Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S. polyrhiza, leading to triacylglycerol and carbohydrate accumulation. In FGD wastewater-treated plantlets, the degraded products of monogalactosyldiacylglycerol, primarily polyunsaturated fatty acids (18:3), were incorporated into triacylglycerols. Genes involved in early fatty acid biosynthesis, ß-oxidation, and lipid degradation were upregulated while genes involved in cuticular wax biosynthesis were downregulated by treatment. The transcription factor gene WRINKLED3 (SpWRI3) was upregulated in FGD wastewater-treated plantlets, and its ectopic expression increased tolerance to FGD wastewater in transgenic Arabidopsis (Arabidopsis thaliana). Transgenic Arabidopsis plants showed enhanced glutathione and lower malondialdehyde contents under stress, suggesting that SpWRI3 functions in S. polyrhiza tolerance of FGD wastewater-induced heavy-metal stress. These results provide a basis for improving heavy metal-stress tolerance in plants for industrial applications.

Probing Arabidopsis chloroplast diacylglycerol pools by selectively targeting bacterial diacylglycerol kinase to suborganellar membranes.

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAG kinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGK is small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to other membranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress.

Altered lipid composition and enhanced nutritional value of Arabidopsis leaves following introduction of an algal diacylglycerol acyltransferase 2.

Enhancement of acyl-CoA-dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.

Freezing tolerance in plants requires lipid remodeling at the outer chloroplast membrane.

Plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. SENSITIVE TO FREEZING 2 (SFR2), a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane. SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors, forming oligogalactolipids and diacylglycerol, which is further converted to triacylglycerol. The combined activity of SFR2 and triacylglycerol-biosynthetic enzymes leads to the removal of monogalactolipids from the envelope membrane, changing the ratio of bilayer- to non-bilayer-forming membrane lipids. This SFR2-based mechanism compensates for changes in organelle volume and stabilizes membranes during freezing.

Lipid transport mediated by Arabidopsis TGD proteins is unidirectional from the endoplasmic reticulum to the plastid.

The transfer of lipids between the endoplasmic reticulum (ER) and the plastid in Arabidopsis involves the TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins. Lipid exchange is thought to be bidirectional based on the presence of specific lipid molecular species in Arabidopsis mutants impaired in the desaturation of fatty acids of membrane lipids in the ER and plastid. However, it was unclear whether TGD proteins were required for lipid trafficking in both directions. This question was addressed through the analysis of double mutants of tgd1-1 or tgd4-3 in genetic mutant backgrounds leading to a defect in lipid fatty acid desaturation either in the ER (fad2) or the plastid (fad6). The fad6 tgd1-1 and fad6 tgd4-3 double mutants showed drastic reductions in the relative levels of polyunsaturated fatty acids and of galactolipids. The growth of these plants and the development of photosynthetic membrane systems were severely compromised, suggesting a disruption in the import of polyunsaturated fatty acid-containing lipid species from the ER. Furthermore, a forward-genetic screen in the tgd1-2 dgd1 mutant background led to the isolation of a new fad6-2 allele with a marked reduction in the amount of digalactosyldiacylglycerol. In contrast, the introduction of fad2, affecting fatty acid desaturation of lipids in the ER, into the two tgd mutant backgrounds did not further decrease the level of fatty acid desaturation in lipids of extraplastidic membranes. These results suggest that the role of TGD proteins is limited to plastid lipid import, but does not extend to lipid export from the plastid to extraplastidic membranes.