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Novel activity-based proteome and lipidome profiling of Arabidopsis in response to a changing environment: An opportunity to identify new key players in plant lipid metabolism

Final Report Summary - LIPIDQUEST (Novel activity-based proteome and lipidome profiling of Arabidopsis in response to a changing environment: An opportunity to identify new key players in plant lipid metabolism)

Publishable summary

LIPIDQUEST addressed the identification of new key players in plant lipid metabolism in response to a changing environment. In addition to their overall functions, lipids are key components of the plant primary metabolism, efficiently storing energy and carbon as plant oil, but also serving as building blocks for the membrane system harboring the complexes of photosynthesis. Our aim was (1) to identify further lipases in (storage) lipid metabolism, and (2) to find activities involved in setting lipid membrane homeostasis in response to a changing environment.
(1) Triacyglycerol (TAG) is the major seed storage reserve in Arabidopsis and fuels postgerminative growth. Blocking lipid turnover results in increased TAG levels in both seeds and vegetative tissue. SUGAR-DEPENDENT 1 (SDP1) was identified as a TAG lipase in Arabidopsis thaliana (Eastmond, 2006), however, other lipases involved in this process such as the DAG or MAG lipases are still under investigation. Also missing are those from the jasmonate biosynthesis or sterolester degradation pathway for example. On the other hand, the Arabidopsis genome carries at least 300 individual genes encoding putative lipases, of which only a few have been functionally characterized so far (Kelly & Feussner, 2016). Lipid enzymes and other lipid-binding proteins are notoriously difficult to handle with respect to their identification and characterization and the huge variety and sometimes very low abundance of lipid molecules (especially intermediates or those involved in signaling) adds to the challenging task of understanding metabolic pathways in lipid biology.

We therefore chose a novel approach by profiling the cellular targets of an analogue of a lipase inhibitor (Orlistat © or tetrahydrolipstatin, an antiobesity drug) in a combined proteomic/mass spectrometry approach. The lipase inhibitor was chemically equipped with a reactive ligand essential for a “click mechanism”, by which novel lipid-binding proteins are bound to the bait and which in turn allows isolation and subsequent identification by proteomics and MS/MS analysis. As expected, more than 100 proteins from 3-days old germinating seedlings from both Arabidopsis and oil-seed rape were found to bind to Orlistat, and some of these are annotated as (putative) lipases. These are currently subjected to further analyses through reverse genetics to understand their role in lipid breakdown during seed germination.
Findings are also evaluated in the context of lipidomic analyses of whole seedlings germinating in the presence of Orlistat. We thus hope to identify novel lipid-binding proteins that have escaped previous efforts to identify lipid-relevant activities in Arabidopsis thaliana.

(2) In a second project we are exploiting the benefits of natural variation to identify candidate genes dealing with adverse growth conditions. The yield of crop plants depends heavily on the plants’ performance in response to the environmental growth conditions, and we are interested in lipid related activities (potentially of economic value) within this context. Natural variation (and with it Quantitative trait loci (QTL) analysis and genome wide association studies (GWAS) of different ecotypes) are increasingly recognized as highly powerful tools to exploit and understand the genetic grounds for naturally existing diverse responses to biotic or abiotic stresses. Through a combination of QTL analysis and GWAS, our group identified a subset of genes, in which single nucleic polymorphisms (SNPs) possibly correlate with changes in seed oil composition. Top of the list is the well-characterized Fatty Acid Desaturase 2 gene (At3g12120) with an SNP in the 5’ untranslated region, which has recently been shown to regulate FAD2 expression (Menard et al., 2017). Other candidate genes on the list include annotated ones, but also some of yet unknown function. To support their predicted involvement in controlling seed oil metabolism, we analyzed the fatty acid phenotype of corresponding insertion mutants. Indeed, the mutants of genes encoding for a ribonucleoprotein family protein and a pentatricopeptide repeat (PPR-like) protein showed differences in their seed fatty acid profile. Interestingly, the PPR-like protein is predicted to be targeted to the chloroplast (which is also the site of FA-biosynthesis). Lastly, the GWAS study also predicted a strong correlation between the seed oil composition and allelic variation of a component of a transcription factor complex. Since no suitable insertion mutant was available, CRISPR-CAS lines were generated to target the candidate gene and homologues, which are currently under investigation. Furthermore, we also studied the lipid composition of different tissues of seedlings from 18 different ecotypes in response to drought or nutrient (phosphate) deprivation. Changes within the lipid phenotype are analyzed in correlation to the observed performance for each ecotype and growth condition. These data now serve as basis for the subsequent (larger) study making use of natural allelic variation that underlies variable phenotypic traits.

Literature cited:

Eastmond PJ. (2006) SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell. 2006 Mar;18(3):665-75.

Kelly AA, Feussner I. (2016) Oil is on the agenda: Lipid turnover in higher plants. Biochim Biophys Acta. 2016 May 4. pii: S1388-1981(16)30117-2.

Menard GN, Moreno JM, Bryant FM, Munoz-Azcarate O, Kelly AA, Hassani-Pak K, Kurup S, Eastmond PJ. (2017) Plant Physiol. 2017 Mar;173(3):1594-1605.

Contact: Dr. Amélie A Kelly, Georg-August-University of Göttingen,Albrecht-von-Haller-Institute for Plant Sciences , Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37077 Göttingen.