Characterization of cassava orange proteins and their capability to increase provitamin A carotenoids accumulation

Authors: JARAMILLO, ANGELICA - International Center For Tropical Agriculture (CIAT); SIERRA, SANTIAGO - International Center For Tropical Agriculture (CIAT); CHAVARRIAGA-AGUIRRE, PAUL - International Center For Tropical Agriculture (CIAT); CASTILLO, DIANA - International Center For Tropical Agriculture (CIAT); GKANOGIANNIS, ANESTIS - International Center For Tropical Agriculture (CIAT); LOPEZ-LAVALLE, LUIS - International Center For Tropical Agriculture (CIAT); ACRINIEGAS, JUAN - International Center For Tropical Agriculture (CIAT); SUN, TIANJU - International Center For Tropical Agriculture (CIAT); Li, Li; WELSCH, RALF - University Of Freiburg; BOY, ERICK - International Food Policy Researc Institute (IFPRI); ALVAREZ, DANIEL - International Center For Tropical Agriculture (CIAT)

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2021
Publication Date: 1/7/2022
Citation: Jaramillo, A., Sierra, S., Chavarriaga-Aguirre, P., Castillo, D., Gkanogiannis, A., Lopez-Lavalle, L., Acriniegas, J., Sun, T., Li, L., Welsch, R., Boy, E., Alvarez, D. 2022. Characterization of cassava orange proteins and their capability to increase provitamin A carotenoids accumulation. PLoS ONE. 17(1):Article e0262412. https://doi.org/10.1371/journal.pone.0262412.
DOI: https://doi.org/10.1371/journal.pone.0262412

Interpretive Summary: Cassava biofortification with provitamin A carotenoids is important to help alleviate vitamin A deficiency as provitamin A carotenoid level in cassava is generally low. To improve provitamin A level in cassava, this study investigated the role of OR protein for carotenoid biosynthesis and stability and its relationship with phytoene synthase (PSY). Various OR variants were identified from cassava roots and higher OR protein level but not gene expression was found in yellow cultivars. Overexpression of PSY greatly enhanced carotenoid accumulation in cassava callus. This study suggests an important role of controlling OR and PSY protein levels for higher provitamin A accumulation in cassava root and establishes an effective callus system to investigate the efficiency of genes for carotenoid enrichment in cassava root.

Technical Abstract: Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.