Biochemical properties of Acyl-CoA-dependent and Acyl-CoA-independent avocado acyltransferases positively influence oleic acid content in nonseed triacylglycerols

Authors: RAHMAN, MD MAHBUBAR - East Tennessee State University; KLUNGA, JOSPHAT - East Tennessee State University; BEHERA, JYOTI - East Tennessee State University; Shockey, Jay; KILARU, ARUNA - East Tennessee State University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2022
Publication Date: 1/10/2023
Citation: Rahman, M., Klunga, J., Behera, J., Shockey, J.M., Kilaru, A. 2023. Biochemical properties of Acyl-CoA-dependent and Acyl-CoA-independent avocado acyltransferases positively influence oleic acid content in nonseed triacylglycerols. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2022.1056582.
DOI: https://doi.org/10.3389/fpls.2022.1056582

Interpretive Summary: Vegetable oils are a major nutritional component of the modern human diet. Increasingly sophisticated consumers have focused more and more on the balance of ‘healthy’ versus ‘unhealthy’ fats in recent years, and vegetable oils are no exception in that regard. Relatively few natural plant oils possess the very proportion of heart-healthy monounsaturated oleic acid found in avocado oil. The avocado fruit produces 60-70% oil by weight, and approximately 70% of the fatty acids are oleic acid. To date, though, extremely little is known about which oil metabolic enzymes help to control avocado oil composition to drive oleic acid content to such high levels in this plant. In the current study, ARS researchers and academic collaborators cloned the genes for two important oil synthesizing enzymes from avocado, and characterized their properties in multiple experimental systems. Both enzymes were found to prefer oleic acid substrates, and may then represent valuable biotechnological tools that can be exploited to produce avocado-like oils in other types of plants or microbes.

Technical Abstract: In higher plants, acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) catalyze the terminal step of triacylglycerol (TAG) synthesis in acyl-CoA-dependent and -independent pathways, respectively. Avocado (Persea americana) mesocarp, a non-seed tissue, accumulates significant amounts of TAG (~70% by dry weight) that is rich in heart-healthy oleic acid. The oil accumulation stages of avocado mesocarp development coincide with high expression levels for type-1 DGAT (DGAT1) and PDAT1, although type-2 DGAT (DGAT2) expression remains low. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional characterization of these three acyltransferases has not been explored to date. We cloned and expressed avocado PamDGAT1, PamDGAT2, and PamPDAT1 in bakers’ yeast and leaves of Nicotiana benthamiana. Heterologous expression of PamDGAT1 successfully complemented the TAG biosynthesis deficiency in the quadruple mutant yeast strain H1246, and substantially elevated total cellular lipid content. Furthermore, in vitro enzyme assays showed that PamDGAT1 prefers oleic acid compared to palmitic acid (16:0). Both PamDGAT1 and PamPDAT1 also increased the lipid content and elevated oleic acid levels when transiently expressed in N. benthamiana leaves. Taken together, these results indicate that PamDGAT1 and PamPDAT1 prefer oleate-containing substrates, and suggest that coordinated expression of these two genes during avocado mesocarp development likely contributes to sustained TAG synthesis that is enriched in oleic acid. This study establishes a knowledge base for future studies focused on exploitation of the biochemical properties of PamDGAT1 and PamPDAT1 to enhance TAG composition in diverse plant tissues.