Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Abstract

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

Regulation of Banana Phytoene Synthase (MaPSY) Expression, Characterization and Their Modulation under Various Abiotic Stress Conditions

“…Phylogenetic analysis was conducted in MEGA6 (Tamura et al, 2013) for deduced protein sequences of arabidopsis, rice, maize and banana. The PSY sequences derived from Asupina (high provitamin A) and Cavendish (low provitamin A) banana cultivars (Mlalazi et al, 2012) were also considered in the study. Neighbor-joining method was used to infer homologs and evolution.…”

Carotenogenesis Is Regulated by 5′UTR-Mediated Translation of Phytoene Synthase Splice Variants

“…The multigene situation often coincides with the development of chromoplasts, implying that additional PSY genes might be required for this purpose, as shown in tomato (Solanum lycopersicum), melons (Cucumis melo), yellow maize, and banana (Musa sp. ; Fraser et al, 1994;Gallagher et al, 2004;Qin et al, 2011;Mlalazi et al, 2012). Conversely, several PSY paralogs are also present in taxa that do not develop chromoplasts, such as in rice and in white maize varieties.…”

Transgenic Pro-Vitamin A Biofortified Crops for Improving Vitamin A Deficiency and Their Challenges

“…The Asupina PSY enzyme exhibited 2-fold higher enzymatic activity than that of Cavendish, and β-carotene contents in Asupina and Cavendish differed by at least an order of magnitude. Thus, utilization of Asupina PSY gene could increase β-carotene in widely consumed banana cultivars, facilitate GMO regulatory approval, and improve consumer acceptance of pVA bananas [60]. Buah et al (2016) further identified key differences between the two bananas; expression of CCD4 in Cavendish which decreased β-carotene in Cavendish, and conversion of amyloplasts to chromoplasts in Asupina which increased β-carotene in Auspina [59].…”

“…However, the fact that the majority of banana cultivars are sterile triploids challenges their conventional breeding. Therefore, genetic modification within the genetic variation in Musa spp., or cisgenics, can be a viable option [60]. Mlalazi et al (2012) characterized banana PSY genes and proposed at least two PSY paralogs, PSY1 and PSY2a are present in Cavendish and Asupina [60].…”

“…Therefore, genetic modification within the genetic variation in Musa spp., or cisgenics, can be a viable option [60]. Mlalazi et al (2012) characterized banana PSY genes and proposed at least two PSY paralogs, PSY1 and PSY2a are present in Cavendish and Asupina [60]. The Asupina PSY enzyme exhibited 2-fold higher enzymatic activity than that of Cavendish, and β-carotene contents in Asupina and Cavendish differed by at least an order of magnitude.…”