Lipidome and Transcriptome Profiling of Omega-3 Fatty Acid Rich Plant Leaves

Lipidome and transcriptome profiling of omega-3 fatty acid rich plant leaves

Thesis submitted to AcSIR for the Award of the Degree of

DOCTOR OF PHILOSOPHY

In the faculty of BIOLOGICAL SCIENCES

V. VENKATESHWARI Registration No. 10BB12A08013

Under the guidance of Dr. Malathi Srinivasan Prof. Ram Rajasekharan

Lipidomic Lab Department of Lipid Science CSIR-CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE Mysuru-570020 India

June 2018

Chapter 5 Discussion

Portulaca oleracea and Talinum fruticosum leaves are rich sources of omega-3 fatty acids. Till date, no attempt has been made to study the expression pattern of the genes that are involved in lipid metabolism, especially related to fatty acid biosynthesis in these plants. In the present study, we found that the level of total lipids and the fatty acids were high in the leaves of both Portulaca and Talinum. The total lipid content in Portulaca, particularly that of ALA is approximately three times more than that of earlier reports in plants. Among the total leaf lipids of Portulaca (7.2 g/100 g), significant amounts of galactolipids (GL) 3.0 g/100 g, phospholipids (PL) 2.5g/100 g, and nonpolar lipids (NL) 1.7 g/100 g were present. The total leaf lipids of Talinum is around 7.7 g/100 g, with a remarkable amount constituted by the galactolipids (GL) 3.2 g, followed by phospholipids (PL) 2.4 g, and nonpolar lipids (NL) 2.1 g. The distribution of fatty acids in all the three lipid classes was also found to vary in both the plants. The predominant fatty acid in the galactolipid fractions is ALA. Studies have shown that omega-3 fatty acids associated with the phospholipid form is better than nonpolar form (triacylglycerol) in obese mice which helps to improve the metabolic profile (Rossmeisl et al., 2012). Omega-3 fatty acid in phospholipid form has capacity to enhance the bioavailability of DHA and EPA in rodents (Cansell et al., 2003) and humans (Schuchardt et al., 2011). Lipidome analysis of both Portulaca and Talinum showed a remarkable increase in the galactolipid (MGDG) levels compared to the other lipids. Further, the lipidome data revealed that the unsaturated fatty acid (ALA) was more in the molecular species of galactolipids and phospholipids, than in nonpolar lipids. Using radiolabel studies we observed that the initial incorporation of label is more in PC compared to other polar lipids. While this slowly reduced with time, a concomitant increase in nonpolar lipids, especially of TAG, was observed. These results suggest that the high level of labeled PC may be directly converted to DAG to form TAG by PDAT (Dahlqvist et al., 2000). In plants, nonpolar TAG constitutes <1% of leaf glycerolipids; however, leaves can accumulate more TAG if they are supplemented with fatty acids or left under stress conditions (Tjellstrom et al., 2015). Research has shown that young leaves are capable of synthesizing TAG within 30 min of the addition of 14C-lauric acid (Koo et al., 2005). These data together suggest that PC serves as a carrier of acyl chains from plastids to ER and plays a crucial role in membrane lipids and TAG biosynthesis.

De novo sequencing of Portulaca transcriptome reveals 10,015 clustered transcripts from 38.75 million high-quality reads. Transcriptome data were annotated using GO, KOG and KEGG, functional categorization of expressed genes showed important pathways like fatty acid and TAG biosynthesis. Transcripts involved in this biosynthesis provide the foundation for further studies on molecular mechanisms underlying PUFA biosynthesis in Portulaca leaves. In particular, genes such as FAD2, FAD3 and FAD7/8 involved in PUFA biosynthesis would provide a key in understanding the mechanism of ALA accumulation in leaves. We found that ∆-15 desaturase (FAD7/8) enzyme responsible for the conversion of LA to ALA was 200 fold up-regulated in the leaves of the portulaca accession that was selected, namely PoRR04. This supports our GC-MS and lipidome data that

Portulaca leaves have high content of ALA due to the high expression of FAD7/8. This is the first report to our knowledge on the transcriptome of Portulaca, which aids in understanding the mechanisms of ALA-rich lipid accumulation in the leaves of PoRR04. Similar studies have been carried out recently; overexpression of FAD7 in tomato increases the ALA level, which led to series of physiological altercations (Liu et al., 2013). Up-regulation of FAD3 and FAD7 in Sacha Inchi seeds also plays an important role in ALA accumulation (Hu et al., 2018). Another enzyme of interest in lipid metabolism is LPCAT which was also identified in the RR04 transcriptome. This sequence was found to share a good identity (76%) with known plant LPCATs, prompting us to further annotate and characterize. LPCAT1 and LPCAT2 overexpression in Arabidopsis showed a significant increase in PUFAs in seed oil (Wang et al., 2012). This LPCAT participates in the turnover of phospholipids to maintain the membrane lipid composition and the asymmetrical distribution of unsaturated fatty acids within the phospholipids. In this study, we purified the microsomal membrane-bound fraction from the leaves of RR04. The solubilized microsomal membrane has a more pronounced role in esterifying LPC with varying chain lengths compared to other lysolipids. Further, the proteins from the active fraction of native-PAGE were identified by mass spectrometry; the major protein being an LPCAT. The recombinant LPCAT from Escherichia coli has been shown to have a lysophosphatidylcholine acyltransferase activity.

It is always debatable as to how and when lipids synthesized in ER pathways are exported to the chloroplast and used as a precursor for galactolipid synthesis. The transcripts involved in fatty acid, galactolipids and triacylglycerol biosynthesis were upregulated; β-oxidation and TAG degradation

89 transcripts were normal compared to control suggesting that these transcripts play an essential role in the lipid accumulation in the leaves. The schematic representation shows the leaf triacylglycerol biosynthetic pathway in the RR04 accession of portulaca.

Among the screened plants for omega-3 fatty acids, Talinum fruticosum was another plant that showed the highest ALA content in the leaves like portulaca. These leaves are inexpensive, easy to cook and used in a normal diet to prevent degenerative diseases. However, there are no reports about the lipid content, fatty acid composition and the lipid biosynthesis of this plant. This work reports the lipid classes, their constituent fatty acids of the leaves of T. fruticosum. Talinum has a 58% ALA in the membrane lipids and ~7.7 g lipid/100 g fresh weight of total leaf lipids. Here we provide lipidome and transcriptome analysis of the leaves of T. fruticosum to identify the genes involved in the fatty acid and triacylglycerol biosynthesis. From Talinum transcriptome, a total of 104776 transcripts were generated with an N50 length of 1605 bp. Amongst KOG analysis, under metabolism category, the major one was lipid metabolism and transport. To further unravel pathway related to lipid metabolism using KEGG analysis, the major routes are glycerophospholipid and glycerolipid metabolism, fatty acid biosynthesis and alpha-linolenic acid metabolism. The expression level of FAD2, FAD3, FAD6 and FAD7/8 genes were high, which supports the high level of ALA accumulation in Talinum leaves. Apart from desaturase genes, the most important enzyme in TAG biosynthesis is DGAT3. This was upregulated in Talinum transcriptome.

From our study, we found an alternative sustainable, economical, cheap leaf source of omega-3 fatty acid (Portulaca and Talinum) and these chloroplast lipids will be the future new food ingredient.