Characterization of a Plastid Triacylglycerol Lipase from Arabidopsis

Abstract

Full-length cDNA corresponding to Arabidopsis (Arabidopsis thaliana) gene At2g31690, which has been annotated in GenBank as a putative triacylglycerol (TAG) lipase, was obtained by reverse transcription-polymerase chain reaction using RNA from senescing rosette leaves of Arabidopsis as a template. The cognate protein was found to contain the lipase active site sequence, and corresponding recombinant protein proved capable of deesterifying TAG. In vitro chloroplast import assays indicated that the lipase is targeted to chloroplasts. This was confirmed by confocal microscopy of rosette leaf tissue treated with fluorescein isocyanate-labeled, lipase-specific antibody, which revealed that lipase protein colocalizes with plastoglobular neutral lipids. Western-blot analysis indicated that the lipase is expressed in roots, inflorescence stems, flowers, siliques, and leaves and that it is strongly up-regulated in senescing rosette leaf tissue. Transgenic plants with suppressed lipase protein levels were obtained by expressing At2g31690 cDNA in antisense orientation under the regulation of a constitutive promoter. Transgenic plants bolted and flowered at the same time as wild-type plants, but were severely stunted and exhibited delayed rosette senescence. Moreover, the stunted growth phenotype correlated with irregular chloroplast morphology. The chloroplasts of transgenic plants were structurally deformed, had reduced abundance of thylakoids that were abnormally stacked, and contained more plastoglobular neutral lipids than chloroplasts of wild-type plants. These observations collectively indicate that this TAG lipase plays a role in maintaining the structural integrity of chloroplasts, possibly by mobilizing the fatty acids of plastoglobular TAG.

Ectopic expression of WRI1 affects fatty acid homeostasis in Brachypodium distachyon vegetative tissues

“…To examine the origin of free FAs in these lines, the expression of possible orthologs encoding enzymes involved in the degradation of TAGs was analyzed by RT-PCR. Arabidopsis SDP1 encodes a TAG lipase that catalyzes the initial step in TAG breakdown (Eastmond, 2006;Padham et al, 2007), while the b-oxidation pathway subsequently breaks down the released FAs (Goepfert and Poirier, 2007). As shown in Figure 6C, both presumed orthologs of SDP1 (BdSDP1-1 [Bradi2g50610] and BdSDP1-2 [Bradi1g04310]) and presumed b-oxidation pathway genes, such as ACYL-CoA OXIDASE1 (ACX1; Bradi1g52320), ACX2 (Bradi4g14090), KETOACYL-CoA THIOLASE1 (KAT1; Bradi3g27960), KAT2 (Bradi3g55420), LONG-CHAIN ACYL-CoA SYNTHEASE6 (LACS6; Bradi4g26610), LACS7 (Bradi4g42950), and MULTIFUNC-TIONAL PROTEIN2 (MFP2; BdMFP2-1 [Bradi2g43020], BdMFP2-2 [Bradi4g28310], and BdMFP2-3 [Bradi4g28310]), were induced, indicating that ectopic expression of BdWRI1 also might cause accelerated TAG turnover.…”

DONGLE and DEFECTIVE IN ANTHER DEHISCENCE1 Lipases Are Not Essential for Wound- and Pathogen-Induced Jasmonate Biosynthesis: Redundant Lipases Contribute to Jasmonate Formation

“…Therefore, a plastidic localization of the lipases involved in JA biosynthesis would be expected. Among the seven PLA-I lipases, plastidic localization has unambiguously been shown for PLAIb1 (DAD1) and PLA-Ia2 (a putative TAG lipase; Ishiguro et al, 2001;Padham et al, 2007). Recently, plastidic localization of the other five PLA-I class lipases, including PLA-Ia1 (DGL), has been shown by transient expression of eGFP-tagged fusion proteins (Hyun et al, 2008;Seo et al, 2009).…”

Living to Die and Dying to Live: The Survival Strategy behind Leaf Senescence

“…Upon senescence, lipid synthesis rates are reduced, while the peroxisomal b-oxidation pathway is up-regulated (Christiansen and Gregersen, 2014). In Arabidopsis (Arabidopsis thaliana), remobilization of chloroplast lipids is essential for normal plant growth, the onset of senescence, and reproductive success (Padham et al, 2007).…”