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.

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.

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