Plants distinguish among the pollen grains that land on the stigma, permitting only compatible pollen to fertilize egg cells. To investigate these cell-cell interactions, Arabidopsis mutations that affect pollen-pistil communication were isolated. A male-sterile mutation that disrupts pollen-pistil interactions by eliminating the extracellular pollen coat (tryphine) is described here. Stigma cells that contact the mutant pollen produce callose, a carbohydrate synthesized in response to foreign pollen. The mutant pollen fails to germinate because it does not absorb water from the stigma, yet germinates in vitro, indicating it is viable. The defect is also conditional; high humidity results in pollen hydration and successful fertilization. Analysis of mature, mutant pollen indicated that it is deficient in long-chain lipids and has none of the lipoidic tryphine normally present on its surface. Immature mutant pollen grains have aberrant tryphine that disappears during pollen development. The sterile plants also lack stem waxes, and pollen from other wax-defective (eceriferum) mutants with reduced fertility has few of the lipid droplets normally present in tryphine. These results demonstrate that tryphine is critical for pollen-stigma interactions and suggest that tryphine lipids are required for fertilization, either by directly signaling the stigma or by stabilizing other tryphine components.[Key Words: Pollen; Arabidopsis; compatibility; lipids; tryphine; eceriferum] Received February 22, 1993; revised version accepted April 7, 1993. Fertilization in flowering plants involves many cell-cell interactions, including adhesion of pollen grains to the -stigma surface, growth of pollen tubes through the pistil, migration of sperm cells within the pollen tube, and, ultimately, fusion of the sperm with egg cells; yet few of the molecules required for these interactions have been identified. As a first step in understanding these cellsignaling events, several sterile Arabidopsis mutants blocked in early steps of fertilization were identified. In this paper, one such mutant, defective in the interaction between pollen grains and stigma cells, is described.The signaling events that occur during fertilization presumably require direct contact between cells and rely on molecules present in low abundance. Few of these molecules have been identified through in vitro reconstitution experiments, in part because manipulation of reproductive tissues is technically difficult. In contrast, genetic approaches depend solely on the function of molecules, rather than on their abundance, and allow cellcell communication events to be identified and characterized in living plants. Because most plant species are ~Corresponding author.hermaphrodites (producing both sperm and eggs), mutant plants defective in either the male or the female reproductive process can be obtained and propagated readily.Arabidopsis thaliana is well-suited to a genetic approach to fertilization. The advantages of its short life cycle, small physical size, and sma...

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

A diverse collection of mutants of Arabidopsis with altered seed lipid compositions was isolated by determining the fatty acid composition of samples of seed from 3,000 mutagenized lines. A series of mutations was identified that caused deficiencies in the elongation of 18∶1 to 20∶1, desaturation of 18∶1 to 18∶2, and desaturation of 18∶2 to 18∶3. In each of these cases the wild type exhibited incomplete dominance over the mutant allele. These results, along with results from earlier studies, point to a major influence of gene dosage in determining the fatty acid composition of seed lipids. A mutation was also isolated that resulted in increased accumulation of 18∶3. On the basis of the effects on fatty acid composition, the nature of the biochemical lesion in three of the mutants could be tentatively attributed to deficiencies in activities of specific enzymes. The other mutant classes had relatively less pronounced changes in fatty acid composition. These mutants may represent alterations in genes that regulate lipid metabolism or seed development. The availability of the mutants should provide new opportunities to investigate the mechanisms that control seed lipid fatty acid composition.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

As in most terrestrial plants, the cuticle on Arabidopsis thaliana (L.) Heynh. forms a continuous lipid membrane over the apical epidermal cell walls of essentially all aerial plant organs. Epicuticular waxes form the outermost layer over this membrane and are visible on Arabidopsis inflorescence stem and silique surfaces as a bluish-white colored coating called glaucousness or waxy bloom. Intracuticular waxes are intermeshed within the cuticle membrane and not visible to the naked eye. Close examination of epicuticular waxes on Arabidopsis stems and siliques using scanning electron microscopy (SEM) at around 3000x magnification best reveals their diverse crystalline structures ( Figure 1A). The stem and silique epicuticular wax morphology is composed primarily of columnar-shaped crystals (of ~1.0 μm diameter), although rods, tubes, vertical plates, dendritic-, and umbrella-like structures are also typically present. The non-glaucous rosette and cauline leaf surfaces of Arabidopsis lack wax crystals detectable at the level of SEM ( Figure 1B). Interestingly, other organs possess epicuticular wax crystals, including those of the pistil (Bowman, 1993). Descriptions of wax crystalline morphology on other flower parts, seeds, seedlings, roots, and other organs of Arabidopsis have not been reported.Whereas the term epicuticular wax is used to describe wax crystals above of the cuticle, cuticular wax is being used here to describe those long chain lipids extracted by submersion of tissues in solvents like hexane and chloroform since this extraction procedure likely removes both epicuticular and intracuticular waxes. The chemical composition of cuticular waxes on Arabidopsis leaves and stems are typical of those on many dicotyledonous plants, being composed primarily of saturated free fatty acids, aldehydes, alkanes, primary alcohols, secondary alcohols, ketones, and wax esters (Figure 2). Within these component classes, homologues occur as aliphatic chains of between 16 and 33 carbons, except the wax esters, which are composed of even more carbons. The dominant wax class on Arabidopsis leaves and stems is the alkanes, although primary alcohols comprise a significant wax fraction on these surfaces. Stems and siliques possess relatively high amounts of ketones and secondary alcohols, whereas rosette and cauline leaves possess these constituents in trace or undetectable amounts. Arabidopsis lacks the wax hydroxy-ß-diketones, ß-diketones, and alkan-2-ol esters found on certain monocots (Bianchi and Bianchi, 1990), the estolides only reported in gymnosperms, and other minor constituents that occur idiosyncratically in plants that have been examined (Walton 1990). Inflorescence stems of wild-type Arabidopsis can produce over ten fold more total wax per area than leaves, and stem wax chain length distribution is shorter than leaves, with the C 29 alkane homologue dominating stem waxes but the C 31 alkane dominating leaf waxes (Jenks et al., 1995). Rosette leaves possess lower relative amounts of primary alcohols than cauline ...