skip to main content


Title: In vivo lipid ‘tag and track’ approach shows acyl editing of plastid lipids and chloroplast import of phosphatidylglycerol precursors in Arabidopsis thaliana
Summary

In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexityin vivo, we developed anin vivolipid ‘tag and track’ method. Essentially, we probed the lipid metabolism inArabidopsis thalianaby expressing a coding sequence for a fatty acid desaturase fromPhyscomitrella patens(Δ6D). This enzyme places a double bond after the 6th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at itssn‐2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to ‘tag and track’ the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence ofER‐derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this ‘tag and track’ method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions.

 
more » « less
NSF-PAR ID:
10064839
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
The Plant Journal
Volume:
95
Issue:
6
ISSN:
0960-7412
Page Range / eLocation ID:
p. 1129-1139
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Summary

    Euonymus alatusdiacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to thesn‐3 position of diacylglycerol to form 3‐acetyl‐1,2‐diacyl‐sn‐glycerol (acetyl‐TAG).EaDAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed thatEaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology ofEaDAcT, we obtained an experimentally determined topology model for a plantMBOAT. TheEaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of theER. In addition, there is a large cytoplasmic loop between the first and secondTMDs, with theMBOATsignature region of the protein embedded in the thirdTMDclose to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase fromEuonymus fortuneipossessed the highest activityin vivoandin vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential forEaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity ofEaDAcT, suggesting that multiple amino acids are important for substrate recognition.

     
    more » « less
  2. Abstract

    Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible.Physaria fendlerinaturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils.

     
    more » « less
  3. Summary

    Long‐chain acyl‐CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil‐producing microalgae, including the diatomPhaeodactylum tricornutum, remain largely unknown.

    InP. tricornutum, a family of five genes (ptACSL1ptACSL5) encodes LACS activities. We generated singlelacsknockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations.

    ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. TheptACSL3mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl‐CoAs for lipid synthesis.

    Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.

     
    more » « less
  4. Summary

    Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green algaC. reinhardtiishowed substantial triacylglycerol (TAG) accumulation and up‐regulation of a gene,GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol‐3‐phosphate dehydrogenase (GPD) domains. CanonicalGPDenzymes catalyze the synthesis of glycerol‐3‐phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone ofTAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. RecombinantChlamydomonasGPD2 showed both reductase and phosphatase activitiesin vitroand it can work as a bifunctional enzyme capable of synthesizing glycerol directly fromDHAP. In addition,GPD2and a gene encoding glycerol kinase were up‐regulated inChlamydomonascells exposed to high salinity.RNA‐mediated silencing ofGPD2revealed that the multidomain enzyme was required forTAGaccumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, aGPD2‐mCherry fusion protein was found to localize to the chloroplast, supporting the existence of aGPD2‐dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities ofPSPGPDmultidomain enzymes may be modulated by post‐translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

     
    more » « less
  5. Summary

    Alternative polyadenylation (APA) is a widespread post‐transcriptional mechanism that regulates gene expression throughmRNAmetabolism, playing a pivotal role in modulating phenotypic traits in rice (Oryza sativaL.). However, little is known about theAPA‐mediated regulation underlying the distinct characteristics between two major rice subspecies,indicaandjaponica. Using a poly(A)‐tag sequencing approach, polyadenylation (poly(A)) site profiles were investigated and compared pairwise from germination to the mature stage betweenindicaandjaponica, and extensive differentiation inAPAprofiles was detected genome‐wide. Genes with subspecies‐specific poly(A) sites were found to contribute to subspecies characteristics, particularly in disease resistance ofindicaand cold‐stress tolerance ofjaponica. In most tissues, differential usage ofAPAsites exhibited an apparent impact on the gene expression profiles between subspecies, and genes with those APA sites were significantly enriched in quantitative trait loci (QTL) related to yield traits, such as spikelet number and 1000‐seed weight. In leaves of the booting stage,APAsite‐switching genes displayed global shortening of 3′ untranslated regions with increased expression inindicacompared withjaponica, and they were overrepresented in the porphyrin and chlorophyll metabolism pathways. This phenomenon may lead to a higher chlorophyll content and photosynthesis inindicathan injaponica, being associated with their differential growth rates and yield potentials. We further constructed an online resource for querying and visualizing the poly(A) atlas in these two rice subspecies. Our results suggest thatAPAmay be largely involved in developmental differentiations between two rice subspecies, especially in leaf characteristics and the stress response, broadening our knowledge of the post‐transcriptional genetic basis underlying the divergence of rice traits.

     
    more » « less