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Functional analysis of β-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae

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Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae.

Abstract

Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the β-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.

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Acknowledgements

This research was supported by grants RTC2017-6461-3 from Ministerio de Economía y Competitividad (MINECO) and RTI2018-097755-B-100 from Ministerio de Ciencia, Innovación y Universidades (MICINN) to MA. Funding from Gobierno de Aragón (A09-20R; Function of genes and plant proteins) is also acknowledged. Authors wish to thank Patricia Lorente for skilful technical assistance.

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MA, MVL and RP conceived the research; AC, RRG, MAL and MdlV, performed the experiments; MA, RRG and MVL analysed the data; MA wrote the manuscript.

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Correspondence to Miguel Alfonso.

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11103_2020_1042_MOESM1_ESM.pptx

Supplementary Fig. 1 Phylogenetic tree of the FAE1 gene from different accessions representing species with different erucic acid content. The tree was obtained with boot-straps 500 using the PHYML software. The names and GENBANK accession numbers of the sequences used for the analysis were: Arabidopsis lyrata (GU929425.1); Arabidopsis thaliana (U29142.1); Brassica carinata (KF664166); Brassica napus (U50771.1); Brassica oleracea (GU325726.1); Brassica rapa (GU325723.1); Camelina sativa (KF030545); Capsella bursa-pastoris (KF664171); Cardamine parviflora (KF664172); Crambe abyssinica (KC565742); Crambe hispanica (KF664157); Isatis tinctoria (AY888038.1); Lepidium apetala (KF030527); Lepidium campestre (FJ907545.1); Lymnanthes douglasii (AF247134); Orychophragmus violaceus (AY888042.1); Raphanus raphanistrum (KF664162); Simondsia chinensis (U37088); Sinapsis arvense (AY888041.1); Sinapsis alba (AY888040.1); Thlaspi arvense (KT223024) and Thlaspi perfoliatum (KF030530). Supplementary file1 (PPTX 57 kb)

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Claver, A., de la Vega, M., Rey-Giménez, R. et al. Functional analysis of β-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae. Plant Mol Biol 104, 283–296 (2020). https://doi.org/10.1007/s11103-020-01042-7

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