Xanthophyll acyl esters in pepper (Capsicum annuum): Delineating the role of two xanthophyll acyltransferases CaPYP1 and CaPYP1-like via gene silencing and genetic complementation.
Pepper fruit accumulated xanthophyll acyl esters during ripening. To characterize the genes involved, cDNAs for two putative xanthophyll acyltransferases named CaPYP1 and CaPYP1-like were cloned. The deduced amino acid sequences of CaPYP1 and CaPYP1-like had conserved hydrolase and acyltransferase domains. Phylogenetic analyses showed that both putative acyl transferases were conserved in…
During the ripening process of pepper fruit, xanthophyll acyl esters are accumulated. To understand the genes involved in this process, cDNAs for two potential xanthophyll acyltransferases, designated as CaPYP1 and CaPYP1-like, were cloned. Both CaPYP1 and CaPYP1-like possess amino acid sequences with conserved hydrolase and acyltransferase domains.
Phylogenetic analysis revealed that these putative acyltransferases are conserved across Viridiplantae, indicating their importance in plant function. When GFP-fusion proteins of CaPYP1 and CaPYP1-like were expressed in leaf tissue, they localized in the plastid. Gene expression analysis showed that both genes exhibited the highest expression levels in ripening fruit, ranging from 52 to 64 days after anthesis, as well as in senescent leaves, with CaPYP1 demonstrating a more significant expression level compared to CaPYP1-like.
In experiments utilizing virus-induced gene silencing in pepper, it was observed that xanthophyll esterification was significantly reduced when CaPYP1 was silenced, resulting in a shift in fruit ripening color from dark red to bright red. Complementation tests demonstrated that overexpression of CaPYP1 in a tomato mutant, impaired for petal coloration and xanthophyll esterification, restored both the petal color and the synthesis of mono, diacyl, and triacyl esters of xanthophylls.
Conversely, overexpression of CaPYP1-like in the same genetic background led to the synthesis of relatively smaller amounts of xanthophyll monoesters. Additionally, in vitro tests revealed that zeaxanthin exhibited greater sensitivity to light compared to zeaxanthin dipalmitate; however, both compounds remained protected when mixed with triacylglycerol, suggesting that acyl moieties could enhance xanthophyll stability.
Collectively, these findings suggest that xanthophyll esterification during pepper fruit ripening plays a crucial role in fruit coloration, xanthophyll accumulation, and stability, and is regulated by both CaPYP1 and CaPYP1-like, with CaPYP1 exhibiting a more significant role in this process.
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