Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT . The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.

Pollen fecundity is crucial to crop productivity and also to biodiversity in general. Pollen development is supported by the tapetum, a metabolically active sporophytic nurse layer that devotes itself to this process. The tapetum in cereals and a vast majority of other plants is of the nonamoeboid type. Unable to reach out to microspores, it secretes nutrients into the anther locule where the microspores reside and develop. Orbicules (Ubisch bodies), studied in various plants since their discovery Ϸ140 years ago, are a hallmark of the secretory tapetum. Their significance to tapetal or pollen development has not been established. We have identified in wheat and rice an anther-specific single-copy gene (per haploid genome equivalent) whose suppression in rice by RNA interference nearly eliminated the seed set. The flowers in the transgenics were normal for female functions, but the pollen collapsed and became less viable. Further characterization of the gene product, named RAFTIN, in wheat has shown that it is present in pro-orbicule bodies and it is accumulated in Ubisch bodies. Furthermore, it is targeted to microspore exine. Although the carboxyl portion of RAFTINs shares short, dispersed amino acid sequences (BURP domain) in common with a variety of proteins of disparate biological contexts, the occurrence RAFTIN per se is limited to cereals; neither the Arabidopsis genome nor the vast collection of ESTs suggests any obvious dicot homologs. Furthermore, our results show that RAF-TIN is essential for the late phase of pollen development in cereals.

SummaryBread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self-pollinating plant with bisexual¯owers that produce short-lived pollen. Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds. We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther-speci®c biochemical function. These eight-exon genes, found at only one copy per haploid complement in this large genome, express speci®cally within the sporophytic tapetum cells. The presence of TAA1 mRNA and protein was evident only at speci®c stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated-microspores. This temporal regulation matched the assembly of wall-impregnated sporopollenin, a phenylpropanoid-lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature. Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum. Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly speci®c sexual interactions at the pollen±pistil interface. This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general.

SummaryA maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identi®ed a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modi®cations. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows »72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.

The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds. The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates. Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase. Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive. We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly. CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect. CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard. In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls. Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells. Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H.Plants synthesize thousands of secondary metabolites from offshoots of primary metabolism (Croteau et al., 2000). In most cases the biosynthetic routes are unknown and even in some of the well-studied pathways many aspects remain uncertain. Phenylpropanoid metabolism generates phenolic intermediates and end products that include lignin monomers, flavonoids, isoflavonoids, lignans, tannins, quinones, and sinapate esters (Strack, 1997; Dixon and Steele, 1999;Nair et al., 2000). Lignin constitutes approximately 15% to 30% of the dry weight in woody plants, and contributes about 30% of the organic carbon in plant biomass in general (Lewis and Yamamoto, 1990; Douglas, 1996; Boudet, 2000). Thus, lignin assembly places a huge demand on phenylpropanoid supply. Lignification is considered a biochemical adaptation to provide mechanical strength and "non-seeping" water transport channels as plants adopted terrestrial habitats. The biosynthetic pathways appear to have been further diversified and recruited to supply metabolites for a variety of other end uses such as attraction of pollinators for promoting sexual propagation, pest deterrence, pathogen resistance, UV radiation protection, and allelopathic exclusion of potentially competing plants (Dixon et al., 1996). The inherent ...