Lysophosphatidyl acyltransferase (LPAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. We examined putative LPAT genes in Arabidopsis thaliana and characterized two related genes that encode the cytoplasmic LPAT. LPAT2 is the lone gene that encodes the ubiquitous and endoplasmic reticulum (ER)-located LPAT. It could functionally complement a bacterial mutant with defective LPAT. LPAT2 and 3 synthesized in recombinant bacteria and yeast possessed in vitro enzyme activity higher on 18:1-CoA than on 16:0-CoA. LPAT2 was expressed ubiquitously in diverse tissues as revealed by RT-PCR, profiling with massively parallel signature sequencing, and promoter-driven b-glucuronidase gene expression. LPAT2 was colocalized with calreticulin in the ER by immunofluorescence microscopy and subcellular fractionation. LPAT3 was expressed predominately but more actively than LPAT2 in pollen. A null allele (lpat2) having a T-DNA inserted into LPAT2 was identified. The heterozygous mutant (LPAT2/lpat2) had minimal altered vegetative phenotype but produced shorter siliques that contained normal seeds and remnants of aborted ovules in a 1:1 ratio. Results from selfing and crossing it with the wild type revealed that lpat2 caused lethality in the female gametophyte but not the male gametophyte, which had the redundant LPAT3. LPAT2-cDNA driven by an LPAT2 promoter functionally complemented lpat2 in transformed heterozygous mutants to produce the lpat2/lpat2 genotype. LPAT3-cDNA driven by the LPAT2 promoter could rescue the lpat2 female gametophytes to allow fertilization to occur but not to full embryo maturation. Two other related genes, putative LPAT4 and 5, were expressed ubiquitously albeit at low levels in diverse organs. When they were expressed in bacteria or yeast, the microbial extract did not contain LPAT activity higher than the endogenous LPAT activity. Whether LPAT4 and 5 encode LPATs remains to be elucidated.

Oxalate is produced by several catabolic pathways in plants. The best characterized pathway for subsequent oxalate degradation is via oxalate oxidase, but some species, such as Arabidopsis thaliana, have no oxalate oxidase activity. Previously, an alternative pathway was proposed in which oxalyl-CoA synthetase (EC 6.2.1.8) catalyzes the first step, but no gene encoding this function has been found. Here, we identify ACYL-ACTIVATING ENZYME3 (AAE3; At3g48990) from Arabidopsis as a gene encoding oxalyl-CoA synthetase. Recombinant AAE3 protein has high activity against oxalate, with K m = 149.0 6 12.7 mM and V max = 11.4 6 1.0 mmol/min/mg protein, but no detectable activity against other organic acids tested. Allelic aae3 mutants lacked oxalyl-CoA synthetase activity and were unable to degrade oxalate into CO 2 . Seeds of mutants accumulated oxalate to levels threefold higher than the wild type, resulting in the formation of oxalate crystals. Crystal formation was associated with seed coat defects and substantially reduced germination of mutant seeds. Leaves of mutants were damaged by exogenous oxalate and more susceptible than the wild type to infection by the fungus Sclerotinia sclerotiorum, which produces oxalate as a phytotoxin to aid infection. Our results demonstrate that, in Arabidopsis, oxalylCoA synthetase encoded by AAE3 is required for oxalate degradation, for normal seed development, and for defense against an oxalate-producing fungal pathogen.

In plants, subcellular triacylglycerol granules in seeds (oil bodies) and floral tapetum (tapetosomes) are stabilized by amphipathic structural protein called oleosin. We hereby report a novel group of oleosins that is present inside the pollen of Arabidopsis thaliana. We have used the conserved sequence of oleosins to locate, via the DNA database, all 16 oleosin genes in the Arabidopsis genome. The oleosin genes can be divided into three groups according to their sequences and tissue-specific expressions, as probed by RNA blot hybridization and reverse transcriptase-PCR. The first group includes eight genes specifically expressed in the floret tapetum. The second group includes five genes specifically expressed in maturing seeds. The third, novel group includes three genes expressed in both maturing seeds and floral microspores, which will become pollen. Transgenic study using the promoter of one of these genes attached to a reporter gene has provided corroborative evidence for the specific expression of the gene in the microspores in the florets. One of the pollen oleosins can be identified by microsequencing and specific immunoblotting. Pollen oleosins synthesized by recombinant bacteria can collaborate with phospholipids in stabilizing reconstituted oil bodies. Thus, pollen has oleosins to stabilize the abundant subcellular oil bodies. Seed oil bodies and floret tapetosomes have been isolated from the miniature Arabidopsis plants, and the success indicates that the organelles can be subjected to future biochemical and genetic studies.