pear fruits, whereas jasmonate increases anthocyanin and flavone/isoflavone biosyntheses. The branching of the jasmonate-induced flavonoid biosynthesis pathway is determined by ethylene. Co-expression network and Mfuzz analyses revealed 4,368 candidate transcripts. Additionally, ethylene suppresses PpMYB10 and PpMYB114 expression via TF repressors, ultimately decreasing anthocyanin biosynthesis. Jasmonate induces anthocyanin accumulation through transcriptional or post-translational regulation of TFs-like MYB and bHLH in the absence of ethylene. However, jasmonate induces ethylene biosynthesis and the associated signalling pathway in pear, thereby decreasing anthocyanin production, increasing the availability of the precursors for flavone/isoflavone biosynthesis and enhancing deep yellow fruit coloration. We herein present new phenotypes and fruit coloration regulatory patterns controlled by jasmonate and ethylene, and confirm that the regulation of fruit coloration is complex.

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear.

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.

Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in 'Red Zaosu' pear fruits by interacting with MYB114.

KEY MESSAGE: Pp4ERF24 and Pp12ERF96 fine tune blue light-induced anthocyanin biosynthesis via interacting with PpMYB114 and promoting the interaction between PpMYB114 and PpbHLH3, which enhances the expression of PpMYB114-induced PpUFGT. The red coloration of pear fruit is attributed to anthocyanin accumulation, which is transcriptionally regulated by the MYB-bHLH-WD40 complex. A number of ethylene response factors (ERF) have been identified to regulate anthocyanin biosynthesis in different plants. In pear, several ERF transcription factor genes were identified to be potentially involved in the light-induced anthocyanin biosynthesis according to transcriptome data. But the molecular mechanism of these ERFs underlying the regulation of anthocyanin accumulation is unknown. In this study, exposure of 'Red Zaosu' pear, a mutant of 'Zaosu' pear, to blue light significantly induced the anthocyanin accumulation by increasing the expression levels of anthocyanin biosynthetic genes. Gene expression analysis confirmed that the expression of Pp4ERF24 and Pp12ERF96 genes were up-regulated in the process of blue light-induced anthocyanin biosynthesis. Yeast two-hybrid and bimolecular fluorescence complementation assay revealed that Pp4ERF24 and Pp12ERF96 interacted with PpMYB114, but not with PpMYB10. Bimolecular fluorescence complementation assay demonstrated that the interaction between these two ERFs and PpMYB114 enhanced the interaction between PpMYB114 and PpbHLH3. Further analysis by dual luciferase assay verified that these two ERFs increased the up-regulation of PpMYB114-mediated PpUFGT expression. Furthermore, co-transformation of Pp12ERF96 with PpMYB114 and PpbHLH3 in tobacco leaves led to enhanced anthocyanin accumulation. Transient overexpression of Pp4ERF24 or Pp12ERF96 alone in 'Red Zaosu' pear fruit also induced anthocyanin biosynthesis in pear peel. Our findings provide insights into a mechanism involving the synergistic interaction of ERFs with PpMYB114 to regulate light-dependent coloration and anthocyanin biosynthesis in pear fruits.

BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear.

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white-light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans-activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one-hybrid assays, the complex of PpBBX16/PpHY5 strongly trans-activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus-induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light-induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light-induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis-related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.

The blue light signal transduction pathway is involved in anthocyanin accumulation in 'Red Zaosu' pear.

MAIN CONCLUSION: A conserved blue light sensing and transduction pathway contributes to blue light-induced anthocyanin accumulation in the peel of red pear. Peel color is an economically important characteristic that influences the appearance quality of red pear, whose red color is due to anthocyanin accumulation. The process of coloration in the fruit peel is strongly influenced by light. However, how light quality influences color development remains unclear. In this study, we analyzed the effects of different light qualities on color development in the red pear 'Red Zaosu', a mutant of the hybrid cultivar 'Zaosu' of Pyrus pyrifolia and P. communis. The results showed that blue light increased anthocyanin accumulation after 72 h of light treatment, while red light had almost no effect. The expression of anthocyanin biosynthesis-related genes showed a similar trend to the anthocyanin accumulation. To clarify the mechanism of blue-light induced coloration, PpCRYs, PpCOP1 and PpHY5 genes were cloned. Gene expression analysis showed that their transcript abundance did not correlate with the expression of anthocyanin-related genes or anthocyanin content, but the yeast two-hybrid system revealed conserved physical interactions among these proteins. In addition, PpHY5 directly bound to the promoters of the anthocyanin biosynthesis genes PpCHS, PpDFR, PpANS and PpMYB10, and activated the transcription of PpCHS in a Nicotiana benthamiana-based dual-luciferase assay. In summary, our results preliminarily revealed that the conserved blue light signal transduction module CRY-COP1-HY5 contributed to the anthocyanin biosynthesis induced by blue light in red pear. However, our results did not provide evidence for why red light had no effect on anthocyanin accumulation, which needs further study.

PpZAT5 suppresses the expression of a B-box gene PpBBX18 to inhibit anthocyanin biosynthesis in the fruit peel of red pear.

BBX (B-box) proteins play a vital role in light-induced anthocyanin biosynthesis. PpBBX18 was an indispensable regulator for the induction of anthocyanin biosynthesis in the peel of red pear fruit (Pyrus pyrifolia Nakai.). However, the upstream regulation of BBX genes has not been well characterized. In this study, PpZAT5, a cysteine2/histidine2-type transcription factor, was discovered as the upstream negative regulator of PpBBX18. The results showed that PpZAT5 functions as a transcriptional repressor and directly binds to the CAAT motif of PpBBX18 and inhibits its expression. PpZAT5 expression was inhibited by light, which is converse to the expression pattern of anthocyanin-related structural genes. In addition, less anthocyanin accumulated in the PpZAT5-overexpressing pear calli than in the wild-type pear calli; on the contrary, more anthocyanin accumulated in PpZAT5-RNAi pear calli. Moreover, the crucial genes involved in light-induced anthocyanin biosynthesis were markedly down-regulated in the transcriptome of PpZAT5 overexpression pear calli compared to wild-type. In conclusion, our study indicates that PpBBX18 is negatively regulated by a C2H2-type transcriptional repressor, PpZAT5, which reduces anthocyanin content in pear. The present results demonstrate an upstream molecular mechanism of PpBBX18 and provide insights into light-induced anthocyanin biosynthesis.

Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation.

Bagging is an efficient method to improve fruit colour development. This work reported a transcriptome analysis using bagging-treated red Chinese sand pear peels. In total, 8,870 differentially expressed genes were further analysed by a weighted gene co-expression network analysis and early-, middle- and late light-responsive genes were identified. An annotation analysis revealed several pathways involved in the different responsive stages. The presence of LONG HYPOCOTLY 5, CRY-DASH and a CONSTANS-like transcription factors among the early light-responsive genes indicated the pivotal role of light, especially blue light, in the biological changes that occurred after bag removal. Other light-responsive transcription factors were also identified from the three light-responsive stages. In addition, the light-responsive pattern of anthocyanin biosynthetic genes differed among the biosynthetic steps. Although yeast-one hybrid assay showed that most of the structural genes were regulated by PpMYB10, their different temporal expressive pattern suggested that besides PpMYB10, other light-responsive transcriptional factors were also involved in the regulation of anthocyanin biosynthesis. In summary, our transcriptome analysis provides knowledge of the transcriptional regulatory network operating during light responses, which results in anthocyanin accumulation and other significant physiological changes in red Chinese sand pear peels after bag removal.