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Glycerolipid synthesis in leaves - ScienceDirect

Polyclonal antibodies were raised in rabbits to two polypeptides, GLEAETGRLLQDVAREALGDQIPLAVISGP and LASTDQTFADDLQQLLHCGKSFRVYSNPDF, synthesized based on the deduced amino acid sequence of E. coli gpsAFR. Immunoblot analysis of gpsAFR protein from total leaf extracts and purified chloroplasts was performed according to Millenaar et al. (). Proteins (10 μg) were separated by SDS-PAGE and subsequently electrotransferred to nitrocellulose filters using blot transfer buffer (25 m Tris, 192 m glycine, 20% (v/v) methanol). Antiserum for gpsAFR was used as the primary antibody with a dilution factor of 1:1000. Anti-rabbit IgG Fab fragments conjugated to peroxidase (Roche Applied Science) were used as the secondary antibody (1:25,000). The blot was detected for 1.5 min using the protein gel blotting detection reagent ECL+ Plus (Amersham Biosciences) and developed with BioMax MR film (Kodak).

Glycerolipid synthesis in leaves - ResearchGate

Gly-3-P is an intermediary metabolite linking several pathways in the metabolic network (, ,). In addition to glycerolipid synthesis, Gly-3-P metabolism concerns glycolysis, gluconeogenesis (), homeostasis of the cytosolic NADH/NAD+ ratio, and mitochondrial respiration (). Routes of Gly-3-P generation exist both in the cytosolic and plastidic compartments through glycerol-3-phosphate dehydrogenases (, ). Direct glycerol phosphorylation by glycerol kinase () also produces Gly-3-P. The cellular Gly-3-P pool may thus be influenced by a multitude of metabolic factors. In comparison to glycerol/Gly-3-P feeding experiments and mutant studies, transgenic enhancement of Gly-3-P level offers the advantage of not disrupting the associated regulatory circuit. The feedback-resistant gpsAFR gene appears to be particularly potent at increasing Gly-3-P levels, because it was recently reported that overexpression of Gly1 (At2g40690), a Gly-3-P dehydrogenase from Arabidopsis, yielded only a wild-type basal level of Gly-3-P (). The apparent lack of impact of gpsAFR on the transcript level of the Arabidopsis genes directly leading to Gly-3-P production is intriguing. However, our data does not rule out the possibility that these metabolic steps are affected at the enzyme activity level, as exemplified by Gly-3-P feedback inhibition of the gpsAFR protein in E. coli.

Glycerolipid synthesis in Avena leaves during greening …

flux control exerted by the enzyme for lipid synthesis in barley or maize leaves

Although data of indicate that DGAT1 and PDAT1 account for the majority of TAG synthesis in seeds, the possible contribution of these other acyltransferase enzymes to TAG biosynthesis in leaves is largely unknown. As an initial approach to assess possible roles of different Arabidopsis acyltransferases in leaf TAG synthesis, this study has examined the ability of two acyltransferases mutants to synthesize TAG.

The Arabidopsis pdat1 mutant has no seed oil phenotype (); however, in vitro assays suggest that PDAT contributes 10–60% of TAG synthesis in sunflower and safflower microsomes (). Notably, AtPDAT1 is more highly expressed in Arabidopsis leaves than seeds (), but the role of PDAT in leaves is largely unknown, although it has been suggested that it may have a role in removing oxygenated fatty acid (FA) from membrane lipids (). Studies on the overexpression of 35S-PDAT1 in Arabidopsis have also resulted in different results (; ; ). reported no changes in lipid phenotype in Arabidopsis seedlings between 35S-PDAT1 and control plants. More recently, reported up to 8.7% TAG DW–1 in tgd1 mutants transformed with 35S-AtPDAT1/35S-OLEOSIN1. However, Banaś and co-workers did not observe TAG accumulation in WT Arabidopsis transformed with 35S-PDAT, although these plants had increased growth and biomass, resulting in increased FA content on a plant basis (). Thus, the involvement of PDAT1 in leaf lipid metabolism remains uncertain, particularly in non-transformed plants.

an integral component of eukaryotic glycerolipid synthesis.

The rate of fatty acid synthesis in leaves is six-fold higher in the light than in ..

Supplying [14C]12:0 to Arabidopsis leaves resulted in incorporation of radioactivity into all major glycerolipid classes (; at JXB online). At the earliest sampling time (20min), TAG was one of the two most highly labelled lipid classes, with a level similar to [14C[PC. Therefore, although TAG represents Arabidopsis leaves, the initial incorporation of 14C into TAG confirms that young leaf tissue has a high capacity for TAG synthesis without expression of additional genes.

Metabolic networks are often studied as systems downstream of the control of the transcriptome. How an intermediary metabolite influences gene expression in the metabolic network has rarely been studied. Although it is not possible to ascertain whether Gly-3-P serves as a direct or indirect signal in affecting the regulatory architecture of the glycerolipid pathways, our study uncovered clear evidence that an increase in the Gly-3-P level was associated with many transcriptional changes in the glycerolipid pathway enzymes. More importantly, the changes at the transcript level were found to be entirely consistent with the biochemical phenotype, i.e. expression of the prokaryotic pathway enzymes was induced, whereas enzymes of the eukaryotic pathway were suppressed. Thus, although biochemical machinery defines the details as to how the two glycerolipid pathways function as units with distinct underlying biological activities, the Gly-3-P level seems to coordinate their components at the transcript level.

25/10/2016 · Tracking synthesis and turnover of triacylglycerol in leaves
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In etiolated leaves glycerolipid synthesis developed ..

In plants, TAG is synthesized by acylation of diacylglycerol (DAG) by diacylglycerol acyltransferase (DGAT) or phospholipid:diacylglycerol acyltransferase (PDAT) using acyl-CoA or phospholipid as acyl donor, respectively (; ; ; ). Arabidopsis mutants of DGAT1 and double mutants of DGAT1/PDAT1 demonstrate that these two enzymes are responsible for the majority of seed TAG synthesis (), but their contribution to TAG synthesis in leaves is less clear. The level of TAG DW–1 is ~500-fold higher in Arabidopsis seeds compared with leaves (350 μg mg versus 0.6 μg mg DW–1; Y. ; ; ). In contrast, DGAT1 transcripts are only 5-fold more abundant in seed compared with leaves (). Thus, transcript levels suggest that leaves may have inherent capacity for synthesizing TAG, and indeed DGAT activity has been measured in leaves (). An Arabidopsis dgat1 mutant displayed a 10-fold reduction of leaf TAG compared with the wild type (WT) in 4-week-old plants (), although a reduction of TAG in the dgat1 mutant was not observed by . Differences have also been observed from overexpression of DGAT1 in Arabidopsis and Nicotiana leaves (; ).

In this study it was possible to trace leaf glycerolipid synthesis ..

Comparative analysis of individual molecular species within subsets of glycerolipids may shed new light on the alterations of lipid metabolism in the gpsAFR lines. To this end, a complete lipid compositional profile of rosette leaf tissues of 3-week-old seedlings was generated from a lipidomic analysis performed at the Kansas Lipidomic Research Center (). The analysis, which is based on ESI-MS/MS, yields information on phospholipid and glycolipid molecules to the level of the head group, carbon chain length, and degree of unsaturation of the acyl groups. Data presented in were uncorrected for specific response factors of individual molecular species, and the analysis was focused on a comparison of relative mol % of the individual glycerolipid species. In MGDG, there was a significant increase in C34:6, which presumably had a composition of C18:3/16:3 and came from the prokaryotic pathway. Some increase of C34:6 DGDG was also observed, but in DGDG the major change was a decrease in C36:6 (C18:3/18:3) originating from the eukaryotic pathway. Together with the aforementioned sn-2 fatty acyl composition analysis, these results further confirmed the reduced channeling of DAG moieties from the eukaryotic pathway to chloroplasts. In PG, the most significant change was increases in C32:1, C32:0, and C34:0.

(G3P) for glycerolipid synthesis in the plastids.

The increased C16:3 and augmented ratio of total 16- and 18-carbon fatty acids in MGDG and DGDG suggested that the prokaryotic glycerolipid pathway was enhanced in the transgenic lines. To verify this, we performed a compositional analysis of fatty acids at the sn-2 position of MGDG and DGDG. As seen in , the molar percent of sn-2 C-16 fatty acid was significantly higher in MGDG and DGDG of the transgenic lines. Our analysis with PG also showed a similar trend. These results thus confirmed that in the transgenic lines, the enhanced Gly-3-P production was associated with a metabolic adjustment augmenting the relative contribution of the prokaryotic glycerolipid pathway. Based on the firmly established concept that diacylglycerol moieties with an sn-2 C16 fatty acid originate from the prokaryotic pathway (), we were able to deduce the relative contribution of the two glycerolipid pathways in the transgenic lines. As shown in , the relative contribution from the prokaryotic pathway was clearly enhanced. Interestingly, our analysis also showed that there was an increased level of C16 at sn-2 of PC, suggesting that DAG formed by the prokaryotic pathway may also be channeled back for PC synthesis ().

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