Increase in lysophosphatidate acyltransferase activity in oilseed rape (Brassica napus) increases seed triacylglycerol content despite its low intrinsic flux control coefficient

Abstract

Summary Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT. We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential. Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14C‐glycerol between phosphoglycerides. Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products.

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.)

“…The increase in TAG content caused by the production of HaLPCAT1 or HaLPCAT3, in the absence of CsFAD2 expression, might be associated with increased acylation of LPA due to the LPAAT activity of these HaLPCATs. These results support the hypothesis that increasing the production of acyltransferases in yeast leads to a greater flux of intermediates and results in enhanced TAG accumulation, as described in plants (Zou et al, 1997;Woodfield et al, 2019). Indeed, yeast expressing HaLPCAT1 or HaLPCAT3 grew faster than yeast expressing HaLPCAT2 in the LPAAT activity assessment assays (Figure 4).…”

Transgenic manipulation of triacylglycerol biosynthetic enzymes in B. napus alters lipid-associated gene expression and lipid metabolism

“…In contrast to GPAT-OE lines, overexpression of the second Kennedy pathway enzyme, LPAT, caused an increase in seed TAG despite its low intrinsic flux control coefficient 24 . Moreover, we show here that LPAT overexpression changed the cellular distribution of PC and TAG molecular species.…”

“…The resultant final plasmid was verified by restriction mapping and sequencing the insert. The construct used to produce LPAT/LPAAT ( TmLPAT2 ) overexpressing lines of B. napus has been described previously 24 .…”

“…This has led to particular plant LPAATs being used to manipulate the final fatty acid content of plant oils 19 and many manipulations have resulted in higher seed oil contents 20 – 23 . We examined the effect of LPAAT overexpression in B. napus in relation to flux control and concluded that the increase in oil occurred despite a low intrinsic flux control coefficient 24 .…”

Working with Randy: The Diacylglycerol Acyltransferase Story

“…In particular, we have been interested in whether any other enzymes in the Kennedy pathway, apart from DGAT, could contribute significantly to the carbon flux into TAG formation. When we looked at lysophosphatidic acid acyltransferase (LPAAT), we found that its overexpression could raise TAG accumulation significantly and our collaborator, David Fell, was able to derive some new (and potentially very useful) equations to allow calculation of flux without using inhibitors to modify the radiolabeling of intermediates (Woodfield et al, 2019). Randy spotted our new publication and e‐mailed me, even before he had received his special “New Phytologist” copy that we had recommended!…”