Transcriptional Analysis of Carotenoids Accumulation and Metabolism in a Pink-Fleshed Lemon Mutant

Abstract

Pink lemon is a spontaneous bud mutation of lemon (Citrus limon, L. Burm. f) characterized by the production of pink-fleshed fruits due to an unusual accumulation of lycopene. To elucidate the genetic determinism of the altered pigmentation, comparative carotenoid profiling and transcriptional analysis of both the genes involved in carotenoid precursors and metabolism, and the proteins related to carotenoid-sequestering structures were performed in pink-fleshed lemon and its wild-type. The carotenoid profile of pink lemon pulp is characterized by an increased accumulation of linear carotenoids, such as lycopene, phytoene and phytofluene, from the early stages of development, reaching their maximum in mature green fruits. The distinctive phenotype of pink lemon is associated with an up-regulation and down-regulation of the genes upstream and downstream the lycopene cyclase, respectively. In particular, 9-cis epoxycarotenoid dioxygenase genes were overexpressed in pink lemon compared with the wild-type, suggesting an altered regulation of abscisic acid biosynthesis. Similarly, during early development of the fruits, genes of the carotenoid-associated proteins heat shock protein 21, fibrillin 1 and 2 and orange gene were overexpressed in the pulp of the pink-fleshed lemon compared to the wild-type, indicating its increased capacity for sequestration, stabilization or accumulation of carotenes. Altogether, the results highlighted significant differences at the transcriptomic level between the pink-fleshed lemon and its wild-type, in terms of carotenoid metabolism and the capacity of stabilization in storage structures between the two accessions. Such changes may be either responsible for the altered carotenoid accumulation or in contrast, a metabolic consequence.

A comprehensive analysis of carotenoids metabolism in two red-fleshed mutants of Navel and Valencia sweet oranges (Citrus sinensis)

“…Exhaustive research has focused on the pattern of carotenoid accumulation in these red-fleshed citrus fruits with the aim of elucidating the molecular mechanisms responsible for this trait. The profile of carotenoids suggests that the basis of the alteration leading to lycopene accumulation may differ among citrus species and varieties ( Liu et al., 2007 ; Alquézar et al., 2008 , Yu et al., 2012 ; Alquézar et al., 2013 ; Zeng et al., 2015 ; Lu et al., 2016 ; Yan et al., 2018 ; Lana et al., 2020 ; Promkaew et al., 2020 ). In the red Hong Anliu, a spontaneous mutant of the Anliu orange, the concentration of carotenoids in the juice sacs was 2-times higher than in the standard variety, mainly by the accumulation of lycopene (~2 μg g -1 ) at expenses of xanthophylls, which are strongly reduced ( Liu et al., 2007 ).…”

“…The comparison of several pummelos varieties with different pulp pigmentation indicated that lycopene is associated with a reduced expression of genes encoding for enzymes operating downstream lycopene ( Liu et al., 2016 ; Yan et al., 2018 ; Promkaew et al., 2020 ). On the contrary, in the pulp of Pink lemon, a spontaneous bud mutant of Eureka lemon, accumulation of lycopene was associated with an up-regulation and down-regulation of the genes upstream and downstream lycopene β-cyclase, respectively ( Lana et al., 2020 ). While in Cara Cara orange, an enhancement of the expression of specific MEP pathway genes and an increased channelling of isoprenoid precursors into the carotenoid pathway, has been hypothesized to be related to its red-fleshed phenotype ( Alquézar et al., 2008 ; Lu et al., 2017 ; Zhang et al., 2021 ), even though other mechanisms may be involved ( Alquézar et al., 2008 ; Ye et al., 2010 ; Lu et al., 2017 ).…”

A genomic approach to the evolution, diversification and domestication of the genus Citrus

“…Our results would not support the existence of a master gene controlling carotenoid accumulation, but rather suggest that this trait would depend on the additive effects of several genes involved in this process. This idea is supported by the large number of somatic mutants that display an altered fruit color, most of which have been linked with mutations affecting genes all along the carotenoid biosynthetic pathway, which ultimately produces the mutant phenotype (Liu et al, 2007;Alquézar et al, 2008;Alós et al, 2008;Rodrigo et al, 2019;Lana et al, 2020). According to our results, some of these genes would have a more determinant role in of the red or yellow fruits.…”

“…Taken together, these observations suggest that Citrus red peel coloration is not a consequence of a single key gene but rather the result of the combined effect of many independent genes. This statement is supported by the multiple pigmentation mutants reported in Citrus, affecting a many different loci Alquézar et al, 2008;Alós et al, 2008;Rodrigo et al, 2019;Lana et al, 2020).…”

Comparative transcriptomics of wild and commercial Citrus during early ripening reveals how domestication shaped fruit gene expression

“…Since other authors suggested the importance of substrate availability in determining the final carotenoid profile in Citrus [ 98 , 104 ], we believe that the expression changes in PSY or ZDS reported in our work might be also affecting it. The idea that citrus peel pigmentation depends on many independent genes has been already postulated [ 87 ], and is vastly supported by the large number of somatic mutants that display an altered fruit color, mostly due to mutations affecting genes in the carotenoid biosynthetic pathway [ 92 , 105 – 108 ].…”