Chloroplasts in plants and algae are organelles that carry out a number of important functions including photosynthesis, fatty acid biosynthesis and oxylipin mediated plant immune responses. Chloroplast membranes with a unique lipid composition are crucial for the functions of chloroplasts. Chloroplast membranes are composed of a specialized glycerolipid matrix with predominately galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), and two anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol. The lipid composition of the chloroplast membranes is finely tuned in response to stresses or during various developmental stages to maintain plant fitness.SENSITIVE TO FREEZING 2 (SFR2) is a galactolipid : galactolipid galactosyltransferase that is highly conserved in land plants. In response to freezing, SFR2 is activated in freezing tolerant plants like Arabidopsis and remodels MGDG to form a class of oligogalactolipids that stabilize the bilayer structures. Phylogenetically, SFR2 is highly conserved in land plants including those that are freezing sensitive. Therefore, I hypothesized that SFR2 in freezing sensitive plants may be involved in plant resilience to other abiotic stresses, like salt or drought stresses because similar to freezing, salt and drought stress cause cell dehydration. In Chapter 2, freezing sensitive tomato was used as a model to test this hypothesis. I generated the SlSFR2 RNAi lines and observed their hypersensitivity to salt and drought responses. Decreased tolerance was correlated with compromised production of oligogalactolipids in the SlSFR2 RNAi lines, suggesting SFR2 plays a role in salt and drought tolerance in freezing sensitive plants.Maintenance of the chloroplast membranes requires a finely tuned lipid anabolic and catabolic machinery. Contrary to the chloroplast lipid biosynthetic enzymes, a large number of the predicted lipid-degrading enzymes in the chloroplasts have unknown functions. In Chapter 3, I identified over 50 chloroplast localized lipases in silico. Using a reverse genetic approach, I first focused on the characterization of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A1. In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific PG that contains 16:1Δ3trans at the sn-2 glyceryl position. PLIP1 is predominately expressed in seeds, and the plip1 mutant seeds contain less oil while the PLIP1 overexpression seeds contain more compared to the wild type. Pulse-chase labeling assays suggest that the acyl groups released by PLIP1 are exported from the chloroplasts, reincorporated into phosphatidylcholine before ultimately entering seed triacylglycerol. Therefore, 16:1Δ3trans uniquely labels a biochemical active PG pool in developing Arabidopsis embryos, which is subject to PLIP1 mediated acyl export and seed oil biosynthesis.The Arabidopsis genome encodes two putative PLIP1 paralogs which are designated PLIP2 and 3. PLIP2 and PLIP3 are also localized in the chloroplasts, but with different subplastid locations. PLIP2 possesses similar biochemical properties in vitro to PLIP1, but in vivo studies suggest that PLIP2 prefers MGDG as substrate, while PLIP3 prefers PG. Overexpression of PLIP2 or PLIP3 severely stunts plant growth and leads to accumulation of oxylipins. Genetically blocking jasmonate signaling restored the growth of the overexpression plants. The expression of PLIP2 and PLIP3, but not PLIP1 is induced by abscisic acid (ABA), and plip1,2,3 triple mutants exhibit compromised jasmonate biosynthesis in response to ABA. Therefore, in Chapter 4, I am proposing that PLIP2 and PLIP3 are involved in linking ABA responses with jasmonate production.
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