Elucidating the function of Sorghum Flavin containing Mono-oxygenase’s (FMOs)

Abstract

The genes being studied here are plant Flavin-containing monooxygenase (FMO’s) which are enzymes that place molecular oxygen on heteroatoms such as nitrogen, and sulfur with the help of cofactors such as NAD(P)H and FAD. Plant FMOs are divided into three major clades: N-hydroxylating, S-hydroxylating and YUCCA. The YUCCA family is conserved in all plants, and performs oxidative decarboxylation of indole-3-pyruvic acid (IPA) to produce Indole-3-acetic acid (IAA), a plant hormone also called auxin. FMO’s have been relatively well characterized in Arabidopsis thaliana, a plant model organism, yet there is deficiency in characterization of cereal crops. Cereal FMOs have been postulated to have traits that enable them to survive against different biotic and abiotic stress; in the future, these cereal FMOs can be inserted into other crops for their survival. This project aims to characterize four sorghum FMOs: three N-hydroxylating FMOs and one YUCCA.

Using gateway cloning, constructs containing the four sorghum genes were created and were electroporated in Agrobacterium tumefaciens AGL10. Agro-infiltration into tobacco (Nicotiana benthamiana) leaves was carried out so that their metabolic response could be studied. The leaves were taken after 5 days and metabolites produced were analyzed with Liquid Chromatography-Mass spectrometry (LC-MS) for expected and unexpected metabolites. The response from the Sorghum YUCCA gene was unique and not found in the other 2 control YUCCAs from Barley and Arabidopsis. The concentration of three metabolites was increased due to the Sorghum YUCCA response in comparison to the other two YUCCA’s. One of the N-hydroxylating FMO, in opposition to its hypothesis, seems to be acting on proline instead of pipecolic acid. One other N-hydroxylating FMO; induced during wounding, shows decrease in tryptophan concentration instead of an increase.

For further examination, the four Sorghum FMO genes and one Arabidopsis gene were cloned into an expression vector using Ligation Independent Cloning (LIC). They were cloned for the purpose of being used in in-vitro assays. Two dyes were produced, blue and orange-reddish dye. The dyes obtained from SbFMO1 were analyzed with thin layer chromatography (TLC) together with standards, to identify the dyes produced. The blue dye was confirmed to be indigo while orange-reddish dye’s nature could not be confirmed, but it was hypothesized to be Indirubin.

The experimental results obtained from in-vivo (metabolic response from LC-MS) indicate that there might be a difference between cereal FMOs and Arabidopsis FMO while in-vitro (color from E. coli) result shows indigo as well as another color. This study gives new insights into the function of Sorghum FMO’s, clearly showcasing that their function cannot easily be deduced only by bioinformatic methods; experimental work also must be done to corroborate their actual function.

Popular Abstract

As the days go by, the effects of climate change are increasingly becoming clearer which affects agricultural yield and will de-stabilize food security. There are some strategies to combat this, such as improving the infrastructure, cross-pollinating different plants to get better crops and using genetic engineering. There are some crops that can grow in difficult conditions such as barley and sorghum. These plants must be further studied to understand how they can grow in these difficult conditions. They are not only studied because of their survival capability but also because of their importance. After barley and sorghum have been studied, we can transfer their traits to other plants to help them survive. And unless we want people in the future to starve, we need to improve crops to survive these terrible conditions.
This is why I'm doing research in plant FMO’s (Flavin containing mono-oxygenases) which are enzymes that have been linked to plant survival and defense. Plant FMO research and applications are a bit behind compared to bacterial FMO and they have not been properly studied in plants. There is data suggesting that FMO’s are responsible for sorghum surviving in difficult conditions, but it has not been confirmed. There might also be other FMO’s that might be responsible for giving other better traits. It's a big unknown.
The goal is to understand these unknown FMO’s and characterize their function so maybe in the future they can be put in other plants which may help them grow in the difficult conditions. My main focus was on Sorghum FMOs because they are one of the plants that can adapt and grow in the harsh conditions of drought and because it is the 4th most important cereal crop. The sorghum genes were studied using molecular biology techniques. The results were encouraging. One of the FMO’s that was studied started producing and storing chemicals that might help sorghum grow in difficult conditions; and this result has not been seen in other plants. It is only found in Sorghum and should be studied further to understand the new functions. This might open the path for more resistant crops for future generations.

• author: Kulkarni, Anirudh
• supervisor: Sofia Marmon; Elizabeth Neilson; Joachim Christensen
• organization: Pure and Applied Biochemistry
• course: KBKM01 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Applied biochemistry, Sorghum Flavin, FMOs, Flavin containing mono-oxygenases
• language: English
• id: 9098925
• date added to LUP: 2022-09-06 12:44:21
• date last changed: 2022-09-06 12:44:21