Downregulation of key genes involved in carbon metabolism in Medicago truncatula results in increased lipid accumulation in vegetative tissue

Alfalfa (Medicago sativa L.), is the most widely grown perennial forage crop, which is a close relative of the model diploid legume Medicago truncatula. However, use of alfalfa lead to substantial greenhouse gas emissions and economic losses related to inefficiencies in rumen fermentation. The provision of supplemental lipids has been used as a strategy to mitigate these issues, but it is a costly approach. The ability to enhance lipid content within the vegetative tissues of alfalfa would therefore be very advantageous. As such, our aim was to assess and select gene candidates to increase total shoot lipid content in M. truncatula using a virus-induced gene silencing (VIGS) approach. We targeted gene homologs of the SUGAR-DEPENDANT 1 (SDP1), ADP-GLUCOSE-PYROPHOSPHORYLASE SMALL SUBUNIT 1 (APS1), TRIGALACTOSYLDIACYLGLYCEROL 5 (TGD5) and PEROXISOMAL ABC TRANSPORTER 1 (PXA1) in M. truncatula for silencing. In this study reduced target transcript levels were confirmed and changes of shoot lipid content and fatty acid composition were measured in replicated experiments. We observed silencing of SDP1, APS1 and PXA1 each resulted in significant increases in shoot total lipid content. This resulted in significantly increased proportions of α-linolenic acid (18:39cis,12cis,15cis) and stearic acid (18:0) levels and significantly decreased levels in the total acyl lipids extracted from vegetative tissues of each of the M. truncatula silenced plants. In contrast, palmitic acid (16:0) levels were significantly decreased in only SDP1 and PXA1-silenced plants.
This study indicate that SDP1, APS1 and PXA1 represent potentially useful targets for a loss-of-function approach in the conventional or molecular breeding of forage legumes to produce genotypes with enhanced lipid content in vegetative tissues. The increased lipid content of these legume forages could increase their energetic value as a feed for cattle and provide an environmental benefit through the reduced production of methane. Enhanced production of 18:3 in these forages could also potentially provide a feed for dairy and beef cattle which leads to milk and meat, respectively, with improved quality. Further research will be required to determine whether the increase in leaf lipid production through the down-regulation of these genes occurs at the expense of protein and or carbohydrate accumulation, or affects other agronomic traits. In the future, it would also be interesting to explore possible synergistic effects on lipid biosynthesis by down-regulating two or more combinations of the four target genes.
Genes of PXA1 and SDP1 would be ideal targets for mutation as a means of improving the quality of alfalfa for increasing feed efficiency and minimizing greenhouse gas emissions from livestock production in the future.