Control of early seed development.

Seed development requires coordinated expression of embryo and endosperm and has contributions from both sporophytic and male and female gametophytic genes. Genetic and molecular analyses in recent years have started to illuminate how products of these multiple genes interact to initiate seed development. Imprinting or differential expression of paternal and maternal genes seems to be involved in controlling seed development, presumably by controlling gene expression in developing endosperm. Epigenetic processes such as chromatin remodeling and DNA methylation affect imprinting of key seed-specific genes; however, the identity of many of these genes remains unknown. The discovery of FIS genes has illuminated control of autonomous endosperm development, a component of apomixis, which is an important developmental and agronomic trait. FIS genes are targets of imprinting, and the genes they control in developing endosperm are also regulated by DNA methylation and chromatin remodeling genes. These results define some exciting future areas of research in seed development.

Maternal control of seed development.

Maternal control of higher plant seed development is likely to involve female sporophytic as well as female gametophytic genes. While numerous female sporophytic mutants control the production of the ovule and the embryo sac true maternal effect mutations affecting embryo and endosperm development are rare in plants. A new class of female gametophytic mutants has been isolated that controls autonomous development of endosperm. Molecular analyses of these genes, known as FIS class genes, suggest that they repress downstream seed development genes by chromatin remodelling. Expression of the FIS genes in turn is modulated by parent specific expression or genomic imprinting which in turn is controlled by DNA methylation. Thus maternal control of seed development is a complex developmental event influenced by both genetic and epigenetic processes.

Polycomb group genes control pattern formation in plant seed.

Transcriptional activators of the Trithorax group (TRX-G) and repressors of the Polycomb group (Pc-G) are involved in multiple aspects of embryogenesis in Drosophila and the mouse [1, 2] and appear to have a conserved role in the zygotic control of the development of the anterior-posterior axis [3, 4, 5]. In the model plant Arabidopsis, three Pc-G genes have been isolated and characterized to date. CURLY LEAF (CLF) represses the expression of a floral homeotic gene in vegetative tissues but does not appear to have a role in plant embryogenesis [6]. Two other Pc-G genes, FIS1/MEDEA [7, 8, 9], and FIS3/FIE [8, 10] have been characterized in studies of mutants that produce seeds in the absence of fertilization. Seeds resulting from autonomous development in fis mutants do not contain an embryo but only endosperm, the second product of double fertilization in flowering plants [11, 12]. Thus, FIS genes are considered to be repressors of endosperm development before fertilization. We report that when fis ovules are fertilized, the endosperm patterning along the major polar axis is perturbed. Posterior structures develop in more anterior domains of the endosperm. This correlates with the ectopic expression of a posterior molecular marker. FIS genes appear to be potent regulators of the establishment of the anterior-posterior polar axis in the endosperm.

Genes controlling fertilization-independent seed development in Arabidopsis thaliana.

We have cloned two genes, FIS1 and FIS2, that control both fertilization independent seed development and postpollination embryo development in Arabidopsis. These genes confer female gametophytic phenotypes. FIS2 encodes a protein with a C2H2 zinc-finger motif and three putative nuclear localization signals, indicating that it is likely to be a transcription factor. FIS1 encodes a protein with homology to the Drosophila Polycomb group gene Enhancer-of-zeste and is identical to the recently described Arabidopsis gene MEDEA. FIS1 is a protein with a number of putative functional domains, including the SET domain present in Enhancer-of-zeste-related proteins. Comparison of the position of the lesions in the fis1 and medea mutant alleles indicates that fis1 is a null allele producing a truncated polypeptide lacking all the protein domains whereas the deduced protein from medea lacks only the SET domain. We present a model of the role of FIS1 and FIS2 gene products in seed development.

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants.

Seven new male-sterile mutants (ms7-ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4',6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified.

Ovule and embryo development, apomixis and fertilization.

Genetic analyses, particularly in Arabidopsis, have led to the identification of mutants that define different steps of ovule ontogeny, pollen stigma interaction, pollen tube growth, and fertilization. Isolation of the genes defined by these mutations promises to lead to a molecular understanding of these processes. Mutants have also been obtained in which processes that are normally triggered by fertilization, such as endosperm formation and initiation of seed development, occur without fertilization. These mutants may illuminate apomixis, a process of seed development without fertilization extant in many plants.

Fertilization-independent seed development in Arabidopsis thaliana.

We report mutants in Arabidopsis thaliana (fertilization-independent seed:fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, approximately 50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.

pEmu: an improved promoter for gene expression in cereal cells.

A recombinant promoter, pEmu, has been constructed to give a high level of gene expression in monocots. It is based on a truncated maize Adh1 promoter, with multiple copies of the Anaerobic Responsive Element from the maize Adh1 gene and ocs-elements from the octopine synthase gene of Agrobacterium tumefaciens. The pEmu promoter was one of 12 different promoter constructs that were linked to the ß-glucuronidase (GUS) marker gene. Promoter activity was measured 48 h after introduction of the constructs into protoplasts of five different monocot species [wheat, maize, rice, einkorn (Triticum monococcum), and Lolium multiflorum] and one dicot (Nicotiana plumbaginifolia). In suspension cell protoplasts, the most highly expressing construct (pEmuGN) gave 10- to 50-fold higher expression than the CaMV 35S promoter in all the monocot species. The pEmu promoter should be valuable where a high level of gene expression is required in monocots. The pEmu promoter showed instability in several widely used Escherichia coli strains but was stable in a recA, recD strain AC001, which is described. Another construct, p4OCSΔ35SIGN, gave a tenfold increase in expression over the CaMV 35S promoter in dicot (Nicotiana plumbaginifolia) protoplasts.

Relative regeneration proficiency ofArabidopsis thaliana ecotypes.

We have compared regeneration proficiency for cultured explants from different tissues of different ecotypes ofArabidopsis thaliana. Proficiency varies widely with both tissue and ecotype, and is highest when the flux of light during regeneration is low. Analysis of F1 hybrids suggests that high proficiency is dominant to low proficiency.

recD: the gene for an essential third subunit of exonuclease V.

Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes. We report here that the enzyme contains in addition a third polypeptide (alpha) with a molecular mass of about 58 kDa. The alpha polypeptide is not synthesized by a class of mutants (previously designated recB) lacking the nuclease activity of exonuclease V but retaining recombination proficiency. The gene, recD, coding for the alpha polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-argA on the E. coli chromosome. The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone. In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.

Role of Escherichia coli RecBC enzyme in SOS induction.

Induction of the SOS genes is required for efficient repair of damaged DNA in Escherichia coli. SOS induction by nalidixic acid or oxolinic acid, two inhibitors of DNA gyrase, requires the RecBC enzyme of E. coli. We report here that the nuclease activity of RecBC enzyme is not needed for SOS induction by these agents. We suggest that the unwinding activity of RecBC enzyme produces single-stranded DNA which activates the RecA protein to stimulate LexA repressor cleavage and SOS induction.

A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination.

Mutants of Escherichia coli sensitive to phage T4 gene 2 mutants were obtained following ethyl methanesulfonate mutagenesis. By mapping and complementation analysis, the mutations in each of the six mutants are in recB and recC. By both in vivo and in vitro analyses, the nuclease activity of RecBC enzyme is undetectable in these mutants. However, by several other criteria, such as proficiency in recombination, relative resistance to UV radiation, and viability of the cells in the culture, these mutants are almost identical to their recBC+ parent. The properties of these mutants indicate that the ATP-dependent double-stranded DNA exonuclease activity of RecBC enzyme is not required for recombination. Chi recombinational hotspots, which stimulate recombination by the RecBC pathway, have no detectable activity in the mutants. This result suggests that the nuclease activity of RecBC enzyme is required for Chi activity and is consistent with the hypothesis that Chi stimulates recombination by directing RecBC enzyme to cut DNA at or near Chi.

Escherichia coli recBC deletion mutants.

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.