PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear

Abstract

Background: Decrease in anthocyanin content results in the loss of red color in leaves, petals and receptacles during development. The content of anthocyanin was affected by the biosynthesis and degradation of anthocyanin. Compared with the known detailed mechanism of anthocyanin biosynthesis, the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear.

Results: Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter.

Conclusions: Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Keywords: Anthocyanin degradation; PbLAC4-like; PbMYB26; Pear.

Fig. 1

Phenotypes, anthocyanin content and PbLAC4-like expression level in pear leaves at different stages. a The phenotypes of pear leaves. b Anthocyanin content and the expression level of PbLAC4-like gene in leaves of various varieties. The significant difference was determined by Tukey test. Error bars represent the means ± SEM of three biological replicates

Fig. 2

Phenotypes, anthocyanin content and PbLAC4-like expression level in pear petals, receptacles at different stages. a The phenotypes of pear petals, receptacles during color fading. b Anthocyanin content and the expression level of PbLAC4-like gene in petals of ‘Zaosu’ and ‘Red Zaosu’ at four stages. c Anthocyanin content and the expression level of PbLAC4-like gene in receptacles of ‘Red Zaosu’ at four stages. The significant difference was determined by Tukey test. Error bars represent the means ± SEM of three biological replicates

Fig. 3

Sequence analysis of PbLAC4-like protein. a Phylogenetic analysis of PbLAC4-like protein and LACs from other plants. b Sequence alignment of PbLAC4-like protein and LACs from other plants (The black, red and green boxes represent three copper oxidation domains, respectively)

Fig. 4

Transient transformation assays in the ‘Palacer’ fruitlet peel to verify the function of PbLAC4-like. a The phenotype of overexpressing PbLAC4-like (OE-PbLAC4-like) in ‘Palacer’ fruit, taking empty vector containing GUS (Empty) as the control. b Anthocyanin content and the expression level of PbLAC4-like gene in OE-PbLAC4-like and Empty. The significant difference was determined by t test for three replicates: *P < 0.05; **P < 0.01. Error bars represent the means ± SEM of three biological replicates

Fig. 5

Anthocyanin degradation activity assay. a The activity assay of the crude enzyme to degrade anthocyanin in leaves of various varieties was carried out with anthocyanin as a substrate. b The SDS-PAGE gel of prokaryotic expression PbLAC4-like protein (M stood for the marker, Eui was pET28a (+) empty vector without induction, Ei was induced pET28a (+) empty vector, Eip and Eis were precipitation and supernatant of induced pET28a empty vector respectively, Lui stood for pET28a (+) -PbLAC4-like vector without induction, Li was induced pET28a (+) -PbLAC4-like vector and Lip and Lis was precipitation and supernatant of induced pET28a (+) -PbLAC4-like vector respectively). c The enzyme activity test of prokaryotic expressed PbLAC4-like protein was carried out with anthocyanin as a substrate. The significant difference was determined by t test for three replicates: *P < 0.05; **P < 0.01. Error bars represent the means ± SEM of three biological replicates

Fig. 6

Expression analysis of candidate upstream regulators in pear leaves. The significant difference was determined by Tukey test for three replicates

Fig. 7

Expression analysis of candidate upstream regulators in petals and receptacles. a Expression levels of candidate upstream regulators in ‘Zaosu’, ‘Red Zaosu’ petals. b Expression levels of candidate upstream regulators in ‘Red Zaosu’ receptacles. The significant difference was determined by Tukey test for three replicates

Fig. 8

Validation of upstream regulators for the PbLAC4-like gene. a Y1H assay showing the interaction between PbMYB26 and the PbLAC4-like. b Effects of PbMYB26 on the promoter activity of PbLAC4-like in a dual luciferase assay. c Gene expression analysis after transient transformation PbMYB26 gene in ‘Zaosu’ fruit peel. The significant difference was determined by t test for three replicates: *P < 0.05; **P < 0.01. Error bars represent the means ± SEM of biological replicates