A Cassava CPRF-2-like bZIP Transcription Factor Showed Increased Transcript Levels during Light Treatment

Abstract

Background: bZIP proteins participate in the regulation of gene expression, playing crucial roles in various biological processes in plants, including response to environmental changes. Luminosity is an environmental factor of extreme importance for plant metabolism, acting as a regulator of its growth and development. Despite advances in the identification of bZIP proteins in several plant species, studies on these transcription factors in cassava are lacking. Cassava (Manihot esculenta Crantz) is one of the most important food crops in tropical and subtropical regions, mainly in developing countries, where its storage root is a major source of calories for low-income people.

Objectives: Our main aim was the isolation of a cDNA sequence encoding a bZIP protein from cassava (MebZIP) as well as the in silico characterization of its nucleotide and deduced amino acid sequences. In addition, we evaluated the expression pattern of the MebZIP gene in response to light, and its possible relationship with regulation of the chalcone synthase (MeCHS) gene.

Methods: RT-PCR and 3' and 5' RACE assays were used to isolate the full-length cDNA sequence of MebZIP. Bioinformatics tools were used to characterize the nucleotide and amino acid sequences of MebZIP. Semiquantitative RT-PCR assays were used to evaluate the expression levels of MebZIP and MeCHS genes.

Results: We isolated the full-length cDNA sequence of MebZIP with a 1320-bp ORF encoding a deduced protein with a predicted molecular weight and isoelectric point of 47 kDa and 5.85, respectively. Comparative analyses with GenBank sequences showed high identity of MebZIP with bZIP CPRF-2 of Hevea brasiliensis (XP_021650934) and Petroselinum crispum (Q99090.2). Besides the basic region and leucine zipper domains, MebZIP contains putative conserved domains (D1- D4), found in parsley CPRF-2 and bZIP proteins closely related to this protein. Since CPRF proteins are known for their function in regulation of the CHS gene by light, we evaluated the expression levels of the MebZIP gene and the possible target gene to be regulated by MebZIP (the MeCHS gene) in cassava under light conditions. Semi-quantitative RT-PCR assays revealed that MebZIP transcription increased in response to white light, with maximum expression levels at 6 h of light exposure. On the other hand, the expression levels of the MeCHS gene were statistically constant in all samples, indicating that they were not influenced by the experimental conditions used here.

Conclusion: The putative MebZIP protein identified in this work contains the conserved domains (bZIP, D1-D4) that indicate its functionality, thus allowing it to be considered a new member of the bZIP transcription factor CPRF-2 family. The expression levels of the MebZIP gene increased during white light exposure, indicating a potential function in light-response in cassava.

Keywords: MebZIP protein; bZIP protein; cassava; light-response; transcription factor; transcriptional regulation.